Highlights from the 2018 International Roundtable (IRT) on Nucleosides, Nucleotides, and Nucleic Acids

• Over 400 Attendees from Around the World Congregate at UCSD
• Four Days Full of Topics Spanning Basic Chemistry Through Therapeutics
• Jerry Comments on His Five Favorites

The 23rd (XXIII) International Roundtable (IRT) on Nucleosides, Nucleotides, and Nucleic Acids was held at the University of California, San Diego (UCSD) in La Jolla, California on August 26 – 30, 2018. This 23rd biannual event, which was sponsored by the International Society of Nucleosides, Nucleotides, and Nucleic Acids (IS3NA) was attended by over 400 researchers in all levels of academia and industry from around the world. Award lectures (2), invited lectures (17), oral presentations (26), and posters (198) spanned a plethora of cutting edge scientific topics, ranging from the origins of life to the development of novel therapeutics. Local organizers were Prof. Yitzhak Tor, Chair (UCSD), Dr. Yogesh Sanghvi (Rasayan, Inc.) and Dr. Rick Hogrefe (TriLink BioTechnologies).

I thoroughly enjoyed attending this 2018 IRT, where I had the opportunity to completely immerse myself in diverse aspects of chemistry, biochemistry, molecular biology, medicinal chemistry, and drug development—all related to nucleosides, nucleotides, and nucleic acids. The meeting gave me the opportunity—and challenge—of selecting five noteworthy presentations, shared here in random rather than rank order. There are several other presentors with fantastic work that I apologize for not being able to discuss at this time. For example, Dr. Alexandre Lebedev at TriLink Biotechnologies, gave an excellent oral presentation titled: Efficient initiation of in vitro mRNA transcription with Cap 0, Cap 1 and Cap 2 oligonucleotide primers (CleanCap®). The presentation highlighted TriLink groundbreaking CleanCap Technology, a chemical solution that provides high mRNA capping efficiency, and avoids self/non-self intracellular responses. If you would like a copy of this presentation, please contact TriLink here.

CRISPR Cloaking

An ongoing “hot topic” is sequence-specific cutting of DNA with CRISPR-Cas9 for gene editing as a research tool or therapeutic modality. I have previously blogged about this here. Given the widespread interest on the subject, it’s apropos to start with a poster titled Reversible RNA acylation for CRISPR-Cas9 gene editing control in cells presented by Maryam Habibian, a postdoc in Eric Kool’s group at Stanford University.

As shown here, Kool’s lab has recently published on the reaction of RNA in aqueous buffer with an azide-substituted acylating agent, which yields several 2'-OH acylations per RNA strand in as little as 10 minutes. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine results in spontaneous loss of acyl groups ("uncloaking") that fully restores RNA folding and biochemical activity.

Data in this poster showed that an azide-substituted reagent efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. These cloaked sgRNAs completely inhibit the endonuclease activity of Cas9 in vitro and in living HeLa cells. However, the sgRNA activity is efficiently recovered both in vitro and in cells by treatment with water-soluble phosphines. This study highlights the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function.

Chemically Modified DNAzymes

Prof. David Perrin at the University of British Columbia in Canada gave an oral presentation titled Chemically modified DNAzymes as sequence-specific ribonuclease-A mimics—from potential therapeutics to the origin of life. He noted that the use of a synthetic RNAzyme or DNAzyme to cut a particular sequence target mRNA for use as a possible therapeutic agent has been a concept for ~40 years, but has not yet come to realization. The principal challenge, he added, is to find a suitably structured nucleic acid that catalyzes efficient phosphodiester bond cleavage in RNA in the absence of Mg⁺² or at the relatively low Mg⁺² concentrations in cells.

As shown here, this presentation described the in vitro selection of novel RNA cleaving DNAzymes that are selected using 8-histaminyl-deoxyadenosine (imidazole-A), 5-guanidinoallyl-deoxyuridine (guanodino-U), and 5-aminoallyl-deoxycytidine (amino-C), along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A.

Remarkably, these exceptional catalysts display classic enzymatic properties of Michaelis-Menten kinetics in the absence of Mg⁺². Interested readers can access complete details on this exciting work here. Perrin added that, in honor of the late Stanley Miller (UCSD), whose pioneering work on the origin of life included the possibility of a highly-decorated RNA world, this work represents a chemist’s approach to biomimicry for testing hypotheses of the origin of life in an RNA-world that must of co-opted synthetic modifications, and underscores the use of modified dNTPs for the selection of modified aptamers. You can read more about aptamers in several of my previous blogs.

Direct Sequencing of N⁶-Methyladenosine in RNA

Enzyme-mediated post-transcriptional RNA modifications are dynamic, and may have functions beyond fine-tuning the structure and function of RNA. Understanding these epitranscriptomic RNA modification pathways and their functions may allow researchers to identify new layers of gene regulation at the RNA level, according to a “grand challenge” discussed in a previous blog. N⁶-Methyladenosine (m6A), shown here, is the most abundant modification in eukaryotic mRNA and long noncoding RNA (lncRNA). It is found at 3-5 sites on average in mammalian mRNA, and up to 15 sites in some viral RNA.

In addition to this relatively low density, specific loci in a given mRNA are a mixture of unmodified- and methylated-A residues, thus making it very difficult to detect, locate, and quantify m6A patterns. Importantly, there is now an elegant solution to this problem. In an invited lecture by Prof. Andreas Marx at the University of Konstanz in Germany titled Elucidating the information layer beyond the genome sequence, an engineered polymerase was said to differentiate between unmodified- and methylated-A residues.

This novel method, which was recently published, involves in vitro evolution and screening to evolve a reverse-transcription (RT)-active KlenTaq DNA polymerase mutant (RT‐KTQ G668Y Y671A) that delivers prominent RT signatures at m6A sites in different sequence contexts. As shown here, this novel polymerase exhibits increased misincorporation opposite m6A compared to unmodified A. Application of this DNA polymerase in next-generation sequencing allowed for identification of m6 A sites directly from the sequencing data of untreated RNA samples.

Phosphorothioate-Modified Oligo Therapeutics

Pioneering investigations of phosphorthioate (PS)-modified nucleic acids by Prof. Fritz Eckstein, followed by fully automated synthesis of PS-modified oligodeoxynucleotides by Prof. Wojciech Stec and yours truly, enabled many other researchers to develop PS-ODNs as therapeutic agents. Although I have previously blogged about this topic, the utility and prevalence of PS-modifications in ODN-based therapeutics was a common theme throughout many presentations at IRT 2018.

Most prominently, in my opinion, Dr. Punit Seth at Ionis Pharmaceuticals in Carlsbad, California gave an invited lecture titled Engineering selectivity into therapeutic oligonucleotides through chemical design. The talk largely dealt with PS-ODNs and included a slide with the following summary:

• PS-ODNs interact with several plasma proteins with a range of binding affinities

  • PS content and single-stranded nature are important for binding
  • Binding can be rationalized by an avidity model wherein each PS contributes a fraction to overall binding

• Interaction with plasma proteins can have functional consequences

  • Binding to α-2-macroglobulin can reduce uptake pathways
  • Strong binding to HRG can reduce activity
  • Lipid conjugation enhances potency in muscle through interactions with albumin and lipoproteins

• PS-ODNs interact with cell-surface proteins such as Stabilin scavenger receptors

  • Stabilins clear anionic polymers of the extra-cellular matrix suggesting a common pharmacophore with anionic PS-ODNs

The influence of antisense PS-ODN Sp and Rp stereochemistry on such pharmacological factors, including RNase-H activity, has been reported by Wave Life Sciences using stereoselective synthesis methodology introduced by Wada (see also Baran). Extending this approach, Troels Koch at the Roche Innovation Center in Copenhagen gave an invited lecture titled Stereodefined LNA Phosphorothioates: Design, synthesis and properties. In particular, he described investigations of 2'-O, 4'-C methylene bridged moieties commonly referred to as “locked nucleic acids” (LNAs), shown here:

Koch stated that LNAs have, over the last 15 years, been intensively used in RNA therapeutics because LNAs offer high affinity that translates into higher potency for RNA targeting. He added that nearly all of these LNA oligonucleotides have PS linkages. His presentation illustrated the diversity of measurable properties of stereodefined PS-LNAs. Importantly, it was shown that identifying the best diastereomers from a large random mixture is not trivial. Several identification tactics were described, including the use of quantum mechanical modelling as a guide towards finding the best use of stereodefined LNA.

In striking contrast to the aforementioned focus on inclusion and improvement of PS linkages in therapeutic oligonucleotides, Prof. Jesper Wengel at the University of Southern Denmark in Odense gave an oral presentation titled Novel DNA-mimicking monomers for gapmer antisense oligonucleotides, wherein the objective is to increase gene knock-down specificity by complete removal or substantial reduction of PS linkages and other strategies. His current design is to use phosphodiester (PO)-linked “3-10-3” LNA-DNA-LNA gapmers with palmitic acid-derived moieties attached, as shown below, and bridging N or O in the LNA residues.

Discovery of a Nucleotide Analog Drug for Ebola Virus

In my blog on the 2014 outbreak of the deadly Ebola virus, I indicated the need for more resources allocated towards the development of a prophylactic vaccine. While such work continues, I was very pleased to learn of promising results obtained for a new drug against Ebola, which would provide treatment for individuals already infected with the virus.

Dr. William Lee at Gilead Sciences, Inc. in Foster City, California, reported in an invited lecture titled Remdesivir (GS-5734): An Antiviral Nucleotide Analog for the Treatment of Ebola Virus that this nucleotide prodrug (shown here) of a novel nucleoside analog has shown broad spectrum in vitro activity against filoviruses, corona viruses, paramyxoviruses, and flaviviruses. Importantly, Remdesivir has demonstrated potent in vivo efficacy against multiple strains of the Ebola virus in the rhesus monkey infection model. His talk reviewed the data in rhesus, the manufacturing challenges, and the limited exposure in patients exposed to the Ebola viruses.

Concluding Comments

In my opinion, IRT 2018 was indeed jam-packed with innovative and interesting presentations by a diverse array of researchers from around the world, all united by the common thread of nucleosides, nucleotides, and nucleic acids. In addition to the science, there was ample opportunity to renew friendships and, more importantly, network and exchange contact information with new people for potential collaborations on mutually interesting projects.

Every IRT meeting includes an announcement (shown here) of the next venue, which for the 24th (XXIV) IRT in 2020 will be Stockholm, Sweden, locally organized by Prof. Roger Stromberg and held at the Karolinska Institutet.

I hope to see you there!

As usual, your comments are welcomed.

Footnote

While walking through the UCSD campus to the “kick off” Keynote Lecture by Prof. Gerald Joyce (Salk Institute) titled RNA-Targeted drug discovery, to be followed by an all-attendees reception sponsored by TriLink, I came across and photographed the large, brightly colored statue shown here.

My curiosity about this eye-catching, fanciful figure led me to learn that it is called Sun God, and is an art object created by Niki de Saint Phalle (1930-2002), who is best known for her oversized figures that embrace contradictory qualities such as good and evil. She lived in New York in the 1960s when she was prominent in the development of "happenings" and other artistic efforts involving the integration of art and life. She lived and worked in La Jolla from 1992 until her death in 2002.

De Saint Phalle's Sun God was the first work commissioned by the Stuart Collection of UCSD and was her first outdoor commission in America. The exuberantly colored, fourteen-foot bird is placed atop a fifteen-foot concrete arch and sits on a grassy area between near the Faculty Club. The students started the Sun God Festival in 1984. It has become one of the largest annual campus events.

The Sun God has become a landmark on the UCSD campus. Students have embellished the statue at various times with giant sunglasses, a cap and gown, a UCSD ID card, and a nest of hay with eggs. Sun God has also been adorned with earphones and a radio/tapeplayer, turning the statue into a "Sony Walkbird," and has sported a machete and headband for its disguise as "Rambird." It appears on T-shirts and mugs. The grassy area beneath it is a popular site for rendezvous and celebrations.

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