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

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

Taken from

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

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

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.








Modified mRNA Mania

  • Biosynthetic modified mRNA for gene-based therapy without the gene!
  • AstraZeneca bets up to $420M on Moderna’s “messenger RNA therapeutics”
  • “Me-too” Pharma frenzy to follow?

In a perspective on gene therapy published in Science this year, Inder M. Verma starts by observing that the concept of gene therapy is disarmingly simple. Introduce a healthy gene in a patient and its protein product should alleviate the defect caused by a faulty gene or slow the progression of the disease. He then asks the rhetorical question: ‘why then, over the past three decades, have there been so few clinical successes in treating patients with this approach?’ The answer in part has to do with challenges for cell or tissue-specific delivery, which admittedly is an issue for virtually any type of therapeutic agent. There is also concern for adverse events generally ascribed to unintended vector integration leading to neoplasias. Nevertheless, according to Verma, the present clinical trials pipeline is jammed with more than 1700 (!) clinical trials worldwide, drawing on a wide array of gene therapy approaches for both acquired and inherited diseases.

In view of this scientifically laudable but undeniable—if not frustratingly—slow progress, it’s not surprising that various groups of investigators—and investors—have recently opted to pursue a strategy that eliminates a DNA-encoded gene entirely! Instead, biosynthetic mRNA is delivered in order to directly produce the desired therapeutic protein product—this is now being referred to as “mRNA therapeutics”.

Having said this, let’s consider some pivotal scientific publications, patents, and the emerging commercial landscape for what looks to be a very hot area for research and corporate competition.

Modified mRNA Therapeutic Vaccines

An excellent review published in 2010 by Bringmann et al. entitled RNA Vaccines in Cancer Treatment covers various approaches to using mRNA encoding for tumor-associated antigens to induce specific cytotoxic T lymphocyte and antibody responses. RNA-transfected dendritic cell vaccines have been extensively investigated and are currently in numerous clinical trials (the details for which can be found at the NIH website by simply searching RNA vaccines).

Interestingly, clinical feasibility and safety assessment for direct intradermal injection of “naked” unmodified mRNA was reported back in 2008 by Weide et al., who removed metastatic tissue from each of 15 melanoma patients for total RNA extraction, reverse-transcription to cDNA, amplification, cloning, and transcription to produce unlimited amounts of copy mRNA.

Stabilizing unmodified mRNA by packaging in liposomes or forming complexes with cationic polymers has been widely investigated, as well as introducing chemical modifications to mRNA to make it more resistant against degradation and more efficient for translation. The latter includes elongation of the poly-A tail at the 3′-end of the molecule and modifications to the cap structure at the 5′-end. For example, if the original 7-methylguanosine triphosphate is replaced by an Antireverse Cap Analog (ARCA), the efficiency of transcription is strongly enhanced. To provide the immune system with even more potent signals, Scheel et al. modified mRNA with a phosphorothioate backbone in early commercial vaccine development work at CureVac GmbH (Tübingen, Germany) that continues today (see image below).

Effects of mRNA vaccines  (taken from an article in Drug Discovery & Development by Ingmar Hoerr, PhD, CEO and Cofounder of CureVac).

Effects of mRNA vaccines (taken from an article in Drug Discovery & Development by Ingmar Hoerr, PhD, CEO and Cofounder of CureVac).

In summary, in a 2013 review entitled RNA: The new revolution in nucleic acid vaccines, Geall et al. from Novartis Vaccines & Diagnostics (Cambridge, MA, USA) stated that “prospects for success are bright.” They site several reasons for this optimistic outlook including the potential of RNA vaccines to address safety and effectiveness issues sometimes associated with vaccines that are based on live attenuated viruses and recombinant viral vectors. In addition, methods to manufacture RNA vaccines are suitable as generic platforms and for rapid response, both of which will be very important for addressing newly emerging pathogens in a timely fashion. Plasmid DNA is the more widely studied form of nucleic acid vaccine and proof of principle in humans has been demonstrated, although no licensed human products have yet emerged. The RNA vaccine approach, based on mRNA, is gaining increased attention and several vaccines are under investigation for infectious diseases, cancer and allergy.

Modified mRNA for Expressing Clinically Beneficial Proteins

Dr. Katalin Karikó, Adjunct Associate Professor of Neurosurgery and Senior Research Investigator, Department of Neurosurgery, University of Pennsylvania (taken from

Dr. Katalin Karikó, Adjunct Associate Professor of Neurosurgery and Senior Research Investigator, Department of Neurosurgery, University of Pennsylvania (taken from

In a landmark publication by Karikó et al. in 2008, it was reasoned that the suitability of mRNA as a direct source of therapeutic proteins in vivo required muting its immunogenicity and boosting its effectiveness. Clues as to how this might be achieved were provided in their earlier work demonstrating the use of base-modified triphosphates to enzymatically synthesize in vitro mRNA having modified nucleosides [such as, pseudouridine (Ψ), 5-methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), or 2-thiouridine (s2U)] had greatly diminished immunostimulatory properties. They reasoned that, “if any of the in vitro transcripts containing nucleoside modifications would remain translatable and also avoid immune activation in vivo, such an mRNA could be developed into a new therapeutic tool for both gene replacement and vaccination”.

Using the aforementioned and other base-modified nucleotide triphosphates—all obtained from TriLink BioTechnologiesKarikó et al. found, surprisingly, that mRNA containing pseudouridine had a higher translational capacity than unmodified mRNA when tested in mammalian cells and lysates or administered intravenously into mice at 0.015–0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, and at each time-point there was more of the reporter protein when pseudouridine-containing mRNA was administered. Moreover, even at higher doses, only the unmodified mRNA was immunogenic. [Note: a fascinating follow-on publication provides a non-obvious—at least to me—molecular-level rationale for the surprising enhanced translation of pseudouridine-modified mRNA]. 


Uridine and pseudouridine differ in bonding to ribose but hydrogen-bond similarly to adenine. Pseudouridine is the most prevalent of the 100+ naturally occurring modified nucleosides found in RNA.

They concluded that, “[t]hese collective findings are important steps in developing the therapeutic potential of mRNA, such as using modified mRNA as an alternative to conventional vaccination and as a means for expressing clinically beneficial proteins in vivo safely and effectively.” Prior to publishing this pivotal report, Katalin Karikó and co-author Drew Weissman filed a patent application in 2006 entitled RNA containing modified nucleosides and methods of use thereof that was issued on October 2, 2012 as US 8,278,036 and is assigned to the University of Pennsylvania.


Blood Boosting with Erythropoietin

EPO stimulates the production of red blood cells (taken from via Bing Images)

EPO stimulates the production of red blood cells (taken from via Bing Images)

In a very persuasive demonstration of the real possibility of mRNA therapeutics, Karikó et al, reported in 2012 that non-immunogenic pseudouridine-modified mRNA encoding erythropoietin (EPO) was translated in mice and non-human primates. Indeed, a single injection of 100 ng (0.005 mg/kg) of HPLC-purified mRNA complexed to a delivery agent elevated serum EPO levels significantly and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10–100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma.

Rhesus macaque (taken from via Bing Images)

Rhesus macaque (taken from via Bing Images)

Using rhesus macaques (aka rhesus monkeys) they could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. Other researchers (Kormann et al.) independently used a single injection of modified murine mRNA to produce EPO in mice.

Kick-Start Cardiac Repair with VEGF-A

That’s the catchy title of a News & Views article in the October 2013 issue of Nature Biotechnology with an equally catchy byline that reads “[t]he survival of mice after experimental heart attack is greatly improved by a pulse of RNA therapy.” The featured report by Zangi et al., which is characterized as “a masterpiece of multidisciplinary studies…that will advance our thinking about therapeutic options in the cardiovascular arena,” is indeed impressive. These investigators report that intra-myocardial injections of vascular endothelial growth factor-A (VEGF-A) mRNA modified with 5-methylcytidine, pseudouridine, and 5’ cap structure resulted in expansion and directed differentiation of endogenous heart progenitors in a mouse model of myocardial infarction. They found markedly improved heart function and enhanced long-term survival of recipients. Moreover, “pulse-like” delivery of VEGF-A using modified mRNA was found to be superior to use of DNA vectors in vivo.

A heart attack (myocardial infarction) occurs when one of the heart's coronary arteries is blocked suddenly, usually by a blood clot (thrombus), which typically forms inside a coronary artery that already has been narrowed by atherosclerosis, a condition in which fatty deposits (plaques) build up along the inside walls of blood vessels (taken from via Bing Images).

A heart attack (myocardial infarction) occurs when one of the heart’s coronary arteries is blocked suddenly, usually by a blood clot (thrombus), which typically forms inside a coronary artery that already has been narrowed by atherosclerosis, a condition in which fatty deposits (plaques) build up along the inside walls of blood vessels (taken from via Bing Images).

Notwithstanding these promising results, the aforementioned News & Views article points out that microgram-scale doses of modified mRNA in mice used by Zangi et al. “would probably correspond to several hundred milligrams…in humans delivered in volumes that might exceed 10 ml per heart. In clinical practice, it would be very difficult to administer such volumes to infarcted hearts.” In my humble opinion, these are legitimate but purely hypothetical issues at this time and, given that it’s very “early days” for therapeutic modified mRNA technologies, it’s not unreasonable to assume that new modifications and/or improved delivery strategies can be developed to enable clinical utility.

From a technical perspective, this work by Zangi et al. involves a form of cell-free reprogramming and, as such, is a good segue into the next section. 

Modified mRNA for Cellular Reprogramming

In 2005, when I first heard of the concept of cellular reprogramming and dedifferentiation—which is to somehow coax a mature, differentiated cell to ‘run in reverse and go backwards biologically’ to a more primitive cell—my immediate impression as a chemist was this was impossible. Surely, I thought, this must violate the Second Law of Thermodynamics or, if not, is completely counterintuitive to how life works. Wow, was I wrong!

Reprogramming of differentiated cells to pluripotency is now firmly established and holds great promise as a tool for studying normal development.  It also offers hope that patient-specific induced pluripotent stem cells (iPSCs) could be used to model disease or to generate clinically useful cell types for autologous therapies aimed at repairing deficits arising from injury, illness, and aging. Induction of pluripotency was originally reported by Takahashi & Yamanaka by enforced retroviral expression of four transcription factors, KLF4, c-MYC, OCT4, and SOX2 (aka “Yamanaka factors”)—collectively abbreviated as KMOS. (TriLink sells these and other factors used to direct cell fate.) Viral integration into the genome initially presented a formidable obstacle to therapeutic use of iPSCs. The search for ways to induce pluripotency without incurring genetic change has thus become the focus of intense research effort.

Consequently, much attention has been given to the 2010 publication by Warren et al. entitled Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. In this work complete substitution of either 5-methylcytidine for cytidine or pseudouridine for uridine in protein-encoding transcripts markedly improved protein expression, although the most significant improvement was seen when both modifications were used together. Transfection of modified mRNAs encoding the above mentioned Yamanaka factors led to robust expression and correct localization to the nucleus. Expression kinetics showed maximal protein expression 12 to 18 hours after transfection, followed by rapid turnover of these transcription factors. From this it was concluded that daily transfections would be required to maintain high levels of expression of the Yamanaka factors during long-term, multifactor reprogramming regimens.

They went on to demonstrate that repeated administration of modified mRNA encoding these (and other) factors led to reprogramming various types of differentiated human cells to pluripotency with conversion efficiencies and kinetics substantially superior to established viral protocols. Importantly, this simple, non-mutagenic, and highly controllable technology was shown to be applicable to a range of tissue-engineering tasks, exemplified by mRNA-mediated directed differentiation of mRNA-generated iPSCs to terminally differentiated myogenic (e.g. heart muscle) cells.

Modified mRNA reprogramming fibroblasts into induced pluripotent cells for directed differentiation into myofibers, according to Warren et al. in Cell Stem Cell (2010)

Modified mRNA reprogramming fibroblasts into induced pluripotent cells for directed differentiation into myofibers, according to Warren et al. in Cell Stem Cell (2010)

Warren et al. concluded that “we believe that our approach has the potential to become a major enabling technology for cell-based therapies and regenerative medicine.”  According to the Acknowledgements section of this 2010 publication, corresponding author Derrick J. Rossi recently founded a company, ModeRNA [sic] Therapeutics, dedicated to the clinical translation of this technology.” That, we shall see below, has had stunning commercial investment consequences.

By the way, and not surprisingly, Rossi & Warren filed a U.S. patent application in 2012 claiming, among other things, iPSCs induction kits using 5-methylcytidine- and pseudouridine-modified mRNA encoding KMOS human cellular reprogramming factors.

AstraZeneca’s Big Bet on Moderna’s Modified-mRNA Therapeutics

AstraZeneca aims to use Moderna Therapeutics’ modified-messenger RNA technology to develop and commercialize new drugs for cancer and serious cardiovascular, metabolic, and renal diseases, under a multi-year deal that could net Moderna more than $420 million. Moderna is also eligible for royalties on drug sales ranging from high single digits to low double digits per product.

AstraZeneca—ranked 7th in sales in 2010 among the world’s pharmaceutical companies—has the option to select up to 40 drug products for clinical development of what the companies are calling messenger RNA Therapeutics™, which could dramatically reduce the time and expense associated with creating therapeutic proteins using current recombinant technologies, the companies say. Moreover, “where current drug discovery technologies can target only a fraction of the disease-relevant proteins in the human genome, we have the potential to create completely new medicines to treat patients with serious cardiometabolic diseases and cancer,” AstraZeneca CEO Pascal Soriot said in a statement. Mr. Soriot, who had been a senior executive at Roche, very recently joined AstraZeneca, which said it would reorganize R&D and eliminate 1,600 jobs by 2016 as part of a plan to address issues related to failures in clinical trials of several drugs just as big sellers like the antipsychotic Seroquel and the heartburn drug Nexium have lost or are about to lose patent protection.

Moderna, based in Cambridge, Massachusetts, is privately held and was founded in 2010 by Flagship VentureLabs in association with leading scientists from Boston Children’s Hospital and Massachusetts Institute of Technology. Moderna has developed a broad intellectual property estate including 144 patent applications with 6,910 claims ranging from novel nucleotide chemistries to specific drug compositions, according to its website.

DARPA also Bets Big on Moderna’s Modified-mRNA Therapeutics

As the saying goes, “when it rains it pours”, and for Moderna it’s pouring money!

On October 2nd, Moderna announced that the U.S. Defense Advanced Research Projects Agency (DARPA)—whose most successful bets so far have been internet technologies—has awarded the company up to $25 million for R&D using its modified-mRNA therapeutics platform as a “rapid and reliable way to make antibody-producing drugs to protect against a wide range of now and emerging infectious diseases and engineered biological threats.” The statement goes on to say that Moderna’s approach can “tap directly into the body’s natural processes to produce antibodies without exposing people to a weakened or inactivated virus or pathogen, as in the case with the vaccine approaches currently being tested.”

The grant could support research for up to 5 years to advance promising antibody-producing drug candidates into preclinical testing and human clinical trial. The company also received a $700,000 ‘seeding’ grant from DARPA in March to begin work on the project.

If you’re interested in some of the possible ideas associated with the project, go to the 2013 patent application by Moderna entitled Methods of responding to a biothreat, which even envisages a portable, battery operated device for synthesizing modified mRNA. Oh well, never let it be said that DARPA fears a risky bet; on the other hand, since DARPA’s “playing with house money” (aka our taxes!), I suppose it’s easy for them. Let’s hope they/we all win.

Other Commercial Players

In addition to TriLink’s mRNA products, related services, and new cGMP facility, there are other companies to mention here, which I’ll do in alphabetical order.

  • Acuitas Therapeutics has compared the effectiveness of its lipid nanoparticle (LNP) carriers in vivo with the most potent delivery systems reported in the scientific literature, and found that Acuitas LNPs demonstrate much greater luciferase expression in the liver after systemic administration.
  • CureVac is combining both the antigenic and adjuvant properties of mRNA to develop novel and effective mRNA vaccines. CureVac is currently developing therapeutic mRNA vaccines in oncology and therapeutic/prophylactic vaccines for infectious diseases. Information on five of its clinical studies is available at
  • Dendreon has a U.S. patent application for a method to make dendritic cell vaccines from embryonic stem cells that are genetically modified with mRNA encoding tumor antigen. However, no mRNA-searchable items are currently listed on Dendreon’s website.
  • In-Cell-Art is investigating new and improved nanocarriers for mRNA vaccines, and has collaborated with Sanofi Pasteur and CureVac in DARPA-funded studies.
  • Mirus Bio offers a TransIT®-mRNA Transfection Kit for high efficiency, low toxicity, mRNA transfection of mammalian cells, as described by Karikó et al.

Also noteworthy, the 1st International mRNA Health Conference recently held on October 23-24 at the University of Tübingen included talks by numerous key scientists in academia and industry that are well worth looking at in the Conference Program.

In conclusion, I hope that you found this emerging area of modified mRNA therapeutics as interesting and exciting as I did in researching this blog posting, and I welcome your comments.


After finishing the above blog, I came across these additional publications on possible mRNA therapies.

Huang and coworkers reported earlier this year that systemic delivery of liposome-protamine-formulated modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy was significantly more effective than plasmid DNA in a therapeutic model of human lung carcinoma in xenograft-bearing nude mice.

Zimmermann et al. reported successful use of mRNA-nucleofection for overexpression of interleukin-10 in murine monocytes/macrophages for anti-inflammatory therapy in a murine model of autoimmune myocarditis. [Note: for a related report on mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation see Levy et al.]

Cystic Fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Bangel-Ruland et al. reported in vitro results indicating that CFTR-mRNA delivery provided a novel alternative for cystic fibrosis “gene therapy”.

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 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 via Bing Images

Sorry, no crystal ball for science! Taken from 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 via Bing Images)

A color enhanced scanning electron micrograph (SEM) of a breast cancer cell. Photograph: Science photo library (taken from 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 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 via Bing Images). Differences between HER family receptors are described here.

Taken from Bing Images

Taken from Bing Images

Ripples from the 2013 TIDES Conference

Nucleic acids are getting small, SMARTT, long and weird

Like the “kid in the candy store,” it was a challenge for me to decide which of the many scheduled talks on oligonucleotide therapeutics and nucleic acids diagnostics at the 15th Anniversary TIDES 2013 conference would provide “tasty” blog content. Even more challenging was how to get that content since I couldn’t be at this event in Boston on May 12-15! Continuing the metaphor, “candy” I eventually selected scientific diversity to hopefully satisfy the different ”tastes” of readers.

Jerry Zon’s standup & co-blogger Rick Hogrefe

Jerry Zon’s standup & co-blogger Rick Hogrefe

The solution for how to be there, yet not be there, had me stumped. Fortunately my cleaver colleagues at TriLink got me there as a life-size cardboard standup for a “Bit of Boston” contest (check out all the pictures of me and TIDES attendees in my May 12 post), while Rick Hogrefe (President/CEO) offered to be my eyes and ears, so to speak. Following is Rick’s report to which I’ve added some visuals, links, and comments (in italics).

Getting Small:  Spherical Nucleic Acids Nanoparticle Delivery

Prof. Chad A. Mirkin, Northwestern University and AuraSense Founder

JZ Comments: Prof. Mirkin’s outstanding scientific accomplishments, pioneering nanotechnology research, and list of numerous awards – including the 2013 Linus Pauling Medal Award, recipients of which have frequently (~50%) gone on to receive a Nobel Prize – may be found at the Mirkin Research Group website.


Prof. Chad A. Mirkin – the most cited chemist in the world (past decade, Thomson Reuters) and the most cited nanomedicine researcher in the world.

The normal course of drug discovery usually starts with a drug concept in search of a discovery vehicle.  Rarely does a discovery vehicle create a new drug concept with its own set of remarkable properties.  Such may have been the luck of researchers at the International Institute for Nanotechnology directed by Prof. Mirkin – a man of many talents and head of a research group larger than many biopharmaceutical companies – who has led a nearly two-decade effort delving into the creation and use of nanoparticles combined with oligonucleotides.  His earlier collaborative work, with many others at Northwestern, has led to commercial successes in the diagnostic world and nanofabrication (see Nanosphere and NanoInk for details). However, recent discoveries regarding therapeutic applications of oligonucleotide-functionalized nanoparticles has led to what may be the development of an entirely new class of therapeutic nucleic acids called spherical nucleic acids (SNA).


As depicted above, SNA are comprised of an inorganic (e.g., gold, silver, silica, etc.) inner core and a nucleic acid outer shell. These compact, 13 nm particles exhibit unusual hybridization properties that are largely based on the cooperativity of densely packed strands and high local concentrations.  Transitions from duplex to single strands that normally occur over a 20°C range take place in a 2-8°C range on SNA.

This property was exploited as a diagnostic tool by preparing fluorescent probes Mirkin called “flares” (aka nano-flares) that are hybridized to the oligonucleotides on the gold SNA and thereby quenched.  Because of the very sharp melting transition, they are able to detect with greater accuracy a wider range of sequences compared to existing microarray technologies.  When the SNA oligonucleotides hybridize to the correct target, the tagged probes are released and light up, like a flare, due to unquenching of the fluorophore. The SNA wiki site provides many interesting details about SNA structure, function, applications and societal benefits of this novel class of materials, including gene regulation, molecular diagnostics, intracellular probes, and materials synthesis.


Depiction of flare-based detection (Mirkin et al) wherein multiplex detection of actin and survivin mRNA expression in cells was demonstrated using survivin-Cy5 and actin-Cy3 flares in conjunction changing the relative levels of survivin and actin mRNA in the cells by treatment with actin or survivin targeted siRNA.

Mirkin also reported that SNA have a range of other favorable properties useful for a therapeutic.  SNA-bound oligonucleotides are nuclease resistant and very non-immunostimulatory in comparison to other delivery vehicles.  Perhaps the most remarkable property is the ability for SNA to permeate almost any cell and distribute to most tissues including the brain.  Approximately 1% of the material administered to an animal crosses the blood brain barrier.  They demonstrated the ability of an SNA to reduce the level of Bcl2L12 expression in a mouse glioblastoma model using systemic delivery.  Read more about this line of research on anti-glioma therapeutics by the Mirkin group using novel siRNA-conjugated gold nanoparticles.

The mechanism of delivery was explored at length, with the most likely mode being uptake through caveolae-mediated endocytosis.  Class A scavenger membrane receptors, which are known to bind various polyanionic ligands that include polynucleotides, were found to likely be associated with this active transport.  Down-regulation of class A scavenger receptors resulted in cessation of uptake.  Also revealing was the fact that the nanoparticles required a nucleic acid shell in order to be taken up.  Controls using other conjugates were not taken up by cells.  In my conversation with Dr. Sergei Gryaznov – recently appointed as CTO of AuraSense Therapeutics, a subsidiary of AuraSense that commercializes SNA – he speculated that perhaps a high density of anionic ligands is all that is required in order to achieve uptake.

Regardless of which applications of SNA meet with success, it’s clear that Prof. Mirkin and his team have done a remarkable job at both developing and commercializing a very interesting class of nanoparticles that we will definitely keep an eye on.

Getting SMARTT:  Block-Polymer Delivery

Dr. Mary Prieve, PhaseRx


Mary Prieve, PhD, is Director of Biology at PhaseRx.

PhaseRx, located in Seattle, entered the nucleic acid delivery fray in 2008.  Their lead technology, called the SMARTT Polymer, is a vinyl polymer delivery system that consists of three domains.  The first is the targeting portion of the molecule.  The second is the payload carrier, to which the nucleic acid is conjugated in the examples shown.  The third is designed to facilitate release from the endosome, which is the mode of uptake of the particle.


SMARTT Polymer Technology (courtesy of PhaseRx)

PhaseRx proof-of-concept studies have focused on hepatocellular cancer (HCC).  Two genes, MET, a cell surface receptor, and CTNNB1 (β-catenin), a cell adhesion protein, are often found to be overexpressed in many HCC patients. siRNA sequences were found that specifically down regulated  those genes.  PhaseRx conducted a very eloquent animal study wherein they introduced the MET and CTNNB1 genes into mice using plasmids, demonstrating that co-expression of these genes can induce HCC, and that they can subsequently turn off the effect using their formulated siRNAs.  The result was a significant reduction in liver tumor size and alpha-fetoprotein (AFP) levels. They then modified the MET gene such that the gene still functioned to induce HCC, but did not match the siRNA sequence developed for wild type MET.  They obtained the result they had hoped for:   the modified MET gene was completely uneffected by the complex.

The audience had several interesting questions, one being whether this vinyl polymer is biodegradable.  Dr. Prieve said those studies are currently ongoing.  Another question was about the payload and the number of siRNAs conjugated to each particle, to which the answer was one per particle. According to PhaseRx’s website, it is “deploying an integrated delivery system based on novel synthetic polymers that delivers RNAi drugs into the cytoplasm by mediating their escape from intracellular vesicles called endosomes, where these drugs are often trapped and sequestered. The synthetic polymers exploit natural cellular processes and pH changes to effect endosome escape, delivering RNAi drugs into the cytoplasm where they can reach and inhibit the desired target of interest. The PhaseRx system offers significant advantages: effective intracellular RNAi delivery, broad applicability, and consistency with pharmaceutical development standards.”

Getting Long:  Targeting Long Non-Coding RNA

Dr. Jim Barsoum, CSO, RaNA Therapeutics


Jim Barsoum, PhD, has over 25 years of experience in biotech at Biogen Synta, and then Theracrine.

RaNA Therapeutics is focusing on the latest in the RNA craze, long non-coding RNA (lncRNA).  The presentation started with a much needed beginners course on lncRNA and what are some of its functions (Wikipedia has some good resources as well if you’re interested). Naturally, new findings will most likely indicate that much of these proposed functions are completely wrong, and that lncRNA operates in some other fashion, but that RaNA’s core technology still works (read on…).  Call that the cynicism of someone who follows the slow unveiling of the roles of microRNAs in the cell.  RNA biology is perhaps at one of its most exciting times.  The range of tools available today for the RNA scientist from commercial sources is remarkable, and “RNA-ologists” are exploiting these tools to make great strides forward.

One new fact that I took home is that lncRNA is often 5’ capped and 3’ polyadenylated, just like mRNA, however they are very poorly expressed, if at all.  The reason for this was not given.  One possibility that came to my mind was that perhaps lncRNAs are degraded by the same mechanism as mRNA.  Another possibility is that they require circularization to function using the same proteins that bind 5’ cap to the 3’ poly-A tail that is required by mRNA in order to be translated. Another interesting fact is that of all the RNA that is transcribed, only ~1% is mRNA for the production of protein.  There is a lot of unknown science in the other ~99%.

JZ Comments: To put Rick’s further remarks in context, I’ve included the mechanism from RaNA’s website for long noncoding RNA.  The description for the mechanism says, “RaNA’s proprietary technology upregulates the expression of desirable genes that can prevent or treat disease. RaNA’s approach operates epigenetically and reverses the endogenous repression of gene expression. RaNA’s therapeutic oligonucleotide can be administered as a subcutaneous injection in saline, and is then taken up by cells in most tissues of the body, crossing the endosome membrane to enter the cytoplasm and nucleus. When transcription of the unique lncRNA target reveals the PRC2 binding domain the RaNA antagonist therapy binds the lncRNA, which blocks PRC2 recruitment and allows for transcription to proceed resulting in mRNA upregulation.”


RaNA is exploiting one function of lncRNA which is the control of transcription in a cell.  The lncRNA silences genes by recruiting transcriptional repressor proteins, such as polycomb repressor 2 (PRC2) by binding to the target gene in a standard hybridization mode with part of lncRNA and to the protein with another part of the molecule in more of an aptamer fashion.  They found they can upregulate expression by blocking the region of the lncRNA that binds to the protein using a short DNA oligonucleotide comprised of 50% LNA.  Their initial target is the binding site for PRC2 on the lncRNA that appears to control transcription of the erythropoietin (EPO) gene.  They demonstrated they were able to upregulate the production of EPO. Preliminary data showing a 25-fold upregulation of Erythrid Krüoppel-like Factor (EKLF; aka KLF-1) in mice was presented as well.

We expect that the story on lncRNA is just beginning and has the potential to far surpass siRNA because of what may soon be found to be a vast number of different functions in the cell.

RaNA Therapeutics was co-founded by Art Krieg, MD, who has more than 20 years of experience in immune stimulatory CpG oligonucleotide R&D in academia, and co-founded Coley Pharmaceutical Group (acquired by Pfizer) based on “CpG-ology,” as well as co-founded the first antisense journal, Oligonucleotides, and the Oligonucleotide Therapeutics Society. According to the RaNA Therapeutics website, “the capitalized “R”, “N”, and “A” in RaNA spell out RNA, the type of nucleic acid underlying our approach.  The lowercase “a” represents activation.  Together the letters stand for RNA activation, the very basis of our drug development approach.”

Getting Weird:  Unnatural Base Pairs

Dr. Ichiro Hirao, RIKEN and President/CEO, TAGCyx Biotechnologies


As is usual for TIDES, there was very little “hard core” chemistry at this meeting.  However, of the few chemistry talks, one of the more interesting was that given by Dr. Hirao of RIKEN.  His research has been focused on the development of unnatural base pairs.  He described the base pair that appears to be the winning combination, which consists of a purine analog (Ds) and a pyrimidine analog (Px) that can bind to each other with high affinity but not to the natural bases.


Taken from Hirao and coworkers NAR 2012.

The authors demonstrated that these unnatural bases can also get incorporated by various polymerases with high fidelity.  Misincorporation of the wrong base against these unnatural bases was only 0.005%, which is only slightly higher than the misincorporation rate of 0.002% for standard bases.  The utility of the Ds base as a novel base for aptamer selection was described.  Several random libraries that contained from one to three substitutions with the Ds base at specific locations were prepared.  The oligos did not contain any Px bases, thus forming unusual structures that were not found in the natural sequence.  This opened up the structural space for aptamer selection greatly.  Using an ingenious method that substituted the Px base pair with a less specific base, Pa allows misincorporation of either dA or T at that site.

According to, RIKEN was founded in 1917 and has ~3000 scientists on seven campuses across Japan, the main one in Wako, just outside Tokyo. RIKEN is an Independent Administrative Institution whose formal name in Japanese is Rikagaku Kenkyūjo and in English is The Institute of Physical and Chemical Research. RIKEN conducts research in many areas of science ranging from basic research to practical applications. It is almost entirely funded by the Japanese government, and its annual budget is approximately $760 million.

JZ Comments: I wish to thank Rick Hogrefe for being a guest blogger and providing  content!  As always, I welcome reader comments!