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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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.”

The Most Interesting Scientist in the World: George M. Church

  • Mind Boggling Breadth and Significance of Scientific Publications
  • Serial Entrepreneur and Science Advisor to Many Companies
  • Radical Advocate of Total Openness for Personal Genomics

While seeing for the umpteenth time a Dos Equis beer commercial featuring The Most Interesting Man in the World, I was suddenly inspired to write a blog about The Most Interesting Scientist in the World. After scrolling and polling my memory to decide who that would be, it was an easy decision to pick George M. Church, professor of genetics at Harvard. As I’ll briefly highlight herein, Prof. Church’s contributions continually span a mind boggling spectrum of science that cuts across academic theory, ground breaking “how to” methods, serial entrepreneurship, and—perhaps most importantly—radical openness for personal genomics.

George M. Church and The Most Interesting Man in the World: ‘I don’t always read science, but when I do it’s by George M. Church.’ (taken from Bing Images)

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We’re Celebrating Click Chemistry In Honor of National DNA Day

  • The Verbification of Click Chemistry 
  • Old Chemistry Morphs into New Applications for DNA and RNA  
  • Amazingly, Phosphorus in DNA and RNA is not Needed for Function 

This post comes only two days after National DNA Day 2015 on April 25th so it’s apropos to feature DNA, but I’d also like to give a nod to the lesser recognized RNA, without which DNA would be akin to music notes in search of a melody.  If you’re a regular reader of this blog, you know my stance on this subject and so I digress…

So-called “Click Chemistry” is trending so “hot” that it has led to a phenomenon known as verbification, which is when a noun becomes a verb by virtue of popularity and linguistic convenience. So, just as Google has become to google for virtually everyone, Click has become to click for synthetic chemists and biotechnologists. Whether or not you’re already familiar with Clicking, I hope to provide herein some interesting snippets about Click, its growing ubiquity, and how it has enabled synthesis of a completely novel, non-phosphorous linkage in DNA that nevertheless functions flawlessly in vivo—a stunning feat never before achieved that has intriguing implications about life. More on that later, but first some snippets about Click.

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DNA’s Forgotten Discoverer: Swiss Scientist Friedrich Miescher 

  • Discovered in 1869 in Pus Cells from Bandages of Crimean War Soldiers
  • Miescher Named this New Matter Nuclein and Intuited that it Played a Fundamental Role in Heredity
  • This put the “N” in DNA—Deoxynucleic Acid
  • Children now Isolate DNA from Fruits & Vegetables in Elementary School 

Truth be told, what led me to writing this post was suddenly realizing one day that, although the vast majority of my professional career involves nucleic acids—and DNA in particular—I did not know anything about the discovery of DNA or its naming. My follow-on thoughts were that this was somewhat embarrassing for a blogger focused on nucleic acids, and should be remedied by some homework! This is also good timing since my mind is currently aflutter with all things DNA in anticipation of National DNA Day coming up on April 25. In the event that you recall my past commentary about the bias toward DNA, yes I am still supporting a National RNA Day to balance the ranks, but I digress…

Friedrich Miescher as young man (taken from cienciaytecnicasuabia.blogspot.com via Bing Images)

Friedrich Miescher as young man (via Bing Images)

In doing my so-called homework, I learned about Swiss scientist Friedrich Miescher’s life story and circumstances surrounding his discovery in the late 1860s of new matter that he named nuclein, which eventually became incorporated into the term nucleic acid. Those circumstances, including Miescher’s unusual source of nuclein, were quite interesting to me so I thought they’d be worth sharing in this post, which draws upon a lengthy article by Ralf Dahm, who has written extensively about Miescher, and has a website worth visiting.

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