Advances in Nucleic Acid-Based Therapeutics Against Alzheimer’s

  • Dementia Develops in Someone in the World Every 3 Seconds
  • Nucleic Acid-Based Approaches for an Alzheimer’s Drug Are Advancing
  • Ionis Pharmaceuticals is First to Begin Clinical Trials with Antisense Oligonucleotide Targeting Tau

The concept of nucleic acid-based therapeutics, as originally conceived by Paul Zamecnik, goes back to his seminal publication in 1978 that I’ve blogged about previously. However, it wasn’t until the advent of automated synthesis of various types of modified oligonucleotide analogs that this “antisense” approach to drug development achieved critical mass of sustained attention. Since then, synthetic oligonucleotides and mechanisms of interference with mRNA expression have greatly expanded to now include a diverse “armory” of alternatives to combat diseases, including:

  • Antisense oligonucleotides (ASOs) that induce cleavage of mRNA by RNase H or other mechanisms
  • Short-interfering RNA (siRNA) that are incorporated into RISC that cleaves mRNA
  • Antagomirs that block microRNA (miR) binding to mRNA
  • Aptamers that bind to target proteins as drugs, or for targeted delivery of other types of drugs
  • Splice-switching oligonucleotides (SSOs) that hybridize with a pre-mRNA and disrupt normal splicing
  • Recently reported RNA-guided RNA-targeted CRISPR-Cas variants that can knockdown RNA systems much more specifically than siRNA

Investigating possible NA-based approaches to currently refractory, or other so-called ‘undruggable’ diseases, has attracted much needed interest from academics and pharma researchers, as well as implementation of GMP procedures. Recognizing this need, and the absence of a conventional drug to treat Alzheimer’s disease (AD)—the leading cause of dementia in adults—kindled my efforts to research the literature and write this blog.

What is AD?

Auguste Deter, who was a patient of German psychiatrist Alois Alzheimer, was the first described case of what became known as Alzheimer’s disease. Taken from Wikipedia.org

According to a U.S. National Institutes of Aging fact sheet, AD is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, and eventually the ability to carry out the simplest tasks. In most people with AD, symptoms first appear in their mid-60s. Statistics vary, but AD accounts for as much as ~80% of all dementia—an umbrella term for loss of cognitive functioning—which will develop in someone in the world every 3 seconds, and afflict close to 50 million people in 2017. This is based on estimates by Alzheimer’s Disease International.

Brains from bodies of deceased persons who had advanced AD exhibit overall “shrinkage,” macroscopically, compared to persons who did not show indications

Taken from fellowshipoftheminds.com

of AD. Microscopically, AD is characterized by accumulation of toxic “amyloid plaques,” which are sticky buildup that accumulate outside nerve cells, or neurons. Amyloid is a protein that is normally found throughout the body, but in AD the protein is cleaved by beta secretase (BACE1) and gamma secretase to yield amyloid beta (Aβ) peptides of 36–43 amino acids. Aβ molecules can aggregate to form flexible soluble oligomers that may exist in several forms. It is now believed that certain misfolded oligomers can induce other Aβ molecules to also take the misfolded oligomeric form, leading to a chain reaction akin to a prion infection.

Amyloid plaques. Taken from slideshare.net

Since 1993, when a variant of the apolipoprotein E (APOE) gene was found to be strongly associated with increased vascular and plaque Aβ deposits in late-stage AD patients, many researchers have probed APOE connections to Aβ. However, in September 2017 this investigative field was reportedly “stunned” by results published by Shi et al. showing that neurotoxic effects associated with APOE may result from a damaging immune response to a different protein named tau, encoded by the MAPT gene. This late-breaking scientific news is part of an ongoing story of AD molecular pathology that interested readers will want to follow.

“Tangles” of tau protein (green) are visible in a brain cell from someone who had Alzheimer’s disease. Taken from Underwood, Science (2017)

Recent Advances in Nucleic Acid-Based Drugs for AD

My strategy for researching this topic began with querying Google Scholar for “Alzheimer’s” articles since 2013 that are coupled with key words for each of the above nucleic acid-based drug modalities, namely, “antisense,” “siRNA,” etc. This led to literature ranked by Google Scholar according to citation counts. I then perused this information to select items for each of these modalities. I linked to the search results for readers interested in further digging into these topics.

Antisense Oligonucleotides

A report that caught my attention was by Lane et al. from Ionis (formerly Isis) Pharmaceuticals. This company (founded and still led by Stanley Crooke, about whom I’ve blogged) is widely acknowledged to be the leader in clinical development of antisense therapeutics. In a nutshell, Lane et al. hypothesized that, given the critical role for tau (see above) in transducing Aβ-linked neurotoxicity, reducing the synthesis of tau could have a therapeutic effect.

Ionis-MAPTRx, a 2′-O-methoxyethyl chimeric ASO, was found to reduce tau expression in transgenic mice and was tested in FDA/IND-enabling toxicology studies in rodents and non-human primates (NHPs). Intrathecal administration of the highest dose in NHPs resulted in a mean MAPT mRNA reduction of 77% in frontal cortex and 74% in hippocampus without dose-limiting side effects. Following up on this, I found that on October 13, 2017, Ionis announced initiation of a Clinical Study of Ionis-MAPTRx in patients with AD, thus earning a $10 million milestone payment from Biogen.

In another ASO approach, Farr et al. have further investigated their previously reported 20-mer phosphonothioate oligonucleotide (GAO) that had been shown to knockdown levels of glycogen synthase kinase (GSK)-3β, which is a multifunctional protein implicated in the pathological characteristics of AD, including neurofibrillary tangles, Aβ, and neurodegeneration. In the present study, they assessed the impact of peripherally administered GAO on learning and memory—measured by a T-maze (see picture)—in two different mouse models of AD, as well as knockdown of protein expression. GAO-treated mice showed improved acquisition and retention, along with decreased protein levels. They concluded that this study “supports peripherally administered GAO as a viable means to mediate GSK-3β activity within the brain and a possible treatment for AD.”

Taken from ratbehaviour.org

To learn about a T-maze, click here.

siRNA

SLN loaded with siRNA (green). Taken from precisionnanosystems.com

While there are several intriguing studies of ASOs associated with AD therapy, siRNA is much more common in the research and treatment of AD. The report that I found most interesting was published by Rassu et al. in 2017, and is titled Nose-to-brain delivery of BACE1 siRNA loaded in solid lipid nanoparticles for Alzheimer’s therapy. My interest stemmed from the focus on developing delivery technology to achieve “nose-to-brain” as a very convenient, non-invasive route for delivery using solid lipid nanoparticles (SLNs), which are viewed as promising technologies for drug-delivery.

In this report, the siRNA targeted the secretase BACE1, which has been widely investigated because of its involvement in forming neurotoxic Aβ peptides, as mentioned above. To increase the transcellular pathway in neuronal cells, a short cell-penetrating peptide derived from rabies virus glycoprotein known as RVG-9R was used, based on previous studies demonstrating that intravenous treatment with an RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Building on this prior knowledge, Rassu et al. optimized the molar ratio of RVG-9R and BACE1 siRNA, and investigated chitosan-coated and uncoated SLNs as a nasal delivery system capable of exploiting both olfactory and trigeminal nerve pathways.

Taken from researchgate.net

The positive charges from protonated amino (NH2) groups of the coating formulation ensured muco-adhesiveness to the particles, and prolonged residence-time in the nasal cavity. They studied cellular transport of siRNA released from the SLNs using the Caco-2 cells, which is a human epithelial colorectal adenocarcinoma cell line, as a model for epithelial-like phenotypes. It was found that siRNA better permeates the monolayer when released from chitosan-coated SLNs vs. uncoated SLNs or “naked” siRNA.

Chitosan. Taken from Wikipedia.org

Antagomir

MicroRNA-146a (miR-146a) is upregulated in the brains of patients with AD and induces activation of tau (see above). To determine whether reducing miR-146a could ameliorate tau-related AD pathologies, Wang et al. assessed its levels and the use of a miR-146a inhibitor (antagomir) in a validated mouse model of AD. The antagomir and solvent control were delivered into the hippocampus of these mice at three months of age and memory was tested in all mice using several types of mazes (see above) before extracting brain samples for quantitative RT-PCR using measurements of miR-146a and protein targets. In a nutshell, the overall results demonstrated that improvement of memory by intrahippocampal miR-146a antagomir was associated with the predicted alterations in the tau-related neural pathway, confirming that inhibition of miR-146a expression has a therapeutic effect in this mouse model of AD. It was concluded that “this data support (sic) the concept that miR-146a antagomir is a potential efficacious therapeutic target for the tau pathology of AD.”

Aptamers

Aptamer A1 structure reported by Liang et al.

In a study published by Liang et al. in 2015, systematic evolution of ligands by exponential enrichment (SELEX) with random-sequence libraries was used to obtain a DNA aptamer (A1). That aptamer is pictured below, and has been shown to have high-affinity binding to purified human BACE1 extracellular domain. They subsequently confirmed that A1 exhibited a marked inhibitory effect on BACE1 activity in an AD cell model, based on decreased concentrations of Aβ fragments and BASE1 protein. These investigators concluded that “these findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.”

Splice-Switching Oligonucleotides

According to Hinrich et al., apolipoprotein E receptor 2 (ApoER2), which is involved in long‐term potentiation, learning, and memory, has been proposed to be involved in AD, though a role for the receptor in the disease is not clear. ApoER2 signaling requires amino acids encoded by alternatively spliced exon 19. To test the role of deregulated ApoER2 splicing in AD, they designed a splice-switching oligonucleotide (SSO) that increases exon 19 splicing. Treatment of AD mice with a single dose of SSO corrected ApoER2 splicing for up to 6 months and improved synaptic function and learning and memory. They concluded that “these results reveal an association between ApoER2 isoform expression and AD, and provide preclinical evidence for the utility of SSOs as a therapeutic approach to mitigate AD symptoms by improving ApoER2 exon 19 splicing.”

CRISPR

Individuals heterozygous for the Swedish mutation of the amyloid precursor protein (APPswe) display an increased β-secretase cleavage leading to higher Aβ levels—both in brain and peripheral tissues, according to Gyorgy et al. They added that the mutation is a double base change adjacent to each other and has a dominant effect, which led them to hypothesize that the CRISPR system would selectively disrupt the mutated allele without affecting the wild-type (wt) allele.

In a nutshell, human APPswe fibroblasts and non-mutated control fibroblasts from subjects of the same family were grown in vitro, and then transfected with a Cas9 plasmid together with different guide RNAs (gRNAs) designed to bind either the mutated or non-mutated site with the mutation in the gRNA recognition sequence. Sanger sequencing was performed on cells that had been successfully transfected with CRISPR plasmids, and on such cells, both the APPswe mutant and wt alleles could be disrupted with gRNAs designed against the mutated and non-mutated sites, respectively. Moreover, these effects appeared to be highly specific as assayed by deep sequencing as they did not find any random mutations on the wt allele with the gRNA targeting the mutated site or vice versa.

This study reported in 2016 was said to “[provide] the first experimental evidence that the CRISPR/Cas9 method could be used to develop a novel treatment strategy against familial forms of Alzheimer’s disease caused by dominant mutations.”

Closing Comments

From the above sampling of publications reporting promising results for nucleic-acid-based therapeutic approaches to AD, I hope you will agree with me that it seems likely a clinically successful drug will prevail. What and when are uncertain, but I’m betting that something will be Ionis-MAPTRx , which is the most advanced clinical candidate to date.

Addendum

Taken from October 31, 2017 GEN

Alzheimer’s disease may move, cancer-like, from place to place in the body, lodging in the brain after originating in peripheral tissues, according to October 31, 2017 news in GEN. This cancer-like mobility was demonstrated through a technique called parabiosis—the surgical union of two specimens to allow them to share a blood supply, as shown here. This technique was used to keep pairs of mice together for several months, wherein normal mice, which don’t naturally develop AD, were joined to transgenic AD mice, modified to carry a mutant human gene that produces high levels of plaque-forming Aβ. It was reported that human Aβ originating from transgenic AD mice entered the circulation and accumulated in the brains of normal mice, forming cerebral amyloid angiopathy and Aβ plaques after a 12-month period of parabiosis.

Jerry’s Favs from the Recent 7th Cambridge Symposium

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

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

Taken from na-cb.co.uk

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

Taken from worldachitecture.org

Overview of the Symposium

Mike Gait. Taken from histmodbiomed.org

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

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

Subject areas this year included:

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

Marv Caruthers. Taken from colorado.edu

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

Jerry’s Favs from the Symposium

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

DNA Can Function as an Enzyme!

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

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

Taken from Silverman Acc Chem Res (2015)

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

RNA Polymerase Activity Without Proteins!

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

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

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

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

Systemic Brain Delivery of Therapeutic Oligos!

Taken from igtrcn.org

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

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

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

Parting Thoughts and The Eagle

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

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

As usual, your comments are welcomed.

Personal photo using a Samsung Galaxy S8

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Advances in Aptamer Applications – Part 2

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

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

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

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

Top 3 Cited Publications in 2014

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

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

Taken from pubs.rsc.org

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

Taken from mct.aacr.org

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

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

Aflatoxin B₁ structure. Taken from wikipedia.org

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

Top 3 Cited Publications in 2015

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

Taken from sciencedirect .com

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

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

Chloramphenicol. Taken from Wikipedia .com

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

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

Taken from mdpi .com

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

Top 3 Cited Publications in 2016

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

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

Taken from sigmaaldrich .com

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

Taken from wikipedia.org

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

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

Taken from Liu et al. Biomaterials (2016)

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

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

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

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

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

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

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

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

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

Aptamer Publications in 2014-Present Citing TriLink Products

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

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

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

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

As usual, your comments are welcomed.

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Curiously Circular RNA (circRNA) Gets Curiouser

  • circRNA Molecules Have, Oddly, No Beginning or End
  • circRNA Are Now Recognized as Regulators of Gene Expression 
  • A Flurry of New Findings Indicate circRNA Are Also Templates for Synthesis of Proteins Having As Yet Unknown Functions

Electron micrograph of ~3,000-nt circRNA. Taken from Matsumoto et al. PNAS (1990).

About a year ago, my blog titled Curiously Circular RNA pointed out that circular RNA (circRNA) in animals are odd molecules in that, unlike the vast majority of other RNA in animals, circRNA have no structural beginning (5’) or end (3’). This very curious feature has, not surprisingly, stimulated considerable scientific interest in knowing more about these molecules, which were serendipitously discovered some 30 years ago.

Application of next-generation sequencing has revealed that circRNA are actually relatively abundant and evolutionarily conserved, which implicates biological importance rather than inconsequential mistakes during RNA splicing mechanisms. Some circRNA have been shown to have function—circRNA can hybridize to complementary microRNA (miRNA), and thus serve as a kind of ‘sponge’ that influences miRNA-based gene expression. Evidence for circRNA involvement in gene expression continues to grow, as there are now >700 items on “circRNA [and] sponges” in Google Scholar.

Very recently published lines of research (that I’ll outline in what follows) implicate circRNA as coding templates for proteins, which heretofore has been exclusively associated with messenger RNA (mRNA). Current dogma holds that translation of mRNA into protein requires recognition of the 7-methylguanylated (m7G) 5’-cap structure to start ribosome binding, while the 3’-poly(A) tail protects the mRNA molecule from enzymatic degradation and aids in stopping translation, as depicted below.

Taken from Shoemaker & Green Nature Structural & Molecular Biology (2012).

Start and stop structural elements characteristic of mRNA are obviously not present in circRNA, which are literally just circles of RNA. Consequently, finding proteins encoded by circRNA has stirred up controversy about whether such proteins are a new and fundamentally important aspect of genetics or just inconsequential biochemical mistakes.

Translation of circRNA in Fly Head Neurons

Fruit fly. Taken from turbosquid.com

Researchers at The Hebrew University of Jerusalem in Israel in collaboration with a team at Max-Delbruck-Center for Molecular Medicine in Berlin, Germany recently reported in Molecular Cell the first compelling evidence that a subset of circRNA is translated in vivo. The study by Kadener & coworkers was carried out using the common fruit fly (Drosophila melanogaster), which is known to have a number of features that lend to investigations of circRNA: (1) >2,500 fruit fly circular RNAs have been rigorously annotated, (2) these are mostly derive from back-splicing (pictured below) of protein-coding genes, (3) hundreds of which are conserved across multiple Drosophila species, and (4) exhibit commonalities to mammalian circRNA.

Direct back-splicing: a branch point in the 5’ intron attacks the splice donor of the 3’ intron. The 3’ splice donor then completes the back-splice by attacking the 5’ splice acceptor forming a circRNA. Taken from Jeck & Sharpless Nature Biotechnol (2014).

This study by Kadener & coworkers involves a plethora of technically complex experimental procedures and associated jargon, from which I’ve extracted what I believe to be some key points to share. After annotating the Drosophila circRNA open reading frames (cORFs), which, by definition,h have the potential for translation, they searched for evidence of their translation utilizing previously published ribosome footprinting (RFP). This led to identification of 37 circRNAs with at least one specific RFP read, referred to as ribo-circRNAs.

Taken from Jeck & Sharpless Nature Biotechnology (2014)

Several representative ribo-circRNAs were then constructed to each have (pictured below) a metallothionine (MT) promoter and V5 tag to facilitate translation and anti-V5 antibody-based detection of the expected protein after transfection into cells.

To determine whether circRNAs are translated in a more relevant tissue, they set up the RFP methodology in fly heads. A genetic locus named mbl that is known to produce a circRNA (circMbl3) at high abundance was selected for targeted mass spectrometry from a fly head immunoprecipitated MBL. They utilized synthetic peptides to determine characteristic spectra for which to search in the fly head immunoprecipitate and found a consistent and very high confidence hit for a peptide that can only be produced by circMbl3.

Kadener & coworkers extended these fly head findings to mammalian mouse and rat systems, but the most interesting part of this study—in my opinion—dealt with what signals ribosome binding and translation in the absence of the 5’ cap structure present in mRNA. They demonstrated circRNA translation under conditions intended to block normal 5’ cap-dependent translation of mRNA, and concluded that “[untranslated regions] of ribo-circRNAs (cUTRs) allow cap-independent translation [and that] further research is necessary to uncover how these sequences promote translation.”

Remarkably, as you’ll now read, another group of investigators have apparently found how such promotion of circRNA translation can occur.

Translation of circRNA is Driven by N6-Methyladenosine (m6A)

The most abundant modification of RNA in eukaryotes is m6A, which has been recently shown by Li et al. to recruit binding proteins that collectively facilitate the translation of specifically targeted mRNAs—i.e. those “marked” with m6A—through interactions with 40S and 60S ribosome subunit “machinery” that actually carry out translation. Contemporaneously, Yang et al. found that m6A likewise promotes efficient initiation of protein translation from circRNAs in human cells. They discovered that consensus m6A motifs are enriched in circRNAs, and a single m6A site is sufficient to drive translation initiation.

As depicted below, this m6A-driven translation requires initiation factor F4G2 and m6A “reader” YTHDF3. Experiments showed that this translation is enhanced by methyltransferase METTL3/14 and inhibited by demethylase FTO, which enzymatically “add” and “subtract” methyl (Me) groups on specific adenosines (A) in circRNAs, respectively.  It has also been shown to be upregulated upon heat shock, which is a commonly employed method to induce “stress” in cells.

Taken from Yang et al.

Further analyses through polysome profiling, computational prediction and mass spectrometry revealed that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Yang et al. concluded by stating that their “study expands the coding landscape of [the] human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.”

Zinc Finger Protein in Muscle Cell Development

Finally, and essentially contemporaneously with above mentioned two publications, a third independent investigation reported by Legnini et al. demonstrated selective circRNA downregulation using short-interfering RNAs (siRNAs). These reagents for RNA interference (RNAi) were used in an image-based functional genetic screen of 25 circRNA species, conserved between mouse and human, expression of which are differentially expressed during myogenesis (i.e. formation of muscular tissue) in Duchenne muscular dystrophy myoblasts.

This siRNA/RNAi-based functional analysis provided one interesting case related to zinc finger protein 609 (circ-ZNF609)—a reported miRNA sponge—the phenotype of which could be specifically attributed to the circular form and not to the linear mRNA counterpart. Consistent with the circ-ZNF609 sequence having an ORF, they found that a fraction of circ-ZNF609 RNA is loaded onto polysomes and that, upon puromycin treatment, it shifted to lighter fractions, similar to mRNAs. The coding ability of this circRNA was proved through use of artificial constructs expressing circular tagged transcripts, and by CRISPR/Cas9—the trendy gene editing method about which I’ve already commented multiple times.

Despite all this evidence, Legnini et al. stated that they “have no hints on the molecular activity of the proteins derived from circ-ZNF609 and as to whether they contribute to modulate or control the activity of the counterpart deriving from the linear mRNA.”

In thinking about closing comments about this update in circRNA, I decided to emphasize that investigations in the field of RNA continue to reveal complexities that will require many more years of global attention to unravel and understand. In just the past decade or so we’ve learned about gene regulation by miRNA/siRNA, reclassification of “junk DNA” as encoding a myriad of long noncoding RNA (lncRNA), mRNA regulation by base-modifications, and curious circRNAs that are more than sponges, and likely encode hundreds (if not thousands) of proteins whose functions have yet to be elucidated. Amazing!

What are your thoughts about all of this?

Your comments are welcomed.

Postscript

After writing this blog, Panda et al. at the National Institute on Aging-Intramural Research Program, National Institutes of Health published a paper titled High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Here’s a snippet of the abstract which adds to the increasingly curious occurrence of circRNAs that begs, if you will, further research aimed at discovering functions of circRNA-derived proteins.

“Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.”

Evolving Polymerases to Do the Impossible

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

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

Floyd E. Romesberg. Taken from utsandiego.com

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

Doing the Impossible

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

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

DNA template   5’ ————————————- 3’

RNA primer                                 ←←← 3’ xxxxxx 5’

mPol ↓ 2’-OMe NTPs

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

2’-OMe template   5’ xxxxxxxxxxxxxxxxxxxxxxxxxxx 3’

DNA primer                                           ←←← 3’ —— 5’

mPol ↓ dNTPs

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

How They Did It

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

Taken from Chen et al. Nature Chemistry (2017)

What is the Significance

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

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

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

As usual, your comments are welcome.

New CRISPR System Reported for Targeting RNA Instead of DNA

  • Current “CRISPR Craze” for DNA Editing is Catalyzing Creativity
  • Early CRISPR Innovator Feng Zhang Now Reports Targeting RNA
  • This New “C2c2” System has Specificity Issues But is Nevertheless Promising

Just when you thought that the “CRISPR craze” would soon transition from the fundamental discovery phase to the improvements phase, something entirely new for CRISPR has come along. That something, recently published by Feng Zhang and others in venerable Science magazine, targets RNA instead of DNA. Consequently, this may lead to transient vs. permanent editing, as well as other RNA- vs. DNA-based applications.

Before further commenting on this exciting new RNA-targeting approach using CRISPR, here are a few snippets about the original DNA version of CRISPR to set the stage, and substantiate my tongue-in-cheek referral to the craze about it.

Backstory

Taken from igtrcn.org

Taken from igtrcn.org

Editing with CRISPR, which is short-form for CRISPR-Cas9, uses sequence-specific guide RNA (gRNA) to target DNA for cutting by Cas9 nuclease, as depicted below. Guide RNA and Cas9 can be introduced into cells either encoded in a vector or as synthetic gRNA and synthetic Cas9 mRNA, which TriLink offers in either wild-type or base-modified forms of Cas9 mRNA. CRISPR for genome editing was publicly described in Science in 2012 by co-corresponding authors Jennifer A. Doudna, a biologist at the University of California, Berkeley, and the French microbiologist Emmanuelle Charpentier. But Feng Zhang, at the Broad Institute, was first to obtain a patent on the technique.

Not surprisingly, given the financial potential for DNA editing by CRISPR, which has been called the ‘biotech discovery of the century,’ there is ownership litigation. This dispute is getting rather ugly, if you will, according to an article in Science titled Accusations of errors and deception fly in CRISPR patent fight.

crispPotential financial gain aside, PubMed stats I found clearly substantiate the craze factor in numeric terms: >4,000 publications to date with a rapidly increasing trajectory, i.e. ~600 in 2014 and ~1,200 in 2015, which is an average of roughly 4 publications every day in that year!

By the way, in the chart above that I made for CRISPR publications in PubMed, there was only one report in 2002, which was the first publication to identify CRISPR. These Dutch investigators used computer analysis to find a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. They noted that “[t]his family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteristic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR).” 

Taken from youtube.com

Taken from youtube.com

CRISPR was also selected as 2015 Science Breakthrough of the Year, and is featured in an interesting YouTube video that is definitely worth watching, in my opinion.

Enough said for CRISPR editing of DNA, let’s move on to RNA editing with CRISPR that offers a fundamentally different editing approach: whereas DNA editing makes permanent changes to the genome of a cell, CRISPR-based RNA-targeting approach may allow researchers to make temporary changes. Moreover, this can be adjusted up or down, and may one day provide greater specificity and functionality than existing methods for RNA interference (RNAi) using either siRNA or antisense oligos.

CRISPR Targeting RNA

Feng Zhang. Taken from mit.news.edu

Feng Zhang. Taken from mit.news.edu

At the risk of seeming to be too trendy, this section heading could have read “Feng Zhang 2.0” in that Zhang at the Broad Institute, along with co-corresponding author Eugene V. Koonin at NIH and uber-famous “Broadster” Eric Langer plus others on a large team, have characterized a new CRISPR system that targets RNA—but not DNA. In their recent Science publication they demonstrated that this new system involves a Class 2 type VI-A CRISPR-Cas effector—aptly abbreviated C2c2 (pronounced “see too, see too”)—that has RNA-guided RNase function.

The researchers originally identified C2c2 in the bacterium Leptotrichia shahii (L. shahii) in a systematic search for previously unidentified CRISPR systems within diverse bacterial genomes. They focused on C2c2 because its sequence contained two copies of a domain called higher eukaryotes and prokaryotes nucleotide-binding (HEPN) that has only been found in RNases. Mutating the putative catalytic site within either of C2c2’s HEPN domains demonstrated that none of the mutated enzyme versions could cut RNA in vitro, suggesting that both HEPN domains are necessary for C2c2 to work.

CRISPR-C2c2 from L. shahii reconstituted in E. coli to mediate interference of the RNA phage MS2 via crRNA facilitated by the two HEPN nuclease domains. Taken from Abudayyeh et al.

CRISPR-C2c2 from L. shahii reconstituted in E. coli to mediate interference of the RNA phage MS2 via crRNA facilitated by the two HEPN nuclease domains. Taken from Abudayyeh et al.

But C2c2 Cleaves Collateral RNA—a Case for “Lemons into Lemonade”?

Unlike Cas9, which cuts DNA only within the sequence dictated by the CRISPR gRNA, C2c2 was found to make cuts within the target sequence and adjacent, nonspecific sequences. While this collateral cleavage obviously presents a specificity problem, Zhang and his colleagues were able to create a deactivated C2c2 (dC2c2) variant by alanine substitution of any of the four predicted HEPN domain catalytic residues. To me this clever trick is like converting “lemons into lemonade” in that undesired non-specific cleavage is transformed into a programmable RNA-binding protein having potential utility.

For example, the investigators speculate that the ability of dC2c2 to bind to specified sequences could be used in the following ways:

  • Bring effector modules to specific transcripts in order to modulate their function or translation, which could be used for large-scale screening, construction of synthetic regulatory circuits, and other purposes.
  • Fluorescently tag specific RNAs in order to visualize their trafficking and/or localization.
  • Alter RNA localization through domains with affinity for specific subcellular compartments.
  • Capture specific transcripts through direct pull-down of dC2c2 in order to enrich for proximal molecular partners including RNAs and proteins.

Listen to Zhang’s Grad Students 

While the details of this seminal work published in Science is not easily summarized, its practical implications have been concisely translated, if you will, by first coauthor Omar O. Abudayyeh, and second coauthor Jonathan S. Gootenberg—both graduate student members of the Zhang lab—in three short videos that I encourage you to watch at this link.

Left: Omar Abudayyeh. Taken from zlab.mit.edu Right: Jonathan Gootenberg Taken from zlab.mit.edu

Left: Omar Abudayyeh. Taken from zlab.mit.edu Right: Jonathan Gootenberg Taken from zlab.mit.edu

Publication protocol generally lists coauthors in order of contribution, so in this C2c2 publication that has many coauthors, these fellows know what they’re talking about because they did lots of the lab work. Congrats to them, Feng Zhang (again), and all of the other contributors.

As always, your comments are welcomed and encouraged.

RNA World Revisited

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

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

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

In Vitro Evolution of an RNA Polymerase

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

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

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

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

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

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

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

The TriLink “Connection”

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

2′-Azido-dUTP (aka 2′-azido-UTP)

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

Properties of RNA Polymerase 24-3

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

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

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

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

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

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

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

Exponential Amplification of RNA

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

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

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

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

Implications for the Ancient RNA World

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

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

Applications for Today’s World of Biotechnology

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

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

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

Death of DNA Dogma?

  • Current Genetic Dogma is DNA → RNA → Protein
  • Two Research Teams Independently Implicate Sperm Short RNA Can Transmit Paternal Genetics
  • More Research Needed to Elaborate the New Dogma

The Central Dogma of all life on Earth is currently understood to be DNA encoding RNA that in turn encodes protein. That genetic inheritance is transferred as DNA was first posited by uber-famous Francis Crick, who coined the term Central Dogma. While dogmatic principles, by definition, should have no exceptions, a few species of viruses can be considered to be exceptional cases in this regard.

The Central Dogma. Taken from biology.tutorvista.com

The Central Dogma. Taken from biology.tutorvista.com

That said, there is now quite a scientific buzz—if not shudder by some—over reports implicating RNA molecules as direct (i.e. non-DNA) agents for mammalian inheritance. My instantaneous mental responses to these surprising—if not shocking—revelations was first, “Wow, who would have thunk?” and then, “I’ve got to share this news in a blog.” So here it is.

Surprising Science in Sperm

Human sperm. Taken from leavingbio.net

Human sperm. Taken from leavingbio.net

While most of us are probably at least passingly familiar with textbook descriptions of the basic structure of sperm and its functional role in reproductive molecular biology, more detailed information on its nucleic acid content is less known. Consequently, shown below is a depiction of the basic structural components of a sperm, DNA content, and primary functions for doing its job, so to speak, in fertilization of an egg.

By way of background, here’s information that I thought was worth sharing. My Google Scholar search results for nucleic acid content of sperm included a very impressive technological accomplishment reported by uber-famous professor/entrepreneur Stephen Quake and co-workers in 2012 on microfluidic separation methods for the first ever genome-wide single-cell DNA sequencing of human sperm. Contrary to what one might intuitively expect, 91 genomes of sperm from a single individual were not identical. Since DNA from only one sperm and one egg combine during fertilization, the exact paternal DNA genotypes in the resultant offspring involves “pot luck,” so to speak.

Regarding RNA, my Google Scholar search led to a paper in 2011 by Krawetz et al. on the first ever report of deep-sequencing of short (18-30 bases) RNA (sRNA) in human sperm (for which TriLink offers a high-performance CleanTag™ kit for sRNA library prep as detailed on this poster). Krawetz et al. found microRNA (miRNA) (≈7%), piwi-interacting RNA (piRNA) (≈17%), and repeat-associated sRNA (≈65%). A minor subset of sRNA within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. However, reproductive roles for this molecular menagerie (what I tongue-in-cheek call these various sRNAs) remain speculative.

Fast forwarding to present time leads us to the two “wow” publications in venerable Science that triggered this blog:

While you’ll need to read these publications for details, they collectively raise the following controversial question vis-à-vis the Central Dogma for strictly DNA-based inheritance.

Are You Inheriting More Than Genes from Your Father?

Yes, is the surprising—if not bombshell—answer to this question, which I borrowed from Mitch Leslie’s Science editorial headline. If this conclusion is supported by further studies, it forces a fundamental revision of reproductive molecular cell biology. That’s a very big deal, so to speak, with ramifications not to be under appreciated.

Using sRNA library preparation methods analogous to TriLink CleanTag™ for Illumina deep-sequencing, the USA-Canadian team analyzed sperm from male mice fed a low-protein diet, progeny of which showed elevated activity of genes involved in cholesterol and lipid metabolism. They found that >80% of sRNA were fragments from several kinds of transfer RNAs (tRNAs). Most notably, 5′ fragments of tRNA-Gly-CCC, -TCC, and -GCC shown below all exhibited an approximately 2- to 3-fold increase in low-protein sperm.

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

To understand when, where, and how these tRNA fragments were formed, as well as unravel functional significance, the researchers describe an experimental tour de force—in my opinion. This included antisense modified-oligonucleotide “knock-out” of these tRNA fragments, as well as “knock-in” injection of <40-nt sRNA populations purified from control and low-protein sperm into control zygotes.

The researchers concluded that the sperm acquired most of these fragments while passing through the epididymis, a duct from the testicle where the cells mature. Functionally, they also link tRNA fragments to regulation of endogenous retro-elements active in the preimplantation embryo.

In the second study, the China-USA team also found tRNA fragments by deep-sequencing of sRNA. After feeding male mice either a high-fat or low-fat diet, the scientists injected the animals’ sperm into unfertilized eggs, and then measured metabolic performance of the offspring, which ate a normal diet. Progeny of fat-eating fathers remained lean; however, they showed two abnormalities often found in their dads and in humans who are obese or diabetic—abnormal absorption of glucose and insensitivity to insulin.

Like the first study, these researchers also did “knock-in” experiments wherein they inserted the tRNA fragments into eggs fertilized with other sperm. Fragments that came from fathers that ate the high-fat diet resulted in offspring that also showed impaired glucose absorption.

Take Home Messages

At the risk of over simplifying or over generalizing, the aforementioned two studies of sRNA in sperm provide compelling—and stunning—evidence for how tRNA fragments in sperm are responsible for inheritance independent of sperm DNA sequences. So much for dogma.

With regard to specifics, researchers now need to investigate how permanent these changes are, and how quickly they can be reversed by changing diet.

The flip-side of a bad diet adversely influencing offspring is to investigate if and how a good diet imparts better health to offspring.

Please share your thoughts about these reports, conclusion, and implications by commenting here.

Postscript

If you enjoy hip hop music—or just want to chuckle—this YouTube video for the Central Dogma song will get your head bobbing in sync with the music, lead you to smile, and give you a cool visual display of the central dogma.

Curiously Circular RNA

  • Circular RNA (circRNA) Formation Serendipitously Discovered in 1991  
  • Next-Generation Sequencing Reveals circRNA to be Ubiquitous
  • circRNA can Function as MicroRNA ‘Sponges’ to Regulate Gene Expression

There’s something seductively simple—and curious—about circles, which are unique in having no beginning or end, unlike most other things. On a less philosophical plane, thinking about circles conjures up incongruent memories of delicious doughnuts and geometric definitions from my youthful days going to the neighborhood bakery and diligently taking notes in my high school geometry class, respectively.

donuts

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Norovirus: Science Behind the Headline

  • The Virus is Quite Common with 267 Million Cases and 200,000 Deaths Annually
  • RT-PCR is the Detection Method of Choice
  • First Cell-Culture System May Speed Drug and Vaccine Development

We’ve all seen TV news stories about disgruntled passengers disembarking cruise ships returning to port early because of an outbreak of nasty gastroenteritis (i.e. inflammation of the stomach and intestines leading to nausea, vomiting, diarrhea, and stomach cramps). Norovirus (NoV) is the causative agent of these frequently reoccurring “nightmare” cruises, of which 13 have been reported since 2012, sickening some 200-600 passengers. It’s not just limited to cruises, the virus affected 100+ students at a school in Eugene, Oregon last year. And now there’s new evidence for transmission of NoV by eating oysters—which I will therefore not eat in the future.

Taken from counselheal.com.

Taken from counselheal.com.

But perhaps the most NoV-related media attention—and investor ire or litigant action—has been recently focused on gastroenteritis outbreaks at Chipotle—a popular restaurant chain. A criminal investigation is under way at Chipotle, and according to an Associated Press report the company has been served with a federal subpoena.

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