Aptamers and Clinical Applications

  • Gov Lists 28 Clinical Studies, Mostly Ocular, for Aptamers
  • Only 3 On This List Are Currently Active Studies
  • NOXXON Pharma’s Mirror-Image L-RNA Aptamer is in the Clinic for Cancers

Devoted readers of Zone In With Zon who have photographic memories—or anyone who simply uses this blog’s search engine—will know that my first blog on aptamers touted these nucleic acids as being better than antibodies in many applications. That deliberately provocative proclamation was followed by a second blog on aptamers featuring top-cited publications, and also included aptamer studies which cited TriLink products in the methods section. The present, third blog on aptamers focuses entirely on clinical applications.

In the interest of full disclosure, picking this blog’s titled topic was catalyzed, so to speak, by my being invited to contribute a chapter devoted to clinical aspects of aptamers for a book on nucleic acids therapeutics to be published in 2019. After this book becomes available, I’ll be able to comment on its well-known co-editors, many contributors, and comprehensive contents. However, for now, here are “sneak previews” of some items that will be covered in my chapter.

Overview of Aptamer Functional Versatility

Taken from genelink.com

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. Clinical applications of aptamers, with or without chemical modifications, are all predicated on target-specific binding.

However, as depicted below for diverse examples, aptamers can function as therapeutic agents per se, or as targeting agents. Direct agency of an aptamer drug can include binding to either extracellular protein, cell-surface protein, or viral surface protein, wherein each target is associated with a specific disease or clinical indication of interest for achieving therapeutic intervention. These three targets are also addressable by conventional small molecules or antibodies; however, it was recognized early on that aptamers might provide advantages in specificity or cost, respectively.

Cartoon depicting various modes for RNA aptamers (shown as stem-loop structures) functioning as therapeutic agents (left panel) or as cell-targeting agents (right panel). Figure from Poolsup & Kim with permission.

Use of aptamers as targeting agents is depicted as conjugates for delivery of either a nanoparticle loaded with drug, therapeutic oligonucleotide, such as short-interfering RNA (siRNA), or small molecule drug, such as a cytotoxic agent. Although the present blog is devoted to the first modality, namely, aptamers as therapeutic (i.e. clinical) agents, a review by Poolsup & Kim can be consulted for information on the second mode, namely, aptamers as targeting agents.

Aptamer Drugs Listed in ClinicalTrials.Gov

ClinicalTrials.gov is my “go to” website for obtaining reliable information on clinical investigations in general. This freely accessible website is a comprehensive registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. This trove of information, which is provided as a service of the U.S. National Institutes of Health, is currently comprised of 261,814 clinical research studies in all 50 states and in 201 countries.

Basic information for each study listed in ClinicalTrials.gov includes the sponsor, participating investigators and hospitals, aptamer drug, targeted disease(s), and type of study (aka Phase), i.e. safety (Phase 1), efficacy (Phase 2), and better than standard of care (Phase 3). Geographical mapping on global, country, or state/regional bases for participating hospitals is also provided.

Taken from aidsinfo.nih.gov

The ClinicalTrials.gov website is relatively easy to navigate, and has built-in help “buttons” as well as dropdown menus as aids to find and filter the extensive amount of available information. My search results from ClinicalTrials.gov several months ago using the term “aptamer” provided information for 32 different clinical studies, which when sorted by medical conditions gave 87 items covering a wide range of diseases, with ocular being the most prevalent general category. However, my reading of procedural details for all 32 studies revealed that, in 4 studies, samples from subjects treated with non-aptamer drugs were to be used for in vitro investigations such as screening for aptamer biomarkers. The remaining 28 clinical studies involve aptamer drugs per se, about which I’ll comment below.

Status of Aptamer Studies in ClinicalTrials.Gov

Among the various ways the search results for 28 aptamer drug studies in ClinicalTrials.gov can be sorted for perusal and commentary, I chose to use study “status,” which ClinicalTrials.gov defines as follows:

  • Completed—clinical study has ended normally, and participants are no longer being examined or treated (that is, the “last subject, last visit” has occurred).
  • Terminated—clinical study has stopped recruiting or enrolling participants early and will not start again and participants are no longer being examined or treated.
  • Withdrawn—clinical study stopped before enrolling its first participant.
  • Active—clinical study is ongoing (that is, participants are receiving an intervention or being examined), but potential participants may not be being currently recruited.

Taken from doctorabidi.com

These categories can be used as filters to easily group studies for perusal, which when I did so, led to several general observations. The overwhelming majority of the 17 Completed clinical studies involve ocular diseases, of which age-related macular degeneration (ARMD) was a frequently studied indication. ARMD is the leading cause of blindness for people over the age of 55 years, in the U.S. and Europe, and occurs as either “dry” or “wet” ARMD, as described elsewhere.

While ARMD is a significant medical problem, my guess as to why it was chosen for initial clinical studies with aptamers is that clinical development had fewer practical challenges. Ocular administration required much smaller amounts of drug, which made manufacturing scale-up less problematic, and there were likely less toxicity issues to worry about. In any case, the sponsors included Eyetech Pharma, Ophthotech Corp, Achemix and NOXXON.

Terminated or Withdrawn studies, which numbered 8, were also mostly ARMD-related but also included aptamer for hemophilia and acute myeloid leukemia sponsored by Baxalta and Antisoma Research. Perhaps most important are the Active studies, which were only 3 in number, and I’ve put into tabular form here with some basic information including the National Clinical Trials (NCT) identification number that is searchable in any search engine. You can click on each NCT number to access detailed information provided in ClinTrials.gov; however, following are some snippets for each of these Active Studies.

Table of Active Studies Listed in ClinicalTrials.Gov in 2017

Sponsor (Study ID) Aptamer Drug Disease(s) Type of Study
Eyetech Pharma (NCT01487044) pegaptanib sodium (Macugen) Diabetic Macular Edema (DME) Phase 1
Ophthotech Corp (NCT02686658) ARC1905 (Anti-C5 Aptamer), Zimura® Dry ARMD Phase 2


Ophthotech Corp (NCT02591914) E10030 (Anti-PDGF Pegylated Aptamer, Fovista®) ARMD Phase 1

Pegaptanib structure taken from DrugBank. Light blue nucleotides are 2’-hydroxyl, dark blue nucleotides are 2’-fluoro, and red nucleotides are 2′-O-methyl.

Pegaptanib sodium (aka Macugen), which originated at NeXstar Pharma and was later taken on by Ophthotech via Eyetech, is a 5’-PEG (40kDa)/3’-inverted dT blocked 2′-fluoro/2′-O-methyl modified 27-mer RNA aptamer targeted against vascular endothelial growth factor (VEGF), and is the only FDA-approved aptamer drug. This Active Study is a single-center trial at the Retina Institute of Hawaii, and involves loading of intravitreal pegaptanib bi-weekly during the initial treatment period for diabetic macular edema (DME) when levels of VEGF, its protein target, are the greatest. This is followed by gradually extending the administration frequency to monthly as homeostasis ensues for the treatment of DME. This study was registered in 2011 and is currently recruiting patients.

ARC1905 (aka Anti-C5 Aptamer and Zimura) is reported to be a 38-mer aptamer sequence that was originally identified from a 2′-fluoro-pyrimidine-modified library, and all but three purines could be replaced by 2′-O-methyl purine nucleotides. For in vivo applications, an inverted dT and a 40 kDa PEG moiety were added to the 3′ and 5′ end, respectively, according to a review. Opthotech’s study of ARC1905 assesses the safety and efficacy of intravitreous administration in subjects with geographic atrophy secondary to dry ARMD. This study was registered in 2016 and is currently recruiting participants in numerous locations in the U. S. as well as some sites in Hungary.

Ophthotech’s E10030 (aka ARC127, Fovista), which is an anti-PDGF-BB aptamer, is reported to be a 36-mer published in 1996 that was derived from a DNA library, and was only modified with a 3′-inverted dT moiety to inhibit exonuclease degradation. Further shortening and post-SELEX modifications (2′fluoro pyrimidines, 2′-O-methyl purines, internal hexyl-linkers, all wherever possible) is reported to improve nuclease resistance and resulted in a 32-mer that was later named ARC126, and then ARC127 when pegylated at the 5′ terminus. Ophthotech’s Active Study of E10030 is an open-label trial located at Retinal Consultants of Arizona for safety, tolerability and development of subfoveal fibrosis by intravitreal administration of altering regimens of this aptamer drug and anti-VEGF therapy in subjects with neovascular ARMD. This study is ongoing but not recruiting patients.

Conclusions and Prospects

Oligonucleotides aptamers as possible therapeutics were scientifically “birthed,” so to speak, nearly 30 years ago, shortly after the “birth” of antisense oligonucleotides for therapeutics. Consequently, on a relative basis, I think it’s telling that my search of ClinicalTrials.gov lists only 28 studies of aptamer drugs compared to 152 studies found for “antisense.” In addition to this difference in total number of studies for these two classes of oligonucleotide drugs, filtering the data by Study Phase gave 7 aptamer studies as Phase 3 (all ocular diseases) compared to 14 antisense studies as Phase 3 (various diseases). The number of FDA-approved or soon-to-be-approved drugs mirrors the same inequality: only one (Macugen) for aptamers and a handful for antisense (see Ionis Pharma, Alnylam Pharma, and Sarepta Therapeutics).

Having said this, I hasten to add that I’m not “anti-aptamers” and instead “pro-antisense.” In fact, I’m “bullish” on both strategies for drug development, and expect more clinical progress with aptamers as new chemistries are pursued. Past focus on 2′-fluoro/2′-O-methyl modified oligonucleotide as aptamers represents, in effect, exploration of very limited structural diversity, which can now be expanded to hydrophobic SOMAmers and thioaptamers, or C5-modified dU X-Aptamers.

Finally, let’s consider the graph shown here, which I constructed using PubMed search results for the number of annual publications indexed to “clinical development of aptamers” as a search phrase. To me, there is clearly a trend toward increased numbers of such publications with time. Factors could arise which negatively impact this trend going forward, but absent that eventuality the future looks promising.

As usual, your comments are welcomed.


There are additional Active Studies not registered in ClinicalTrials.gov, such as those with NOXXON’s “Spiegelmers,” which are mirror-image L-nucleic acids stable toward nuclease degradation in vivo. NOXXON states that its lead candidate, NOX-A12, which is an L-RNA aptamer, “is under development as a combination therapy for multiple cancer indications

Taken from iomers.net

where its impact on the tumor microenvironment is intended to significantly enhance the effectiveness of anti-cancer treatments without adding significant side effects for patients. NOX-A12 is currently in a Phase 1/2 trial in metastatic pancreatic and colorectal cancer patients who are not expected to respond to checkpoint inhibition alone. NOX-A12 has also completed two Phase 2a trials, one in chronic lymphocytic leukemia and the other in in multiple myeloma.”






Jerry’s Favs from the Recent 7th Cambridge Symposium

  • DNA Can Function as an Enzyme
  • RNA Polymerase Activity Without Proteins
  • Systemic Brain Delivery of Therapeutic Oligos

The 7th Cambridge Symposium on Nucleic Acids Chemistry and Biology took place September 3-6, 2017 at historic Queens’ College, Cambridge, which was founded in 1448 by Margaret of Anjou (who was then Queen of England by marriage to King Henry VI). Yours truly had the honor of participating in this event and presenting one of TriLink’s posters on the company’s new types of chemically modified mRNA for mRNA therapeutics. As done for other conferences I’ve attended on behalf of TriLink, I wish to share here my personal favorites among the many lectures, which are still fresh in my mind. However, I hasten to emphasize that, while choosing these “favs” is biased a bit by my scientific interests, all the lectures topics are worth looking at later by perusal of the symposium program of nearly forty presentations.

Taken from na-cb.co.uk

By the way, the symposium’s logo artistically depicts a DNA helix under a wooden bridge, better seen in the accompanying picture of the actual bridge over the River Cam at Queens’ College. Built in 1749, it has become known as the Mathematical Bridge for reasons you can read later, and appears to be an arch but is composed entirely of straight timbers. The historical connection between the DNA double helix and Cambridge is that in 1953 Watson & Crick proposed this now famous deoxynucleic acid structure as the molecular basis for genetics, which I’ll comment on again at the end of this post.

Taken from worldachitecture.org

Overview of the Symposium

Mike Gait. Taken from histmodbiomed.org

This symposium is the 7th in a popular conference series going way back to 1981 that brings together nucleic acids scientists across a broad area but with emphasis on chemistry, biochemistry and structure. Michael (Mike) Gait, who is at the Medical Research Council Laboratory of Molecular Biology in Cambridge, originated this series and has been a key organizer for all seven conferences. Participants come from all over the world and include professors, students, and companies—as well as Nobel Laureates (this year Jack Szostak of telomeres fame).

In addition to Mike, the organizing committee included Sir Shankar Balasubramanian, who was recently knighted for his contributions to next-generation sequencing and research on G-quadruplexes, the latter of which I featured here a few years ago. Other committee members were Rick Cosstick, Phil Holliger, and Chris Lowe.

Subject areas this year included:

  • Nucleic acids as therapeutics (including antisense, RNAi, aptamers, immune recognition, cell delivery)
  • RNA and DNA structures and their protein complexes (duplexes, quadruplexes, RNA and DNA enzymes, riboswitches, protein complexes + assemblies)
  • Nucleic acids chemistry applied to cells and cell mechanisms (genomes, evolution, repair, cell manipulation)
  • Nucleic acids as tools, structural assemblies and devices (nanostructures, cages, arrays, supra-molecular chemistry)

Marv Caruthers. Taken from colorado.edu

The showcased and highly prestigious Nucleic Acids Award was presented to Marvin (“Marv”) Caruthers in recognition of his seminal contributions to the synthesis of oligodeoxynucleotides (aka “oligos”) based on the use of phosphoramidite chemistry. This mechanistically elegant chemistry enabled much faster and more efficient coupling for automated synthesis of oligos, which fundamentally transformed all manner of basic and applied research with DNA. His award lecture was titled Synthesis, Biochemistry, and Biology of New DNA Analogues, some of which has been recently published. My previous several posts commenting on Marv, who has been a professor at the University of Colorado in Boulder since 1973, can be read later here.

Jerry’s Favs from the Symposium

To encourage inclusion of unpublished results and other types of “late breaking news” from the lab, the organizers forbade use of Twitter or other real-time social media, blogging, or taking pictures of slides being shown. Consequently, what I can say here is restricted to published papers related to my favs. Keeping this limitation in mind, here are my personal top-three talks that I consider to be tied (i.e., have equivalent scientific importance).

DNA Can Function as an Enzyme!

This lecture by Scott Silverman at the University of Illinois, Urbana-Champaign, dealt with DNA enzymes (aka deoxyribozymes), which were first reported in 1996 by Carmi et al., and are of interest because they expand enzymatic functionality from naturally occurring proteins to synthetic nucleic acids. DNA enzymes can be evolved in vitro starting with random sequences of DNA and applying suitable selection.

In a published account titled Pursuing DNA Catalysts for Protein Modification, Silverman has provided a lengthy and chemically detailed description of his use of in vitro selection to develop DNA catalysts for many different covalent modification reactions of peptide and protein substrates. While interested readers can consult Silverman’s account for various examples, it’s illustrative to consider the molecular design strategy depicted below that was used to evolve a synthetic DNA functional-equivalent of naturally occurring protein kinases that, by definition, carry out protein phosphorylation.

Taken from Silverman Acc Chem Res (2015)

In this case, modular deoxyribozyme design involved a stretch of 40 randomized bases (N = A/G/C/T) having a hairpin loop conjugated to a tyrosine (Tyr)-containing peptide on one end, and an ATP-binding aptamer on the other end. This was intended to experimentally assess whether it would be helpful to provide a predetermined small-molecule binding site in the form of an aptamer, which would cooperate functionally with an initially random catalytic region (N40) from the onset of selection. The selection outcome established that while modular deoxyribozymes that utilize a distinct predefined aptamer domain can indeed be identified, such DNA catalysts do not have any functional advantage relative to nonmodular analogues selected simultaneously for binding and catalysis, at least for this test case of tyrosine kinase activity using an ATP phosphoryl donor.

RNA Polymerase Activity Without Proteins!

By analogy to use of in vitro selection to evolve DNA enzymes from complex pools of random sequences of DNA, complex mixtures of unrelated RNA sequences can also be subjected to in vitro selection to evolve RNA enzymes (aka ribozymes). Indeed, as noted and cited in a talk by co-organizer Philipp Holliger, the emergence of an RNA catalyst capable of self-replication is considered a key transition in the origin of life in the prebiotic “RNA World” first hypothesized by Walter Gilbert in the 1980s. How such self-replicating (replicase) ribozymes emerged from the pools of short RNA oligomers arising from prebiotic chemistry and non-enzymatic replication, however, is unclear.

In a published version of Holliger’s talk addressing this important open question, his laboratory carried out an elegant series of experiments demonstrating that RNA polymerase ribozymes can assemble from catalytic networks of RNA oligomers that are each no longer than 30 nucleotides. Additionally, they found that entropically disfavored assembly reactions are driven by iterative freeze-thaw cycles. Such cooling (to freeze)-warming (to melt) cycles for aqueous solutions of RNA oligo reactants are notionally opposite to heating (to dissociate)-cooling (to hybridize) cycles used for amplification by PCR.

Interested readers can peruse Holliger’s publication for details about these novel findings, but for the purposes of this blog the schematic shown below depicts and describes the mechanism for assembly wherein relatively short RNA oligomers undergo serial ligations and “grow” into a self-replicating RNA polymerase. To me, these results provide an amazing glimpse backward in time to how the RNA World may have evolved!

Assembly of a RNA polymerase ribozyme (RPR 1234) from oligonucleotides devoid of pre-activation. (a) Schematic representation of the assembly trajectory involving (anti-clockwise from top left), ribozyme (blue) cleavage of a short 3′ tail (red) generating a 2′, 3′ cyclic phosphate (>p) (red dot), dissociation of the cleaved tail and strand exchange to cognate substrate (orange) followed by ligation of substrate 5′ OH with >p. (b) Network diagram of RPR 1234 assembly from 4 tailed fragments 1, 2, 3 and 4. Tailed input fragments can ligate to their cognate 5′fragments but must be cleaved (red lines) before ligation to 3′fragments. Taken from Holliger Nat Chem (2015).

Systemic Brain Delivery of Therapeutic Oligos!

Taken from igtrcn.org

A talk by Fazel Shabanpoor titled Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy described work that he had just published with a group of collaborators that included symposium co-organizer Mike Gait, whose lab interests have recently focused on cell-penetrating peptides. This was a fav for me because it had multiple interesting elements: (1.) systemic brain delivery, which is a widely recognized challenge; (2.) “weirdly” structured morpholino oligos, which have backbone structures quite unlike DNA that I’ve commented on here previously; and (3.) splice-switching antisense oligos (SSOs). The latter class of molecules (SSOs) base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Readers interested in SSOs—currently a “hot topic”—can consult a recent comprehensive review, while SSOs for spinal muscular atrophy (SMA) has been featured in previous blog here.

Shabanpoor’s lecture highlighted the fact that development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, Shabanpoor et al. investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching 20-mer phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. They identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript.

Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. It was concluded that this work provides proof of principle for the ability to select new “peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform” for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.

Parting Thoughts and The Eagle

I hope that you can now appreciate why these three lectures were my favs from the 7th Cambridge Symposium. Silverman’s conversion of DNA into an enzyme that can phosphorylate a protein is an exciting demonstration of the power of bio-organic chemistry to manipulate DNA to do things it can’t do naturally. Holliger’s demonstration of how RNA polymerase ribozymes may have evolved gives credence to the RNA World hypothesis, and indicates that this supposed prebiotic environment may have provided critical freezing and thawing cycles over many millennia of molecular evolution. In the present biotic world, humans afflicted with neuromuscular and neurodegenerative diseases may benefit from Shabanpoor & Gaits’ new peptide paradigms to enhance CNS delivery and activity of therapeutic oligos.

In conclusion, I should mention that researchers who work with DNA will invariably visit The Eagle when in Cambridge, which is a pub only a short walk from Queens’ College. This pub, which dates back to 1667, is quite famous because it is known with certainty to be the place where Francis Crick interrupted patrons’ lunchtime on February 28, 1953 to announce that he and James Watson had ‘discovered the secret of life’ after they had come up with their proposal for the structure of DNA. Today the pub serves a special ale dubbed “Eagle’s DNA” to commemorate the discovery. Trust me when I say that this ale is mighty tasty, because I enjoyed a pint of it, and while standing in the que for that brew, was inspired to capture this image to share here.

As usual, your comments are welcomed.

Personal photo using a Samsung Galaxy S8




Advances in Aptamer Applications – Part 2

  • Top Cited Aptamer Publications Over the Past Three Years
  • Jerry’s Picks for Top 3 Aptamer Publications So Far This Year
  • TriLink Products Cited in Numerous Aptamer Publications

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. My first blog on aptamers was titled Aptamers: Chemistry Bests Mother Nature’s Antibodies. This purposefully provocative claim was intended to emphasize the growing body of evidence that collectively indicates aptamers can perform better than antibodies in many applications.

NMR-derived structures of aptamers binding to either a large protein or small molecule. Taken from genelink .com

Because it has been nearly four years since that boastful blog in 2013, I thought it was time to survey aptamer applications published since then to comment on what has been trending or is otherwise notable. I found more than 1,500 articles in PubMed for 2014 through 2017 (estimate) that have the search term “aptamer” in the title or abstract. Given this huge number of publications, I used Google Scholar citation frequency as a numerical indicator of interest, importance and/or impact for these publications in each year. I also decided to focus on original publications that, by definition, excludes review articles. 

Top 3 Cited Publications in 2014

  1. Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet–aptamer nanoprobe (109 citations)

This Chinese team of researchers led by uber-prolific Weihong Tan, about whom I’ve previously blogged, designed a novel methodology for imaging tumor cells using quenched-fluorescent aptamers. In the presence of target cells, the binding of these “dark” aptamers to cell surface markers weakens the adsorption of aptamers on MnO2 nanosheets causing partial fluorescence recovery (i.e., unquenching), thus illuminating the target cells, as well as facilitating endocytosis into target cells. After endocytosis, reduction of MnO2 nanosheets by glutathione further activates the fluorescence signals and generates large amounts of Mn2+ ions as a contrast agent for magnetic resonance imaging (MRI).

Taken from pubs.rsc.org

  1. A phase II trial of the nucleolin-targeted DNA aptamer AS1411 in metastatic refractory renal cell carcinoma (88 citations)

Taken from mct.aacr.org

The anticancer mechanism of action for DNA aptamer AS1411, which has multiple G-quadruplex moieties that disrupt cancer cell replication following nucleolin-mediated uptake, is depicted below and detailed elsewhere. In this clinical study, it was found that AS1411 appears to have limited activity in patients with metastatic renal cell carcinoma. However, rare, dramatic and durable responses can be observed and toxicity is low. Further studies with AS1411 and other nucleolin-targeted compounds may benefit from efforts to discover predictive biomarkers for response.

  1. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1 (61 citations)

Aflatoxin B₁ structure. Taken from wikipedia.org

Aflatoxin B₁ (AFB1) produced by Aspergillus flavus and A. parasiticus is considered the most toxic aflatoxin and it is highly implicated in hepatocellular carcinoma in humans. In this work by Korean researchers, a rapid and simple dipstick assay based on an aptamer has been developed for determination of AFB1 contamination in food. The dipstick assay format was based on a competitive reaction of a biotin-modified aptamer specific to AFB1 between target and Cy5-modified DNA probes. Streptavidin and anti-Cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min.

Top 3 Cited Publications in 2015

  1. Aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of staphylococcus aureus (63 citations)

Taken from sciencedirect .com

Staphylococcus aureus (S. aureus) is one of the most important human pathogens and causes numerous illnesses. This report by Iranian researchers describes a sensitive and highly selective dual-aptamer-based sandwich immunosensor for the detection of S. aureus. As depicted below, a biotinylated primary anti-S.aureus aptamer was immobilized on streptavidin coated magnetic beads (MB), which serves as a capture probe. A secondary anti-S.aureus aptamer was conjugated to silver (Ag) nanoparticles such that, in the presence of target bacterium, a sandwich complex is formed on the MB surface and the electrochemical signal of Ag is measured by anodic stripping voltammetry.

  1. Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles (59 citations)

Chloramphenicol. Taken from Wikipedia .com

Chloramphenicol (CAP) shown below is a naturally occurring antibiotic that is artificially manufactured for use in veterinary and human medicine. Due to its adverse effects in humans, use of the antibiotic is restricted and, in Europe, ‘zero tolerance’ for CAP in food products has been legislated. In this report by Chinese researchers, detection of CAP uses aptamer-conjugated magnetic nanoparticles for both recognition and concentration, together with upconversion nanoparticles for detection. The method was validated for measurement of CAP in milk vs. a commercially available enzyme-linked immunosorbent assay (ELISA) method.

  1. A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus (48 citations)

Taken from mdpi .com

This work by Chinese investigators describes a novel aptamer/graphene interdigitated gold electrode piezoelectric sensor for detecting S. aureus by binding to the aptamer, which is immobilized on the graphene via the π–π stacking of DNA bases, as depicted below. When S. aureus is present, aptamer dissociates from the graphene and thus leads to change of oscillator frequency of the piezoelectric sensor.

Top 3 Cited Publications in 2016

  1. Aptamer–MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen (38 citations)

This study in the UK uses a thiolated DNA aptamer for prostate specific antigen (PSA) immobilized on the surface of a gold electrode. Controlled electropolymerization of dopamine around the complex served to create an imprint of the complex following removal of PSA. This molecularly imprinted polymer (MIP) cavity was found to act synergistically with the embedded aptamer to provide recognition properties superior to that of aptamer alone. A generalized depiction for producing a MIP is shown below.

Taken from sigmaaldrich .com

  1. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device (34 citations)

Taken from wikipedia.org

Monitoring the periodic diurnal variations in cortisol (aka hydrocortisone, show below) from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. This publication from China reports use of aptamer-functionalized gold nanoparticles pre-bound with electro-active triamcinolone for detection of cortisol based on its competitive binding to the aptamer by monitoring a signal from the displaced triamcinolone using square wave voltammetry at graphene-modified electrodes. The assay was benchmarked vs. ELISA and radioimmunoassays.

  1. Multifunctional aptamer-based nanoparticles for targeted drug delivery to circumvent cancer resistance (32 citations)

Taken from Liu et al. Biomaterials (2016)

In yet another publication from China, Liu et al. report use of a G-quadruplex nanostructure to target cancer cells by binding with nucleolin, in a manner analogous to that mentioned above. A second component is double-stranded DNA (dsDNA), which is rich in GC base pairs that can be applied for self-assembly with doxorubicin (Dox) for delivery to resistant cancer cells. These nanoparticles were found to effectively inhibit tumor growth with less cardiotoxicity.

Jerry’s Top 3 Publication Picks for 2017-to-Date

Here are my Top 3 “fav” aptamer articles published during the first half of 2017, and my reasons for these aptamer selections—pun intended. Interested readers can consult the original publication for technical details.

  1. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

CRISPR/Cas9 is unquestionably—in my opinion—the hottest topic in nucleic acid-based R&D these days, as I have previously blogged about. Off-target effects of CRISPR/Cas9 can be problematic, so using targeted delivery to cells of interest is an important approach for mitigating this problem. In this study, an aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery with adaptability to other situations. The chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand, and the approach “provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.”

  1. A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva

This report claims the first ever development of a split aptamer that achieves enhanced target-binding affinity through cooperative binding. In this instance, a split cocaine-binding aptamer incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. This system afforded specific, ultra-sensitive, one-step fluorescence detection of cocaine in saliva without signal amplification. This limit of detection meets the standards recommended by the European Union’s Driving under the Influence of Drugs, Alcohol and Medicines program.

  1. Detection of organophosphorus pesticide–Malathion in environmental samples using peptide and aptamer based nanoprobes

Environmental contamination with pesticide residues has necessitated the development of rapid, easy and highly sensitive approaches for the detection of pesticides such as malathion, a toxic organophosphorus pesticide, widely used in agricultural fields. These Indian investigators employed an aptamer, cationic peptide and unmodified gold nanoparticles. The peptide, when linked to the aptamer renders the gold nanoparticles free and therefore, red in color. When the aptamer is associated with malathion, however, the peptide remains available to cause the aggregation of the nanoparticles and turn the suspension blue. The sensitivity was tested in real samples and the results implied the high practicability of the method.

Aptamer Publications in 2014-Present Citing TriLink Products

I was pleasantly surprised to find more than 250 publications on aptamers in Google Scholar citing the use of TriLink products since 2014. This volume of literature is way too large to summarize succinctly, so I decided to do a quick scan to select the following items that provide an indication of the broad diversity of applications partially enabled by TriLink products:

2’-F-UTP. Taken from trilinkbiotech .com

In closing, I should first mention that, while scanning the aptamer/TriLink publications mentioned above, it was evident that the most frequently cited TriLink products were 2’-F-CTP and 2’-F-UTP, which are incorporated into aptamers to impart nuclease resistance, as discussed on a TriLink webpage.

My second and last comment is that, as you may have noticed, there seems to be a high proportion of aptamer publications coming out of China and/or coauthored by Chinese investigators collaborating with researchers in other countries. This despite the fact that Chinese publications in Life Sciences are ~6-times fewer that those from the US, according to reliable statistics. I have no idea why this is so, but thought it’s an intriguing factoid.

As usual, your comments are welcomed.








Evolving Polymerases to Do the Impossible

  • Polymerases Aren’t What They Used to Be! 
  • Scripps Team Evolves Polymerases That Read and Write With 2’-O-Methyl Ribonucleotides
  • Key Reagents for Romesberg’s “Molecular Moonshots” Are Supplied by TriLink BioTechnologies

Long-time devotees of these posts will likely remember a blog several years ago about Prof. Floyd Romesberg at the Department of Chemistry, The Scripps Research Institute who achieved a seemingly impossible feat. Namely, designing a new pair of complementary bases such that DNA replicating in E. coli would be comprised of six bases, thereby creating a six-base genetic code that is expanded from Nature’s four-base code.

Floyd E. Romesberg. Taken from utsandiego.com

More recently, Romesberg has cleverly outfoxed Nature once again, this time by evolving nucleic acid polymerases into mutant polymerases that can do what heretofore seemed impossible. He and his research team’s publication (Chen et al.) is a tour de force of experimental methodology that is not easily read, and is even harder to simply summarize in a short space like this blog. Consequently, I’ll first tell you what was accomplished, then give a short synopsis of principal new methodology, and close by commenting on the significance of this fascinating work.

Doing the Impossible

Romesberg’s lab successfully achieved what I think of as “multiple molecular moonshots,” wherein a Taq polymerase (which normally reads and writes DNA during PCR), was evolved by novel selection (SELEX) methods into mutant polymerases that are able to transcribe DNA into 2’-O-methyl (2’-OMe) RNA, and reverse transcribe 2’-OMe RNA into DNA for PCR/sequencing.

As depicted below, this was exemplified using a 60-mer DNA template and 18-mer 2’-OMe RNA primer to produce a fully-modified 48-mer 2’-OMe RNA by means of an evolved mPol and all four A, G, C and U 2’-OMe NTPs, which I’m proud to say were bought from TriLink BioTechnologies! This type of molecular evolution of a polymerase has no precedent.

DNA template   5’ ————————————- 3’

RNA primer                                 ←←← 3’ xxxxxx 5’

mPol ↓ 2’-OMe NTPs

Determining the fidelity of this seemingly impossible molecular transformation was addressed by achieving a feat of comparable impossibility! As depicted below, the aforementioned 48-mer 2’-OMe RNA product was hybridized to a DNA primer for reverse transcription into a 48-mer complementary DNA (cDNA) strand, using an evolved mPol, together with all four A, G, C and T unmodified dNTPS, which were also purchased from TriLink. This unprecedented conversion of 2’-OMe RNA into cDNA was followed by conventional PCR/sequencing, the results of which demonstrated relatively high fidelity.

2’-OMe template   5’ xxxxxxxxxxxxxxxxxxxxxxxxxxx 3’

DNA primer                                           ←←← 3’ —— 5’

mPol ↓ dNTPs

cDNA                        3’ ————————————– 5’

How They Did It

In the selection cycle shown below, (1) phage-display libraries were used to expose individual polymerases (Pol) on E. coli. cells in proximity to chemically attached primer/template complexes of interest, which are mixed with natural or modified triphosphates including biotin (green; B)-labelled UTP to extend the primer. (2) Phage that display active mutant polymerases (mPols) are isolated with streptavidin (SA) beads. After washing to remove nonspecific binders, phage cleaved from the beads are used to re-infect E. coli. (3) Heat-treated lysates of E. coli that express the recovered mPols are next subjected to plate-based screening using 96-well plates coated with primer/template complex and extension buffer that contained natural or modified triphosphates and B-UTP, incorporation of which is chromogenically detected. (4) Mutants that give rise to the most activity are selected for individual gel-based analysis, from which (5) promising candidates are selected for further diversification (e.g., by gene shuffling, as depicted) and then subjected to additional rounds of evolution.

Taken from Chen et al. Nature Chemistry (2017)

What is the Significance

In a previous blog, I’ve commented on increasing interest in the utility of aptamers, which are oligonucleotides that can specifically bind small molecules or motifs in proteins, and thus be used to build electronic sensors or studied as potential therapeutic agents rivaling antibodies. Therapeutic aptamers, like antisense oligonucleotides, require incorporation of chemical modifications to impart stability toward nucleases in blood or cellular targets.

Burmeister et al. have previously reported methods for mPol transcription of a DNA template into a fully modified, nuclease-resistant 23-mer 2’-OMe RNA aptamer—also using TriLink’s 2’-OMe NTPs! However, they encountered considerable experimental difficulties in generating this therapeutically promising 23-mer against vascular endothelial growth factor. These technical issues have now been surmounted by the mPol-evolution approaches in the present work by Romesberg’s team, which enabled improved access to longer 2’-OMe RNA aptamers with reasonable efficiency and fidelity.

Moreover, the present study is the first to evolve an mPol for reverse transcription of fully modified 2’-OMe RNA into DNA, which can then be amplified by PCR and/or sequenced, thereby opening the door for a variety of new analytical methods. Most importantly, the molecular mechanism by which these remarkable mPol activities was evolved, namely, the stabilization of an interaction between the “thumb and fingers domains,” may be general and thus useful for the optimization of other Pols. In that case, we can look forward to further advances in evolving other Pols to do the impossible—hopefully using modified nucleotide triphosphates from TriLink!

As usual, your comments are welcome.

RNA World Revisited

  • Scripps Researchers ‘Evolve’ an RNA-Amplifying RNA Polymerase 
  • It’s Used for First Ever All-RNA Amplification Called “riboPCR”
  • TriLink Reagent Plays a Role in this Remarkably Selective in Vitro Evolution Method 
Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from scripps.edu

Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from scripps.edu

Those of you who regularly read my blog will recall an earlier posting on “the RNA World,” which was envisioned by Prof. Walter Gilbert in the 1980s as a prebiotic place billions of years ago when life began without DNA. That post recommended reading more about this intriguing hypothesis by consulting a lengthy review by Prof. Gerald Joyce. Now, Prof. Joyce and postdoc David Horning have advanced the hypothesis one step further by reporting the first ever amplification of RNA by an in vitro-selected RNA polymerase, thus providing significant supportive evidence for the RNA World. Following are their key findings, which were enabled in part by a TriLink reagent—read on to find out which one and how!

In Vitro Evolution of an RNA Polymerase

Horning & Joyce designed an in vitro selection method to chemically “evolve” an RNA polymerase capable of copying a relatively long RNA template with relatively high fidelity. The double emphasis on “relatively” takes into account that the RNA World would have many millions of years to evolve functionally better RNA polymerases capable of copying increasingly longer RNA templates with increasingly higher fidelity.

As depicted below, they started with a synthetic, highly structured ribozyme (black) wherein random mutations were introduced throughout the molecule at a frequency of 10% per nucleotide position to generate a population of 1014 (100,000,000,000,000) distinct variants to initiate the in vitro evolution process. Step 1 involved 5’-5’ click-mediated 1,2,3-trazole (Ø) attachment of an 11-nt RNA primer (magenta) partially complementary to a synthetic 41-nt RNA template (brown) encoding an aptamer that binds guanosine triphosphate (GTP). In Steps 2 and 3, the primer hybridizes to template and is extended by polymerization of A, G, C and U triphosphates (cyan).

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Step 4 involves binding of aptameric structures to immobilized GTP (green), then photocleavage of the 1,2,3-triazole linkage in Step 5, followed by reverse transcription to cDNA and conventional PCR in Step 6 for transcription into ribozymes in Step 7. Twenty-four rounds of this evolution by selection were carried out, progressively increasing the stringency by increasing the length of RNA to be synthesized by decreasing the time allowed for polymerization. By the 24th round, the population could readily complete the GTP aptamer shown below. Subsequent cloning, sequencing and screening were then used to characterize the most active polymerase, which was designated “24-3.”

The TriLink “Connection”

2'-Azido-dUTP (aka 2'-azido-UTP)

2′-Azido-dUTP (aka 2′-azido-UTP)

The aforementioned in vitro evolution process actually involves tons of experimental details that interested readers will need to consult in the published paper, which is accompanied by an extensive Supporting Information section. In the latter, a subsection titled Primer Extension Reaction describes 3’ biotinylation of the template RNA strand (brown in above scheme) using TriLink “2’-azido-UTP” (more properly named 2’-azido-dUTP) and yeast poly(A) polymerase, followed by click connection of the RNA template’s 3’-terminal 2’-azido moiety to biotin-alkyne. This very clever functionalization of the RNA template strand allowed for subsequent capture of the double-stranded primer extension reaction products on streptavidin-coated beads, followed by elution of the desired nonbiotinylated strand for GTP aptamer selection (Step 4 above).

Properties of RNA Polymerase 24-3

Needless to say—but I will—enzymologists and RNA aficionados will undoubtedly be interested in musing over the kinetic and fidelity properties of RNA polymerase 24-3.

The rate of 24-3 polymerase catalyzed addition to a template-bound primer was measured using an 11-nt template that is cited extensively in the literature to evaluate various ribozymes. It was found that the average rate of primer extension by 24-3 is 1.2 nt/min, which is ∼100-fold faster than that of the starting ribozyme polymerase randomly mutagenized for in vitro selection.

The NTP incorporation fidelities of the starting and 24-3 ribozyme polymerases on this 11-nt test template, at comparable yields of product, are 96.6% and 92.0%, respectively. Horning & Joyce noted that the higher error rate of 24-3 is due primarily to an increased tendency for G•U wobble pairing.

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Other longer RNA templates having various base compositions or intramolecular structures were also studied, with the stated “final test of polymerase generality” being use of 24-3 to synthesize yeast phenylalanyl tRNA from a 15-nt primer (in red right). The authors humorously describe the results as follows:

“Despite the stable and complex structure of the template, full-length tRNA was obtained in 0.07% yield after 72 h. This RNA product is close to the limit of what can be achieved with the polymerase, but is likely the first time a tRNA molecule has been synthesized by a ribozyme since the end of the RNA world, nearly four billion years ago.”

Exponential Amplification of RNA

PCR is the most widely used method for amplifying nucleic acids, and involves repeated cycles of heat denaturation and primer extension. The 24-3 RNA polymerase was used to carry out PCR-like amplification, but in an all-RNA system (named riboPCR by Horning & Joyce) using A, G, C, and U triphosphates and a 24-nt RNA template composed of two 10-nt primer-binding sites flanking the sequence AGAG. Somewhat special conditions were employed:

  • The concentration of Mg2+ was reduced to minimize spontaneous RNA cleavage
  • PEG8000 was used as a “molecular crowding” agent to improve ribozyme activity at the reduced Mg2+ concentration
  • Tetrapropylammonium chloride was added to lower the melting temperature of the duplex RNA

Under these conditions, 1 nM of the 24-nt RNA template was driven through >40 repeated thermal cycles, resulting in 98 nM newly synthesized template and 106 nM of its complement, corresponding to 100-fold amplification. Sequencing of the amplified products revealed that the central AGAG sequence was largely preserved, albeit with a propensity to mutate the third position from A to G, reflecting the low barrier to wobble pairing.

Amplification of a 20-nt template (without the central insert) was monitored in real time, using FRET from fluorescently labeled primers, and input template concentrations ranging from 10 nM to 1 pM. The resulting amplification profiles shown in the paper are typical for real-time PCR, shifted by a constant number of cycles per log-change in starting template concentration. A plot of cycle-to-threshold vs. logarithm of template concentration, also shown in the paper, was linear across the entire range of dilutions indicating exponential amplification of the template RNA with a per-cycle amplification efficiency of 1.3-fold.

Implications for the Ancient RNA World

It would be an injustice to Horning & Joyce if I would try to paraphrase their concluding discussion of this investigation, so here is what they say:

The vestiges of the late RNA world appear to be shared by all extant life on Earth, most notably in the catalytic center of the ribosome, but most features of RNA-based life likely were lost in the Archaean era. Whatever forms of RNA life existed, they must have had the ability to replicate genetic information and express it as functional molecules. The 24-3 polymerase is the first known ribozyme that is able to amplify RNA and to synthesize complex functional RNAs. To achieve fully autonomous RNA replication, these two activities must be combined and further improved to provide a polymerase ribozyme that can replicate itself and other ribozymes of similar complexity. Such a system could, under appropriate conditions, be capable of self-sustained Darwinian evolution and would constitute a synthetic form of RNA life.

Applications for Today’s World of Biotechnology

The aforementioned report by Horning & Joyce has received wide acclaim in the scientific press and world-wide public media as supporting the existence of a prebiotic RNA World, billions of years ago, from which life on Earth evolved.

While the academic part of my brain, if you will, fully appreciates the significance of these new insights on “living” RNA eons ago, the technical applications part of my brain is more piqued by possible practical uses of all-RNA copying or all-RNA riboPCR.

I, for one, plan to muse over possible applications of such all-RNA systems in today’s world of biotechnology, and hope that you do too, and are willing to share any ideas as comments here.

Reflections on Advances in Medicinal Oligonucleotides

  • Oligos Are Not “Magic Bullets”
  • Oligos Have, Nevertheless, Enabled New Drug Paradigms
  • Oligos Continue to Attract Significant Corporate Investments


The 10th Annual Meeting of the Oligonucelotide Therapeutics Society (OTS) is in full swing today in San Diego, CA where it began on Oct 12 and concludes on Oct 15. Having worked on antisense oligos since the early days (~30 years ago) participating in this meeting led me to several thoughts that I wish to share with you in this post.

First of all, contrary to many sceptics in those early days, the concept of using synthetic oligonucleotides as an entirely new class of medicinal agents has not only survived but also greatly expanded in terms of the biological target/mechanism of action and types of oligo constructs used—each with a seemingly endless array of chemical modifications to evaluate. In approximate chronological order of discovery, these targets and the types of oligos they have now come to include are listed below coincidentally, most of these are represented in the 2014 OTS agenda.

transcription factors
splice junctions
RNA interference
Oligo Type
dsDNA decoys






Secondly, while it has been possible for oligo chemists to design and synthesize a plethora of modified oligos to achieve optimized nuclease stability, binding to target, etc., efficient delivery has remained the single most challenging problem to deal with. In talks on medicinal oligos, this situation is oftentimes eluded to as something to the effect of “there are only three remaining problems to solve: delivery, delivery, and delivery.”

Lastly, contrary to early hopes of being Dr. Ehrlich’s “magic bullets” (see caption below), oligos didn’t quite prove to be the new paradigm for a speedy concept-to-clinic solution. As all of us in the oligo world know, oligo-based therapeutics have encountered long and costly R&D timelines and clinical development paths typical of all other classes of therapeutic compounds.

Continue reading

Aptamers: Chemistry Bests Mother Nature’s Antibodies

• Discovered ~20 years ago, aptamer applications are booming
• Modified oligos and nucleotide triphosphates play pivotal roles
• World’s first chemical guided missile could be the answer to wiping out cancer
• Are antibodies passé?

Aptamers are nucleic acids (or peptides) that bind to a specific target molecule. RNA or DNA aptamers are usually created by selection from a large pool (aka library) of random sequences; however, natural RNA aptamers also exist in riboswitches. As discussed below, aptamers have been used for an impressively wide variety of applications in either basic research or, especially, health-related diagnostics and therapeutics. This remarkable utility is clearly reflected in the publication statistics—since their discovery in 1990, there have been ~11,000 publications indexed to DNA or RNA aptamers in SciFinder with a projected average rate of ~5 per day in 2013! More than 1,600 of these publications are patents, which is a stunning testament to the commercial potential of aptamers.

Most published authors: Analyzing these SciFinder aptamer publications by author gave the following top-5 ranking for number of publications indexed to aptamers…amazing productivity!

Tan, Weihong:  306
Ellington, Andrew D.:  173
Ikebukuro, Kazunori:  112
Famulok, Michael:  99
Sullenger, Bruce A.:  97

Trying to get what you want sometimes leads to getting what you need…
One concept, two labs, and a pair of seminal publications 

By remarkable coincidence in 1990, two labs independently published conceptually similar achievement of RNA selection in vitro—without using an RNA replicase, as in the past—but by quite different approaches.

Craig Tuerk and Larry Gold described in Science a procedure they cleverly named SELEX, which sounds a bit like select, and was an acronym for systematic evolution of ligands by exponential enrichment. This now classic paper has nearly 5,000 citations in Google Scholar. The authors noted that “[t]he method relies on mechanisms usually ascribed to the process of evolution, that is, variation, selection, and replication.” Briefly, as depicted in the figure below taken from this paper, a segment of RNA was transcribed from DNA template that had been synthesized so as to have eight contiguous, “randomized” positions (N = A, G, C and T) by means of “mixed couplings” using all four phosphoramidite reagents. This pool of 65,536 ( = 48) different RNAs was incubated with gp43 and the subset of RNAs selected by gp43 were then collected, reverse-transcribed into cDNA, amplified by PCR, and then transcribed into RNA for a second round of SELEX. After six rounds of SELEX, sequencing 20 clones of cDNA revealed that, in addition to the wild type 8-nt RNA sequence, a major variant mutated at 4-of-8 positions was also selected, along with several minor variants with fewer mutations.


Taken from Tuerk and Gold in Science 1990.

Those interested in the scientific context of the above work, and the molecular biological interpretations, are encouraged to read the original paper, which is freely available as a pdf through a Google Scholar by searching for “Tuerk Gold 1990”. Here, however, are selected—pun intended—prescient comments by Tuerk and Gold in their concluding section simply entitled “Applications.”

• “Among many general applications of this technique, SELEX analyses can be used to determine the optimal binding sequences for any nucleic acid binding protein.”

• “Small molecules…can be bound to insoluble supports to partition RNAs that interact specifically with these substrates.”

• “We expect that, at the very least, nucleic acid ligands that inhibit replicative proteins of epidemiologically important infections can be likewise evolved. Other, more sophisticated effects of evolved RNA molecules on target activities may be possible.”

• “Finally, SELEX may be just the beginning of evolution in a test tube…[and] provide unpredictable and unimaginable molecular configurations of nucleic acids and proteins with any number of targeted functions.”

Andrew D. Ellington and Jack W. Szostak reported in Nature Methods the synthesis of large numbers of random sequence RNA molecules for binding to specific ligands to investigate fundamental questions related to formation of stable polynucleotide 3-dimensional structures in the context of theories of the origin and early evolution of life. Briefly, they synthesized ~150-nt DNAs comprised of central ~100-nt random sequence—via mixed phosphoramidite couplings (see above)—flanked by defined-sequence 5’ and 3’ ends as primer and restriction sites for cloning. As depicted below in a figure from this paper, transcribed random-sequences of RNA were applied to a column of agarose having an immobilized dye as a model ligand.

Taken from Ellington and Szostak in Nature 1990.

Taken from Ellington and Szostak in Nature 1990.

They chose six dyes that have “many possible hydrogen-bond donor and acceptor groups as well as planar surfaces for stacking interactions.” Bound RNAs were eluted and reverse transcribed for PCR amplification before another cycle of selection. After four to five cycles and sequencing, it was concluded that binding of selected RNAs is specific with regard to cross-reactivity of each selected pool for each column. However, each dye-binding pool was estimated to be a complex mixture of 102 – 105 different sequences, i.e. “there are many independent sequence ‘solutions’ to each ligand-binding ‘problem.’“

For some reason, reading this analogy of many solutions for one problem brought to mind these lyrics from the song in the Rolling Stones 1969 album Let It Bleed:

No, you can’t always get what you want
But if you try sometime, you just might find
You get what you need

Those interested in the details of clonal sequence information, etc. extracted from the results are encouraged to read the original paper, which is freely available as a pdf through a Google Scholar search for “Ellington Szostak 1990.” These authors went on to say that “[w]e have termed these individual RNA sequences ‘aptamers’, from the Latin ‘aptus’, to fit. Furthermore, they suggested that “it may be possible to isolate novel ribozymes from pools of random-sequence RNAs” using transition state analog affinity columns, by analogy with the isolation of catalytic antibodies.

The enabling power of aptamers

The vast body of literature covering basic research and numerous applications of nucleic acid aptamers is a stunning testament to the enabling power of these molecules, and the myriad of expanded types of aptamers and aptamer-generation methods. The Ellington Lab has provided a very useful tool for obtaining information about apatamers in the form of The Aptamer Database, which is a comprehensive, annotated repository for information about aptamers and in vitro selection. This searchable resource is provided to collect, organize and distribute all the known information regarding aptamer selection. For example, when I searched for “toxin” in Title Keywords, three citations were provided with active links, e.g. this 2006 article by Tang et al. in Electrophoresis: The DNA aptamers that specifically recognize ricin toxin are selected by two in vitro selection methods.

Additional search tools for aptamer-related publications, such as PubMed, Scirus, and SciFinder, readily provide access to whatever aspects may be of interest. In perusing this wealth of information, I came across the following items that I selected—pun intended (again!)—as representative examples of recently available literature for various categories.

Selections Methods:

Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer
Multiplexed microcolumn-based process for efficient selection of RNA aptamers
In vivo SELEX for Identification of Brain-penetrating Aptamers

Targeting Cells:

Development of an Efficient Targeted Cell-SELEX Procedure for DNA Aptamer Reagents
Selection of DNA Aptamers against Glioblastoma Cells with High Affinity and Specificity
• Rapid Identification of Cell-Specific, Internalizing RNA Aptamers with Bioinformatics Analyses of a Cell-Based Aptamer Selection


Nucleic acid aptamers: clinical applications and promising new horizon
Therapeutic RNA aptamers in clinical trials
An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells


Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood
Harnessing aptamers for electrochemical detection of endotoxin

Food Safety:

Aptamer-Based Molecular Recognition of Lysergamine, Metergoline and Small Ergot Alkaloids
First report of the use of a saxitoxin?protein conjugate to develop a DNA aptamer to a small molecule toxin
Nucleic acid aptamers for capture and detection of Listeria spp

“World’s first chemical guided missile could be the answer to wiping out cancer”

This attention grabbing headline of an article in Deakin University Newsroom led me to contact Prof. Wei Duan regarding the current status of this promising research that he and his team first described in a Cancer Sci. 2011 publication entitled RNA aptamer against a cancer stem cell marker epithelial cell adhesion molecule. In this study, SELEX was used to identify a 40-base RNA aptamer that binds to epithelial cell adhesion molecule (EpCAM), which is overexpressed in most solid cancers and is reported to be a cancer stem cell marker. Interestingly, the aptamer was further truncated to 19 bases yet still interacts specifically with a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM, but not with those not expressing EpCAM. Importantly, this EpCAM RNA aptamer is efficiently internalized after binding to cell surface EpCAM. These authors believed that this is the first RNA aptamer against a cancer stem cell surface marker being developed, and that such cancer stem cell aptamers will greatly facilitate the development of novel targeted nanomedicine and molecular imaging agents for cancer theranostics.

Prof. Wei Duan, Director of Nanomedicine, Deakin University (Geelong, Victoria, Australia); taken from Deakin University website via Bing Images.

Prof. Wei Duan, Director of Nanomedicine, Deakin University (Geelong, Victoria, Australia); taken from Deakin University website via Bing Images.

As for the current status of this line of research, Prof. Duan provided me with the following comments. “With a few rare exceptions, most of the chemotherapy drugs and molecularly targeted agents in oncology clinics kill only non-cancer stem cells.  In order to improve the cancer treatment outcome, we must target cancer stem cells. Our lab has developed the world’s first RNA aptamer against cancer stem cell marker protein. We are currently working on novel treatment strategies by targeting cancer stem cells in vivo using our aptamers. For example, using cancer stem cell targeting aptamers, we can transform conventional chemotherapy drugs to robust cancer stem cell busters in xenograft tumour models.  With some smart engineering and strategic chemical modifications, we can now deliver siRNA into cancer stem cells in vivo to achieve an unprecedented %ID (percentage of injected dose). I believe that furture development of aptamer for cancer stem cell targeting will transform the way we think and approach cancer treatment.”

Expanding chemical modifications to an expanded genetic alphabet

Moving beyond sequence pools comprised of natural A, G, C, and T/U to chemically modified nucleotides in aptamers has been extensively investigated to either increase diversity of structural elements (e.g. hydrophobicity, polarity, 3D shape, etc.) to obtain improved binding, or improve biological properties (e.g. nuclease resistance, cellular uptake, etc.). Because of limitations in both enzyme activity and the modified NTPs readily available, the vast majority of modified aptamers have been developed using 2′ fluoro dC (fC) and 2′ fluoro dU (fU), although the 2′ amino and 2′ OMe analogs of the pyrimidines have also been used. More “exotic” modified dNTPs with pendant amino acid or hydrophobic moieties that have been investigated can be perused by clicking here for a 2012 review by Marcel Hollenstein entitled Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids.

Modified nucleotides used in SOMAmer™ selection (taken from Technical Note on SomaLogics website)

Modified nucleotides used in SOMAmer™ selection (taken from Technical Note on SomaLogics website)

Especially noteworthy, however, is SomaLogic’s development of novel SOMAmer™ (Slow Off-rate Modified Aptamer) protein-binding technology capable of measuring thousands of proteins in small volumes of biological samples with low limits of detection, a broad dynamic range, and high reproducibility. This approach, which was first published in 2010 in PLoS One, uses chemically-modified nucleotides shown below to mimic amino acid side chains together with new SELEX strategies. SOMAmers have already been developed for more than 1,100 different protein targets critical to normal and disease biology, with many more said to come soon.

More recently, a truly remarkable milestone in aptamer technology was reported earlier this year in Nature Biotechnology by Ichiro Hirao and coworkers at the RIKEN Systems and Structural Biology Center and TagCyx Biotechnologies in Japan. These investigators note that DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. They then describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base Ds, which specifically pairs with the unnatural base Px during PCR, as depicted below and highlighted in an earlier related blog.

Taken from a news article in BioTechniques 2013 via Bing Images.

Taken from a news article in BioTechniques 2013 via Bing Images.

Up to three Ds nucleotides were incorporated in a random sequence library. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. The authors concluded that “[t]hese results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.

Verified Randomized Sequences

I highly recommend reading Ellington and coworkers Current Protocols in Nucleic Acid Chemistry (2009) for detailed guidance on design, synthesis, and amplification of DNA pools for in vitro selection. These experts note that the “quality of randomization” is critically dependent on various aspects of automated DNA chain assembly using empirically determined ratios of A, G, C and T phosphoramidites to generate each N. Completely random pools should theoretically have 25% base composition at each N position, and therefore an unbiased distribution of di-, tri-, etc. nucleotide sequences.

If you’re looking for a supplier for verified random sequences, check out TriLink Biotechnologies. TriLink is a leader in verified manufacturing of randomized sequences that are now offered as convenient, stocked DNA or RNA random-libraries having 20, 30 or 40 randomized Ns and restriction sites for use with corresponding PCR primers and biotin labeling if desired. The mix of individual bases has been optimized to ensure as close to a 1:1:1:1 ratio of bases (i.e. 25% base composition) as possible during oligo synthesis. Each lot is tested by enzymatic digest to determine overall base composition and to verify that it falls within TriLink’s rigorous specifications, ensuring lot-to-lot reproducibility.

To my knowledge, TriLink is the only supplier of randomized sequences that have been verified by high-throughput applications (aka next-generation sequencing) to more deeply analyze randomization. NeoVentures BioTechnology has independently sequenced 61 clones of a 40-N library to obtain base composition at each position and, importantly, tri-nucleotide distribution, as detailed in this white paper. By contrast, NeoVenture’s analysis of a sample from an anonymous vendor revealed substantial deviation from random base composition and, consequently, unacceptable skewing of tri-nucleotide distribution.

Does complete randomness matter? Experts say yes: Ellington and coworkers opine that “[c]ompletely random sequence pools are used to initiate selection experiments when no functional nucleic acid sequence or structural motif is known in advance.” Furthermore, they add, “completely random sequence pools explore a much wider swath of sequence space and are more useful for the isolation of novel binding species (aptamers) or catalytic species.” 

Commercial Landscape

In July 2013 a comprehensive and up-to-date report entitled Aptamers Market – Technology Trend Analysis By Applications – Therapeutics, Diagnostics, Biosensors, Drug Discovery, Biomarker Discovery, Research Applications with Market Landscape Analysis – Global Forecasts to 2018 was made available by Market Research.com for purchase. The following is included in the free abstract of this report.

• Aptamers is an emerging market, widely considered as a rival or substitute to antibodies in the scientific industry. It is poised to grow rapidly in various application areas, including therapeutics and diagnostics. The global aptamers market is valued to be $287 million in 2013 and is expected to reach $2.1 billion by 2018.
• The list of promising aptamers in the clinical trials pipeline estimates that these synthetic chemical antibodies will soon surpass monoclonal antibodies in therapeutics, diagnosis, and imaging. With technological merits over antibodies, the aptamers market is poised to grow at par with antibodies in the next 10-15 years.
• The advantages of chemically modified aptamers and low production cost compared to antibodies aid the growth of the market.
• There are ~60 biotech and pharmaceutical companies working on aptamers.

A review of nanotechnology and aptamers noted an important linkage between “expiration of the early patents…and a simultaneous decentralization of aptamer-technology commercialization”.

Given the range of applications, growth trajectory, and estimated market potential of aptamers, I’m guessing this is a field many of you currently are or will want to monitor.  I’ve put together a list of notable players to keep your eye on. These include, in alphabetical order: AM Biotechnologies, LLC (U.S.) Aptagen, LLC (U.S.) Aptamer Sciences, Inc. (S. Korea) Aptamer Solutions, Ltd. (U.K.) Base Pair Biotechnologies (U.S.), Bioapter S.L. (Spain), Integrated Biotechnologies, Inc. (U.S.), NeoVentures Biotechnology, Inc. (Canada), NOXXON Pharma AG (Germany), OTC Biotech (U.S.), Regado Biosciences, Inc. (U.S.), and TriLink BioTechnologies (U.S.)—hum, maybe I should have done this list in reverse alphabetical order.

What lies ahead?

Sorry, no crystal ball for science! Taken from wired.com via Bing Images

Sorry, no crystal ball for science! Taken from wired.com via Bing Images

I don’t believe in a crystal ball that can predict the future for aptamers, or anything else for that matter. After searching the literature for aptamers and keywords such “future”, “perspective”, “horizon” or other forward-looking terms, I decided that there wasn’t any publication that I could quote from and cite as providing insights as to new directions. Consequently, I’ll offer my half-thoughts on some things that might occur at some time in the future.

Overall, I think that applications of aptamers will out-pace antibodies based on inherently greater structural diversity that can be obtained chemically, as opposed to biologically, and with a much more limited set of amino acid/analog “building blocks”. Other advantages, as noted by Base Pair Biotechnologies, include more economical production—especially in GMP grade—flexible “fine tuning” for desired bio-distribution, stability or shelf-life.

High-throughput sequencing and related bioinformatics will likely continue to enable new and improved aptamer selection methods, as exemplified by the recent work of Hoon et al., who used one such approach to develop a novel method for selecting high-affinity DNA aptamers based on a single round of selection.

Aptamers—not antibodies—will advantageously be selected for small molecule targets to develop new or improved label-free electronic sensors, such as those reported by Ohno et al. for an aptamer-modified graphene field-effect transistor. Incidentally, ethanolamine (HOCH2CH2NH2), which is associated with several diseases and has only four non-hydrogen atoms, was reported by Mann et al. to be perhaps the smallest aptamer target so far.

Finally, I expect that molecular modeling will get faster, cheaper, better and more accessible to folks wanting an aptamer against a molecular target, and thereby enable more computationally guided aptamer selection. One example of advances in this direction can be found in a 2013 publication entitled Computational approaches to predicting the impact of novel bases on RNA structure and stability by Harrison et al.

What do you think? Your ideas or other comments on this post are welcomed.

Postscript:  Latest breaking aptamer news

News from Naples


This first ever international meeting on Aptamers In Medicine Perspectives, which was supported in part by TriLink, brought together many experts in, and practitioners of aptamer technology whose presentations are listed in the Final Program. TriLink’s CEO, Rick Hogrefe was in attendance and provided the following insights:

“Dr. Hoinka, of the National Center for Biotechnology Information, described the use of a novel high- throughput (HT) SELEX method called HTSAptamotif, which is coupled to an NGS platform and probably a much bigger computer than most labs have, to crunch out sequence and motif patterns that allow you to rationally design your aptamer. It is reasonable to expect that some year all that will be needed to design the perfect aptamer will be the crystal structure of the target and your somewhat smarter smart phone. An iPhone app will be soon to follow, no doubt.”

Rick also observed that “[o]ne reoccurring topic of discussion was the advantages of using chemically synthesized aptamers versus those made by transcription. One of the main disadvantages of using long RNA aptamers (>60mer) is that they must be prepared by transcription. Methods are still in development to prepare these compounds economically and eventually at scale. Many researchers truncate their original sequence to something more manageable—a 19-mer was reported in one talk. The greatest fear regarding the use of enzymatic synthesis of aptamers was the removal of one very deleterious contamination—5’-triphosphate oligonucleotides, which are potent activators of RIG-I. This is done with a phosphatase after transcription. During the Q&A session that followed the talk of Dr. Zhou of John Rossi’s lab at the City of Hope, Dr. Sullenger said he very carefully studied this reaction and was unable to optimize it sufficiently to remove all the triphosphates from the aptamer. Another attendee believed that they were able to sufficiently remove the triphosphate. More work is needed to develop a good method to detect the presence of triphosphate modifications on long oligos.”

Rick’s concluding opinions are that “[o]verall, it is apparent that aptamers are not only here to stay, but are rapidly expanding into what may become the most universally applicable oligonucleotide technology to date. The excellent talks in this meeting covered much of the breadth of what aptamers are capable of doing. It is time to buy your random library kit from TriLink and start your own aptamer research. Most likely you will find an aptamer that fits the bill—and your target—perfectly.” 

Where are they now?

drgoldDr. Gold founded SELEX-based NeXagen, which later became NeXstar Pharmaceuticals. Gilead acquired NeXstar in 1999 and teamed up with (OSI) EyeTech to develop Macugen (aka Pegaptanib), which is an anti-angiogenic, chemically modified RNA aptamer for the treatment of neovascular (wet) age-related macular degeneration. Gold is the founder, chairman of the board, and CEO of SomaLogic, where he continues his work on aptamers. Dr. Gold was made a member of the American Academy of Arts and Sciences in 1993, and the National Academy of Sciences in 1995.

tuerkCraig Tuerk was a graduate student with Dr. Gold, and then worked at NeXagen (a SELEX-based company founded by Gold). Dr. Tuerk left NeXagen in 1994, many years before his co-invention of SELEX would ultimately lead to Macugen (see Gold above). He currently teaches biochemistry and genetics at Kentucky’s Morehead State University. The Tuerk Lab research interests include in vitro evolution strategies, combinatorial libraries and drug design.

szostakThe Szostak Lab is affiliated with the Center for Computational & Integrative Biology, Massachusetts General Hospital, the Department of Molecular Biology, Massachusetts General Hospital, the Department of Chemistry and Chemical Biology, Harvard University, the Department of Genetics, Harvard Medical School, and the Howard Hughes Medical Institute. The Nobel Prize in Physiology or Medicine 2009 was awarded jointly to Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak “for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.”

ellingtonDr. Ellington received his graduate degree in Biochemistry and Molecular Biology from Harvard University (working with Dr. Steve Benner), and was a post-doctoral Fellow with Jack Szostak at Massachusetts General Hospital. He was an assistant faculty member at Indiana University in the Department of Chemistry until 1998, and has since been a full Professor in the Department of Chemistry and Biochemistry at the University of Texas at Austin. Throughout his career Dr. Ellington has worked on the directed evolution of molecules and organisms, and has developed both insights into the origin of life and applications in biotechnology. The Ellington Lab motto is “what evolves here changes the world.”

TagCyx Biotechnologies 

According to its website, TagCyx Biotechnologies “core technology, the ‘unnatural base pair system’, has been developed by Dr. Hirao, founder and CEO of TagCyx Biotechnologies, during the course of his research at ERATO project (Exploratory Research for Advanced Technology) of Japan Science and Technology Agency, followed by the research at the University of Tokyo, and presently at RIKEN. The ‘Unnatural base pair system’ is an innovative technology which enables the site-specific incorporation of functional component into DNA, RNA and proteins. TagCyx Biotechnologies was founded in March 2007 as a RIKEN authorized venture business, with the mission of dissemination and application of this technology to various fields.”

From the clever corporate name joining natural base pairs TA and GC with variable, unnatural base pair YX, we can expect to see more from this company in the future.

Incidentally, according to a press release earlier this year, Dai Nippon Printing Co., Ltd. and TagCyx Biotechnologies have jointly developed a printing ink with a highly effective anti-counterfeit feature, through the integration of artificial DNA that is extremely difficult to replicate. This artificial DNA based ink is virtually impossible for third parties to replicate, and as it makes it possible to achieve powerful judgments of authenticity, can be used in anti-counterfeiting efforts connected with high-value printing requiring strong security, such as bank notes or cash vouchers.

MRI by DNA aptamers in vivo

After writing this blog, Philip Liu and his coworkers at Massachusetts General Hospital published the first-ever demonstration of protein-guided magnetic resonance imaging (MRI) by DNA aptamers in vivo using, as a model system, transcription factor (TF) protein AP-1, a heteroduplex protein comprising members of Fos and Jun immediate early gene families that plays an essential role in modifying gene expression by signal transduction. This work was published in FASEB J. A preprint of this report—kindly provided to me by Dr. Liu—describes details for conjugation of TF AP-1 or control synthetic double-stranded phosphorothioate end-capped DNA aptamers to superparamagnetic iron oxide nanoparticles (SPIONs), gold, or fluorescein for imaging by MRI, transmission electron microscopy studies, and optical methods, respectively. The SPION-enabled MRI results revealed that neuronal AP-1 TF protein levels were elevated in neurons of live mice after amphetamine exposure, as expected, while transgenic mice with neuronal dominant-negative A-FOS mutant protein, which has no binding affinity for the AP-1 sequence, showed a completely null MRI signal. These investigators concluded that further clinical development is warranted.

Shortest aptamer yet?

Also after finishing this blog, an 11-mer aptamer was reported by Nadal et al. to have been obtained by truncation of an aptamer that specifically binds to β-conglutin (Lup an 1), an anaphylactic allergen. The highest affinity was observed with a truncation resulting in a G-quadruplex-containing 11-mer sequence that had an apparent equilibrium dissociation constant (KD) of 1.7 × 10-9 M. This 11-mer sequence was demonstrated to have high specificity for β-conglutin and showed no cross-reactivity to other lupin conglutins (α-, δ-, γ-conglutins) and closely related proteins such as gliadin.

Another Resource

Nucleic Acid Therapeutics can be searched for “aptamers” with other keywords for full-text access to numerous publications on various aspects of RNA or DNA aptamer selection, targeting, etc. and reviews thereof.

Language Trivia

The word “aptamer” is used regardless of what language you happen to be reading or speaking. The same is true for “blog”, although in German someone might instead use “digitale Netztagebücher”. Since even for German speakers that doesn’t exactly flow off the tongue, the latest edition of Duden—the German equivalent of the Oxford English Dictionary, completely unrelated to “dude”—added 5,000 new words, many of them in English, such as “blog” and “flashmob”, according to Anna Sauerbrey, a German, in the New York Times Op-Ed on September 26, 2013.

Nanomedicine: Using ‘Tiny Little FedEx Trucks’ to Target Breast Cancer Tumors


As we enter October, national breast cancer awareness month, I thought it an appropriate time to review some of the latest advances in breast cancer research. Let’s start with a review of the latest statistics published by the NCI and a look at current treatments, then we’ll explore some of the most promising new research in this field.

According to the National Institutes of Health (NIH) National Cancer Institute (NCI) Cancer Topics for Breast Cancer, the projected statistics on breast cancer for women in the United States in 2013 are grim: 232,340 new cases and 39,620 deaths. Moreover, global statistics for breast cancer were estimated to be over 1.5 million new diagnoses and 500,000 deaths in 2010—with the majority of these being in low and middle income countries, which presents a major challenge of a different kind.

The NIH NCI website provides an informative online booklet entitled What You Need To Know About™ Breast Cancer to learn about breast cancer types, staging, treatment, and questions to ask the doctor. There is also NCI’s overview of Cancer Advances in Focus: Breast Cancer that provides the following perspectives for “today” and “tomorrow.”


  • Among women diagnosed with breast cancer during the period from 1999 through 2006, 90% were expected to survive their disease at least 5 years. Among white women, the 5-year relative survival rate was 91%; among African American women, it was 78%. The increase in breast cancer survival seen since the mid-1970s has been attributed to both screening and improved treatment.
  • Breast-conserving surgery (lumpectomy) followed by local radiation therapy has replaced mastectomy as the preferred surgical approach for treating early-stage breast cancer.
  • Routine mammographic screening is an accepted standard for the early detection of breast cancer. The results of eight randomized trials, the NIH-ACS Breast Cancer Detection Demonstration Projects, and other research studies showed that mammographic screening can reduce the mortality from breast cancer.
  • Hormonal therapy with selective estrogen receptor modulators (SERMs), such as tamoxifen, and aromatase inhibitors is now standard in the treatment of women with estrogen receptor-positive breast cancer, both as adjuvant therapy and in the treatment of advanced disease. Estrogen receptor-positive breast cancer cells can be stimulated to grow by the hormone estrogen. SERMs interfere with this growth stimulation by preventing estrogen from binding to the estrogen receptor. In contrast, aromatase inhibitors block estrogen production by the body. Food and Drug Administration (FDA)-approved aromatase inhibitors include anastrozole, exemestane, and letrozole.
  • Tamoxifen and another SERM, raloxifene, have been approved by the FDA as treatments to reduce the risk of breast cancer in women who have an increased risk of developing the disease.
  • The monoclonal antibody trastuzumab is an accepted treatment for breast cancers that overproduce a protein called human epidermal growth factor receptor 2, or HER2. This protein is produced in abnormally high amounts by about 20% of breast tumors. Breast cancers that overproduce HER2 tend to be more aggressive and are more likely to recur. Trastuzumab targets the HER2 protein specifically, and this antibody, in conjunction with adjuvant chemotherapy, can lower the risk of recurrence of HER2-overproducing breast cancers by about 50% in comparison with chemotherapy alone.
  • Several breast cancer susceptibility genes have now been identified, including BRCA1, BRCA2, TP53, and PTEN/MMAC1. Approximately 60% of women with an inherited mutation in BRCA1 or BRCA2 will develop breast cancer sometime during their lives, compared with about 12% of women in the general population.


  • We will use our rapidly increasing knowledge in the fields of cancer genomics and cell biology to develop more effective and less toxic treatments for breast cancer and to improve our ability to identify cancers that are more likely to recur. Moreover, we will use this knowledge to tailor breast cancer therapy to the individual patient.
  • We will use our increasing knowledge of the immune system to enhance the body’s ability to recognize and destroy cancer cells. The knowledge we have acquired thus far has facilitated the development of several promising breast cancer treatment vaccines that are currently under clinical evaluation.
  • We will use advanced technologies, including genomic technologies, to improve our ability to detect breast cancer at its earliest stages, when it is most treatable, and to better define individual risk for this disease.
  • We will strive to understand, address, and eliminate factors that contribute to the higher mortality from breast cancer experienced by African American women compared with women of other racial and ethnic groups.

Managing the Nation’s Cancer Research Portfolio

As a scientist and taxpayer, you’ll likely be very interested—as I was—in knowing much more about the specifics of what the NCI proposed for managing the Nation’s cancer research portfolio in 2013. This information is nicely laid out at an NCI website that includes a downloadable pdf version with programmatic details and a summary budget ($5.833 Billion).


National Cancer Institute Research Campus in Bethesda, MD (via Bing Images)

Among the details that I found particularly interesting is an introductory section called “Provocative Questions.” NCI launched an initiative late in 2010, seeking to go beyond the questions that are self-evident or that have been studied for many years. NCI “asked investigators to propose intriguing questions that need attention but might not otherwise get it or that have stumped us in the past but may be answered by new technologies. The initiative, which elicited a strong and exciting response from the research community, has recently funded its first 56 investigators.” In addition to a link therein for the Provocative Questions Project website, there is a link to a short video wherein NCI Director Harold Varmus, MD, discusses this project.

How do Normal v. Breast Cancer Cells Appear Microscopically?

Courtesy of NCI via Bing Images

Courtesy of NCI via Bing Images

From the descriptors given in the summary slide, provided by NCI, it is evident that breast cancer cell shape, size, and other properties reflect cellular dysfunctions, relative to normal cells, as is generally characteristic for other types of cancer. The accompanying high-resolution color enhanced scanning electron micrograph (SEM) of a breast cancer cell is visually stunning yet scary in view of the aforementioned death rate statistics.

A color enhanced scanning electron micrograph (SEM) of a breast cancer cell. Photograph: Science photo library (taken from theguardian.com via Bing Images)

A color enhanced scanning electron micrograph (SEM) of a breast cancer cell. Photograph: Science photo library (taken from theguardian.com via Bing Images)

Spotlight on an Expert

Prof. Esther H. Chang, MD PhD

Prof. Esther H. Chang, MD PhD

Prof. Esther H. Chang, MD PhD, is a member of the Departments of Oncology and Otolaryngology at the Lombardi Comprehensive Cancer Center of Georgetown University Medical Center. Before joining Georgetown University, Dr. Chang held positions at the National Cancer Institute (NCI), Stanford University, and the Uniformed Services University of Health Sciences. Currently, she is serving as the Interim President of the American Society for Nanomedicine and she is also an Executive Board Member of the International Society for Nanomedicine (ISNM) in Basel. Dr. Chang is the founding scientist of, as well as a Senior Consultant for, SynerGene Therapeutics, Inc. She has over 130 publications and has served as a member of a number of scientific advisory boards for NCI, NASA, the US Military Cancer Institute, and the Department of Energy.

I had the good fortune to become a collaborator of Dr. Esther H. Chang when she was working at the Uniformed Services University of Health Sciences, and I was literally across the street working at the FDA at NIH. We found that we shared a common interest in antisense therapeutics, which at that time (1980s) was a relatively new and—importantly—entirely novel drug development paradigm envisaged as affording more specific, less toxic anticancer agents. My lab could synthesize various chemically modified oligos, but had no molecular or cellular biology expertise. Dr. Chang’s lab at the time was working on Ras, which is a common oncogene in human cancer. We eventually published those initial collaborative investigations, and later reported a series of three papers on tumor-specific targeting and delivery of modified hybrid (DNA-RNA) anti-HER-2 siRNA analogs developed by TriLink (click here for details).

Dr. Chang’s achievements in specifically targeting tumors using antibody-fragment-tagged liposomal nanoparticles are indeed notable—and quotable—as outlined below.

“Tiny little FedEx trucks”

Tiny FedEx electric vehicles for delivery in Paris (Bing Images)

Tiny FedEx electric vehicles for delivery in Paris (Bing Images)

As scientists, we can appreciate the need to occasionally over simplify very complex technical concepts, and scientific jargon, in order to talk to nonscientists about our research. Consequently, I had to smile with appreciation when Dr. Chang referred to tumor cell-specific, targeted delivery of liposomal nanoparticles as involving “tiny little FedEx trucks” when interviewed on National Public Radio that can be listened to by clicking here.

Drug-carrying nanoparticles may be targeted to specific cell types. Illustration created by Amadeo Bachar for UCSD Center of Excellence in Nanomedicine; first published in Morachis et al., Pharm Rev 2012 (taken from Bing Images).

Dr. Chang’s use of this catchy metaphor is apropos for a couple of reasons. Just as trucks carry various types of cargo, liposomal nanoparticles (spheres pictured) can be loaded with various classes of therapeutic agents, ranging from traditional small-molecule entities to high-molecular weight genes, mRNAs, and mi/siRNA. Also, just as FedEx employs advanced technologies to locate ZIP-code intended-recipients, nanoparticles can utilize antibodies, fragments of antibodies (Y-shaped molecules pictured above) or other agents (ligands) for specific delivery to intended target cells via binding to cell surface receptors (pink molecules pictured above).

In an early publication entitled Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy, Dr. Chang noted that a long-standing goal in gene therapy for cancer is a stable, low toxic, systemic gene delivery system that selectively targets tumor cells, including metastatic disease. In particular, ligand-directed tumor targeting of cationic liposome-DNA complexes (lipoplexes) showed promise for targeted gene delivery and systemic gene therapy. She demonstrated that Lipoplexes developed in her lab directed by ligands such as folate, transferrin or anti-transferrin receptor scFv antibody fragment, showed tumor-targeted gene delivery, expression, and anti-cancer effect in mouse models of human breast cancer, as well as prostate, head and neck cancers, seeming to meet these goals.

Based upon these promising results, this nanodelivery system has moved in human clinical trials. When I asked Dr. Chang about the status of such trials, and whether these results were applicable is some way to breast cancer, she offered the following comments:

“Tumor specificity is very crucial to a cancer therapy’s efficacy,” Chang asserts. “Because we have a targeting moiety, the nanocomplex will travel through the bloodstream and whenever it encounters a tumor cell — whether a primary tumor or metastasis — the nanoparticle will bind to it, enter the tumor cell and destroy it.”

This tumor targeted nanomedicine delivering the p53 gene (SGT-53) has already completed a Phase I safety trial, the results of which were described in an article entitled Phase I Study of a Systemically Delivered p53 Nanoparticle in Advanced Solid Tumors published in May of this year. The purpose of this trial was to give back to cancer patients the tumor-busting p53 gene they have lost.

She continued, “We were very encouraged by the results since the patients handled SGT-53 very well.  Only minimal side effects occurred. Even more exciting, at the end of treatment the majority of patients showed at least stable disease. We were also able to demonstrate targeted delivery of SGT-53 to metastatic cancer, and not to normal cells. These agents are intended to increase the effects of standard therapies by sensitizing the tumor cells to their killing effects. Thus, cancer is less likely to recur. We have seen fantastic anti-cancer responses in animal models with breast cancer, and we have seen this treatment working in various types of solid tumors in patients. Therefore, we think it is very likely that it will also be successful in treating breast cancer.”

Regarding future development of this agent, Chang stated “Three Phase II clinical trials are imminent in patients with pancreatic cancer, glioblastoma and lung cancer. We hope to evaluate this agent in patients with breast cancer in the near future.”

More on Nanomedicine for Cancer Therapy

Researchers in nanomedicine (a term first used in the late 1990s) aim to develop a variety of revolutionary tools:

  • Drug carriers that focus drug action at the site of disease to limit side effects.
  • Vaccines that are more feasible for use in areas with limited health care access (more stable, less expensive, and with minimal adverse reactions).
  • Imaging agents that produce signal detectable from significant depths only in diseased tissues.
  • Scaffolds for culturing engineered tissues that mimic the corresponding extracellular matrix and enable modulation of development over time and real-time monitoring of their activity and biochemistry.

As with any scientific field, its boundaries blur with those of related fields, including pharmaceutics, bio- and materials engineering, nuclear medicine, tissue engineering, and many others.

Schematic of localized magnetic delivery (taken from Nature Nano 2009 via Bing Images)

Schematic of localized magnetic delivery (taken from Nature Nano 2009 via Bing Images)

Magnetically Triggered, Local On-Demand Release of Drugs

Tran & Wilson highlight a particularly fascinating approach in nanomedicine aimed at making controllable magnetic drug delivery possible for the treatment of breast cancer. They note that a recent study published by Kong et al. in Nano Letters documents the synthesis and performance of porous silica nanocapsules filled with magnetic nanoparticles as a controllable magnetic drug delivery vector. Under a remotely applied radiofrequency magnetic field, these nanocapsules demonstrate on-off switchable release of the internally loaded drug payload. Both in vitro and in vivo studies using mouse breast cancer cell models demonstrate that the magnetic targeting of these nanocapsules allows for deep tumor penetration and subsequent on-demand release of the drug cargo, significantly reducing tumor cell viability.

“Chemical Antibodies” – Improving Nature with RNA Aptamers

Earlier this year, Shigdar et al. reported the use of sensitive “chemical antibodies” for diagnosis in breast cancer. They state that Epithelial cell adhesion molecule (EpCAM) is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70–90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not robust enough to be able to detect EpCAM expression in all pathological tissues. Shigdar et al. go on to report a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded (FFPE) primary breast cancers.

Schematic of dye- or drug-containing nanoparticle targeted to cancer cells by Y-shaped RNA aptamers (taken from Chemical & Engineering News 2012 via Bing Images)

Schematic of dye- or drug-containing nanoparticle targeted to cancer cells by Y-shaped RNA aptamers (taken from Chemical & Engineering News 2012 via Bing Images)

Twenty-mer aptamers were chemically synthesized with 2′-fluoro- or 2′-O-methyl-pyrimidine bases, a 3′-inverted deoxythymidine, and 5′-fluorescent tags. One of these aptamers showed no non-specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast cancer xenograft where an anti-EpCAM antibody showed limited or no reactivity. They conclude that these results show the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy.

I hope you leave this blog inspired by the promising developments that have been made in the field of breast cancer research and optimistic about the strides we, as a research community, continue to make toward finding a cure.

Breaking News:  FDA backs Roche drug as first-of-a-kind therapy to treat breast cancer before surgery

On September 12th, the Washington Post reported online that the FDA’s panel of cancer experts voted 13-0, with one abstention, that the benefits of Perjeta (a monoclonal antibody developed at Genentech) as an initial treatment for breast cancer outweigh its risks. The recommendation is not binding, but sets the stage for the FDA to clear the drug as the first pharmaceutical option approved to shrink or eliminate tumors before surgery. “We are supporting the movement of a highly active drug for metastatic breast cancer to the first-line setting, with the hope that women with earlier stages of breast cancer will live longer and better,” said Dr. Mikkael Sekeres, an associate professor of medicine at the Cleveland Clinic. Doctors hope that using cancer drugs earlier could help shrink tumors, making them easier to remove. In some cases, that could allow women to keep their breasts, rather than having a full mastectomy.

Perjeta works by blocking signals inside cancer cells that would tell them to divide and grow Photo: Roche (taken from itv.com via Bing Images).  Differences between HER family receptors are described here.

Perjeta works by blocking signals inside cancer cells that would tell them to divide and grow Photo: Roche (taken from itv.com via Bing Images). Differences between HER family receptors are described here.

Taken from Bing Images

Taken from Bing Images