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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Highlights of 2015 Publications Using TriLink BioTechnologies Products

  • Publications Citing TriLink Products Exceed 6,000
  • TriLink Products Showed up at a Rate of One Publication per Work Day
  • Among These Customer Publications, Modified mRNA is Trending
Taken from thetrymovement.com

Taken from thetrymovement.com

From my college classes decades ago, I can still clearly recall—thankfully—many “ah ha” moments. Most importantly is when I crystalized to purity and then confirmed structure by NMR the first compound I synthesized in Organic Chemistry Lab. Another ah ha moment—but on a completely different level—was during a philosophy class when the professor partially paraphrased a quote by Aristotle as “we are what we do.” The full quote given above is even more thought provoking because it ties in the notion of excellence, which I took to heart then, and have attempted to live by ever since.

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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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60th Anniversary of the Discovery of DNA’s Double Helix Structure…Diamond Jubilee for the “Monarch of Molecules”

Welcome to my inaugural blog post!  My intention is to provide timely nucleic acid-related scientific content that is informative and, hopefully, will be of interest to a broad readership. New posts will be published biweekly so please check back often.  I encourage thoughtful commentary as well as constructive suggestions.  To find out more about me and my relationship with TriLink Biotechnologies, please visit the ‘About Jerry’ tab at the top of the page.

Considering TriLink’s focus on providing nucleic acid-based products, it seemed appropriate that this inaugural post feature the upcoming 60th anniversary of Watson & Crick’s proposed structure for DNA published in Nature on April 25, 1953. It is widely acknowledged that insights provided therein had a fundamentally transformative impact on science and society. Anyone working with DNA should take a bit of time to read this historically important publication that is freely available at Nature.com. Doing so reveals an extremely brief report—one page and one figure—with perhaps the most understated—and similarly brief, one sentence!—conclusion suggesting the structural basis for genetic encoding by DNA sequence and its replication:

watson-crick“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”

Those interested in an annotated and illustrated autobiographical account of this making of a scientific revolution are referred to a recent book by James D. Watson, Alexander Gann and Jan Witkowski, The Annotated and Illustrated Double Helix that has already received numerous excellent reviews.  There is also a YouTube video of a delightful presentation by Dr. Watson that is well worth watching to see and hear the story of events leading to the 1953 publication and subsequent recognition that DNA is transcribed into mRNA for translation into protein. This new knowledge coupled with the availability of various molecular biological “tools,” and initial advances in oligonucleotide synthesis, set the stage for an exciting race during the late 1970s to synthesize for the first time a medically important human gene—insulin—involving three main groups—Harvard University, University of California at San Francisco, and City of Hope National Medical Center. This fascinating story involving science, egos, and the beginning of bio-venture “start-up” financing that collectively spawned Genentech and Biogen—and subsequent “genetic engineering” companies—is engagingly chronicled by Stephen S. Hal in the book Invisible Frontier-The Race to Synthesize a Human Genome through interviews with numerous individuals who were directly involved.

Through many advances in DNA Sanger sequencing methods and automated instrumentation during 1990-2003, base-by-base sequence determination of the entire human genome was realized by parallel public (government funded) and private (Celera Corporation) efforts.1 During the next decade, taking us to 2013, there has been stunning progress in developing various methods of massively parallel sequencing2 (aka “next-generation sequencing”) that, in part, has enabled further elucidation of epigenomics and RNA-mediated regulation as well as factor-mediated reprogramming of cells and pursuit of regenerative medicine. Such topics were discussed at a recently held Cold Spring Harbor Laboratory meeting entitled “From Base Pair to Body Plan – Celebrating 60 Years of DNA.” Below are just a few selected author names (in alphabetical order) and titles of presentations taken from the list of nearly 50 talks or posters found at the website for this event.3

Baylin, S. Celebrating the discoveries of the “hard drive” of DNA and its “software package,” the epigenome—Basic and translational implications
Young, R.A. Control of gene expression programs
Zaret, K.S. Programming and reprogramming cell fate

In closing this inaugural blog post, one can only wonder what new and exciting aspects of life sciences, diagnostics and medicine—including regenerative—will be realized during the next 10 years on the way to the 70th Anniversary of the Discovery of DNA’s Double Helix Structure. My view of future advances based on DNA fall into three interrelated areas:

  • “faster, better, cheaper” synthesis to create useful DNA-directed, self-assembling  “smart materials”
  • “bigger and deeper” experimentation aimed at complete molecular-level models for organisms and memory
  • “bio-factories” for Green Manufacturing

These thoughts about the next decade for DNA will be elaborated in my future posts.  What do you see happening during this time?

 

References

1. http://en.wikipedia.org/wiki/Human_Genom_Project

2. http://en.wikipedia.org/wiki/Massive_parallel_sequencing

3. http://meetings.cshl.edu/abstracts/dna602013_absstat.html