In Search of RNA Epigenetics: A Grand Challenge

  • Methylated riboA and riboC are the most commonly detected nucleobases in epigenetics research
  • Powerful new analytical methods are key tools for progress
  • Promising PacBio sequencing and novel “Pan Probes” reported   

In a Grand Challenge Commentary published in Nature Chemical Biology in 2010, Prof. Chuan He at the University of Chicago opined that “[p]ost-transcriptional RNA modifications can be dynamic and might have functions beyond fine-tuning the structure and function of RNA. Understanding these RNA modification pathways and their functions may allow researchers to identify new layers of gene regulation at the RNA level.”

Like other scientists who get hooked by certain Grand Challenges, I became fascinated by this possibility of yet “new layers” of genetic regulation involving RNA, either as conventional messenger RNA (mRNA) or more recently recognized long noncoding RNA (lncRNA). Part of my intellectual stimulation was related to the fact that some of my past postings have dealt with both lncRNA as well as recent advances in DNA epigenetics, so the notion of RNA epigenetics seemed to tie these together.

After doing my homework on recent publications related to possible RNA epigenetics, it became apparent that this posting could be logically divided into commentary on the following three major questions: what are prevalent epigenetic RNA modifications, what might these do, and where is the field going? Future directions were addressed by interviews with two leading investigators: Prof. Chuan He, who is mentioned above, and Prof. Tao, who has been involved in cutting edge methods development.

RNA Epigenetic Modifications

More than 100 types of RNA modifications are found throughout virtually all forms of life. These are most prevalent in ribosomal RNA (rRNA) and transfer RNA (tRNA), and are associated with fine tuning the structure and function of rRNA and tRNA. Comments here will instead focus on mRNA and lncRNA in mammals, wherein the most abundant—and far less understood—modifications are N6-methyladenosine (m6A) and 5-methylcytidine (m5C).

structures

Three Approaches to Sequencing m6A-Modified RNA

Discovered in cancer cells in the 1970s, m6A is the most abundant modification in eukaryotic mRNA and 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 were a mixture of unmodified- and methylated-A residues, thus making it very difficult to detect, locate, and quantify m6A patterns. Fortunately, that has changed dramatically with the advent of various high-throughput “deep sequencing” technologies, as well as other advances.

(1.) Antibody-based m6A-seq 

An impressive breakthrough publication in Nature in 2012 by a group of investigators in Israel reported novel methodology called m6A-seq for determining the positions of m6A at a transcriptome-wide level. This approach, which is a variant of methylated DNA immunoprecipitation (MeDIP or mDIP), combines the high specificity of an anti-m6A antibody with Illumina’s massively parallel sequencing of randomly fragmented transcripts following immunoprecipitation. These researchers summarize their salient findings as follows.

“We identify over 12,000 m6A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks—around stop codons and within long internal exons—and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m6A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signaling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m6A has a fundamental role in regulation of gene expression.”

Moreover, their concluding sentence refers back to He’s aforementioned Grand Challenge Commentary about RNA epigenetics in 2010, just two years earlier.

“The m6A methylome opens new avenues for correlating the methylation layer with other processing levels. In many ways, this approach is a forerunner, providing a reference and paving the way for the uncovering of other RNA modifications, which together constitute a new realm of biological regulation, recently termed RNA epigenetics.”

(2.) Promising PacBio Single-Molecule Real-Time (SMRT) Sequencing of m6A

In a previous post, I praised PacBio (Pacific Biosciences) for persevering in development of its SMRT sequencing technology that uniquely enables, among other things, direct sequencing of various types of modified DNA bases via differentiating the kinetics of incorporating labeled nucleotides. Attempts to extend the SMRT approach to sequencing m6A have been recently reported by PacBio in collaboration with Prof. Pan (see below) and others in J. Nanobiotechnology in April 2013. Using model synthetic RNA templates and HIV reverse transcriptase (HIV-RT) they demonstrated adequate discrimination of m6A from A, however, “real’ RNA samples having complex ensembles of tertiary structures proved to be problematic. Alternative engineered RTs that are more processive and accommodative of labeled nucleotides were said to be under investigation in order to provide longer read lengths and appropriate incorporation kinetics.

The authors are optimistic in being able to solve these technical problems, and concluded their report by stating:

  “[w]e anticipate that the application of our method may enable the identification of the location of many modified bases in mRNA and provide detailed information about the nature and the dynamic RNA refolding in retroviral/retro-transposon reverse transcription and in 3’-5’ exosome degradation of mRNA.”

Let’s hope that this is achieved soon!

(3.) Nanopore Sequencing of m6A?

It’s too early to be sure, but continued incremental advances in possible approaches to nanopore sequencing suggest applicability to m6A. As pictured below, Bayley and coworkers describe a method that uses ionic current measurement to resolve ribonucleoside monophosphates or diphosphates (rNDPs) in α-hemolysin protein nanopores containing amino-cyclodextrin adapters.

Taken from Bayley and coworkers in Nano Lett. (2013)

Taken from Bayley and coworkers in Nano Lett. (2013)

The accuracy of base identification is further investigated through the use of a guanidino-modified adapter. On the basis of these findings, an exosequencing approach for single-stranded RNA (ssRNA) is envisioned in which a processive exoribonuclease (polynucleotide phosphorylase, PNPase) presents sequentially cleaved rNDPs to a nanopore. Although extension of this concept to include m6A has yet to be demonstrated, earlier feasibility studies by Ayub & Bayley have shown discrimination of m6A (and other modified bases) from unmodified ribobases.

Two Probe-Based Methods for Detecting Specific m6A Sites

1.) “Pan Probes”

As the saying goes, “what goes around comes around”, and in this instance its repurposing 2’-O-methyl (2’OMe) modified RNA/DNA/RNA oligos. This general class of chemically synthesized chimeric “gapmers” was originally used for RNase H-mediated cleavage of mRNA in antisense studies. Very recently, however, Pan and coworkers have cleverly adapted these probes—which I like to alliteratively refer to as “Pan Probes”—to m6A detection in mRNA and lncRNA.

For details see SCARLET workflow; taken from Pan and coworkers RNA (2013)

Pan Probes are comprised of “7-4-7 gapmers” having seven 2’OMe RNA nucleotides flanking four DNA nucleotides, the latter of which straddle known (or suspected) m6A sites, as depicted in the cartoon shown. The indicated series of steps, which involve site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography, is thankfully called SCARLET by these investigators.

SCARLET was used by Pan and coworkers to determine the m6A status at several sites in two human lncRNAs and three human mRNAs, and found that the m6A fraction varied between 6% and 80% among these sites. However, they also found that many m6A candidate sites in these RNAs were not modified. Obviously, while much more work needs to be done to collect data for deciphering dynamic patterns and implications of m6A RNA epigenetic modifications, these investigators note that SCARLET is, in principle, applicable to m5C, pseudouridine, and other types of epigenetic RNA modifications.

Readers interested in designing and investigating their own Pan Probes can obtain these 7-4-7 gapmers by using TriLink’s OligoBuilder® and simply selecting “PO 2’OMe RNA” from the Primary Backbone dropdown menu, typing the first 7 bases in the Sequence box, selecting the 4 DNA bases from the Chimeric Bases menu and then typing the remaining 7 2’OMe RNA bases.

(2.) Probes for High-Resolution Melting

In a new approach very recently reported by Golovina et al. at Lomonosov Moscow State University, the presence of m6A in a specific position of mRNA or lncRNA molecule is detected using a variant of high-resolution melting (HRM) analysis applicable to, for example, single-nucleotide genotyping. The authors suggest that this method lends itself to screening many samples in a high-throughput assay following initial identification of loci by sequencing (see above). The method uses two labeled probes—one with 5’-FAM and another with 3’-BHQ1 (both available from Trilink’s OligoBuilder®)—that hybridize to a particular query position in a total RNA sample, as shown below for a 23S rRNA model system. The presence of m6A lowers the melting temperature (Tm), relative to A, with a magnitude that is sequence-context dependent.

Taken from Golovina et al. Nucleic Acids Res. (2013).

Taken from Golovina et al. Nucleic Acids Res. (2013).

The authors studied various probe-target constructs, and recommend 12–13-nt-long probes containing a quencher, and >20-nt long probes containing a fluorophore.  They also could advise that the quencher-containing oligonucleotide hybridizes to RNA such that m6A be directly opposite the 3′-terminal nucleotide carrying the quencher. The authors point out that relatively low-abundant, non-ribosomal targets need partial enrichment by, for example, simple molecular weight-based purification or commercially available kits. In this regard, they estimate that, if a particular type of mRNA was present at 10,000 copies per mammalian cell, 107 cells would be required to analyze m6A by this HRM method.

m5C Analysis by Sequencing of Bisulfite-Converted RNA

Selective reaction of bisulfite with C but not m5C in RNA, analogous to that long used for DNA, provides the basis for determining C-methylation status by sequencing. As detailed by Squires et al. in Nucleic Acids Res. in 2013, bisulfite-converted RNA can be sequenced by either of two methods: conversion to cDNA, cloning, and conventional sequencing, or conversion to a next-generation sequencing library. These authors described their salient findings as follows.

“We confirmed 21 of the 28 previously known m5C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions… Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.”

“Writing, Reading, and Erasing” RNA Epigenetic Modifications

Enzyme-mediated post-transcriptional RNA methylation (aka “writing”) and demethylation (aka “erasing”) are critical processes to identify and fully characterize in order to elucidate RNA epigenetics, and are formally analogous to those operative for DNA epigenetics.

RNA epigenetic “writing” mechanisms have focused on N6-adenosine-methyltransferase 70 kDa subunit, an enzyme that in humans is encoded by the METTL3 gene, and is involved in the posttranscriptional methylation of internal adenosine residues in eukaryotic mRNAs to form m6A. According to Squires et al., two m5C methyltransferases in humans, NSUN2 and TRDMT1, are known to modify specific tRNAs and have roles in the control of cell growth and differentiation.

As for “erasing”, in 2011, He’s lab discovered the first RNA demethylase, abbreviated FTO, for fat mass and obesity-associated protein, which has efficient oxidative demethylation activity targeting m6A in RNA in vitro. They also showed for the first time that this erasure of m6A could significantly affect gene expression regulation. In 2013, He’s lab discovered the second mammalian demethylase for m6A, ALKBH5, which affects mRNA export and RNA metabolism, as well as the assembly of mRNA processing factors, suggesting that reversible m6A modification has fundamental and broad functions in mammalian cells.

So, if Mother Nature evolved these mechanisms for writing and erasing RNA epigenetic modifications, what about the equally important, in between process of “reading” them? He and Pan and collaborators have very recently reported insights to such reading. They showed that m6A is selectively recognized by the human YTH domain family 2 (YTHDF2) “reader” protein to regulate mRNA degradation. They identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but also include non-coding RNAs, with a conserved core motif of G(m6A)C. They further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites. The carboxy-terminal domain of YTHDF2 selectively binds to m6A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2–mRNA complex to cellular RNA decay sites. These findings, they say, indicate that the dynamic m6A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

Expert Opinions of the Future for RNA Epigenetics

As I’ve said here before, there is no crystal ball for accurately predicting the future in science, although scientists do enjoy imagining that there is. Opinions of two “hands on” experts in the emerging field of RNA epigenetics are certainly of interest in this regard. Below are some comments offered by the aforementioned Prof. Tao Pan and Prof. Chuan He provided via an email interview in which I posed the question, ‘What do you see as the most important developments for RNA epigenetics?’ These experts have  thrown down the gauntlet, so to speak, by asserting RNA epigenetics as a Grand Challenge.

Prof. Tao Pan

Prof. Tao Pan

“In my opinion, the biggest current challenge for the field is to develop methods that can perturb m6A modification at specific sites to assess m6A function directly in specific genes. RNA interference or overexpression of an mRNA may simply decrease or increase modified and unmodified RNA alike. In a few cases, mutation of a known m6A site in an mRNA resulted in additional modification at a nearby consensus site, so that one cannot simply assume that mutation of a known site would not lead to cryptic sites nearby that may perform the same function. Further, functional understanding of a specific site should also take into account that all currently known m6A sites in mRNA and viral RNA are incompletely modified, so that one may need to explain why cells simultaneously maintain two RNA species that differ only at the site of m6A modification.”   

Prof. Chuan He

Prof. Chuan He

The m6A modification is much more abundant than other RNA modifications in mammalian and plant nuclear RNA and is currently the only known reversible RNA modification. The m6A maps of various organisms/cell types need to be obtained. High-resolution methods to obtain transcriptome-wide, base-resolution maps are important. A future focus should be to connect the reversible m6A methylation with functions, in particular, the studies of the reader proteins that specifically recognize m6A and exert biological regulation. The first example of the YTHDF2 work just published in Nature (above) is a good example. We believe many other reader proteins exist and impact almost all aspects of mRNA metabolisms or functions of lncRNA. 

Besides m6A, there are m5C, pseudoU, 2′-OMe, and potentially other modifications in mRNA and various non-coding RNAs (such as the recently discovered hm6A and f6A). The methods to map these modifications (except m5C) need to be developed and their biological functions need to be elucidated. 

Lastly, potential reversal of rRNA and tRNA modifications needs to be studied. As I stated in the Commentary in 2010, dynamic RNA modifications could impact gene expression regulation resembling well-known dynamic DNA and histone modifications. I think now we have enough convincing data to indicate this is indeed the case. The future is bright.”

Very bright, indeed! Your comments about this posting are welcomed.

No Junk DNA…It’s All Good!

A Short Walk in the Wondrous, Wacky World of Long—and now Circular—Noncoding RNAs

I’m pleased by how much I learn when researching topics for new content, and this was certainly the case for long noncoding RNA (lncRNA), which was briefly mentioned in my last blog post, “Ripples from the 2013 TIDES Conference.”  The topic piqued my interest so I set out to find out more. Plowing through lncRNA (aka lincRNA = large intergenic non-coding RNA) literature I quickly realized that there was an enormous amount of information, and the big challenge would be to capture some intriguing aspects without getting bogged down in “technical weeds” or being overly simplistic. In what follows there is a super brief introduction to what lncRNAs are and what they do—the latter is controversial—along with an appreciation for why lncRNAs are indeed a structurally and functionally wondrous class of nucleic acids that now encompass circular molecules. Maybe—to borrow from Forrest Gump—lncRNAs are like “a box of chocolates” for molecular biologists.

Taken from Geospiza via Bing Images.

Taken from Geospiza via Bing Images.

Introduction of lncRNA

The basic definition of lncRNA as “non-protein coding transcripts longer than 200 nucleotides” distinguishes this class of RNAs from various types of shorter RNAs, such as microRNA (miRNA) and well-known, Nobel Prize-related short interfering RNA (siRNA). But that’s where the simplicity ends and things get complicated. You might be thinking…hold on…why should I want to know more about lncRNAs? My reply to that is to consider the following points, each of which struck me as truly stunning.

  • Across the human genome there are four-times more lncRNAs than traditional coding RNAs. This large fraction of transcribed genome that extends beyond the boundaries of known genes is referred to as “pervasive transcription”—pervasive is defined in dictionaries as existing in or spreading through every part of something; why would cells do this?
  • Whereas it was long held that only ~1% of human DNA is transcribed and therefore ~99% of the genome is “junk” DNA, new analytical methods have revealed quite the opposite: ~94% of the genome is transcribed, giving rise to a plethora of long (and short) ncRNAs.
  • More than 1,500 lncRNAs are transcribed from two or more adjacent genes, and in some instances span chromosomal loci separated by more than 2 Mb! If you thought splicing of exons within a single gene is amazingly complex, then genesis of lncRNAs borders on bizarre.
Paradigms for how lncRNAs function kindly provided for teaching by Cold Spring Harbor Press; for the explanatory caption click here.

Paradigms for how lncRNAs function kindly provided for teaching by Cold Spring Harbor Press; for the explanatory caption click here.

The Controversial Role of lncRNA

Lest you think that all is well in the proposed world of pervasive transcription, I hasten to add that this is definitely not the case. Bakel et al. have challenged the validity of tiling array data based on comparisons with data from RNA-Seq (aka whole transcriptome shotgun sequencing). They conclude that, “while there are bona fide new intergenic transcripts, their number and abundance is generally low in comparison to known exons, and the genome is not as pervasively transcribed as previously reported.” Clark et al. offer a lengthy and technically detailed rebuttal entitled The reality of pervasive transcription, to which Bakel et al. respond in lengthy technical detail.

Scientific debate is a healthy process, but non-experts face a difficult situation:  how does one decide which of these opposing expert-views is correct? Doing more literature searching led me to a publication with a title—metaphorically linked to cosmologic “dark matter” — that directly addresses this issue, namely, Transcribed dark matter:  meaning or myth by Ponting & Belgard. These authors propose that resolution of the debate requires “demonstration, or otherwise, that organismal or cellular phenotypes frequently result when noncoding RNA loci are disrupted.” In other words, if a lncRNA exists and has function, its knockdown by some method should ideally result in an effect that is somehow measurable. While this will obviously take much time and effort, Ponting & Belgard further opine that “[o]nly then, when we have these data and the results of detailed mechanistic studies at hand, will dark matter transcription be revealed as either ‘sound and fury, signifying nothing’ [as it has been described] or else as functional elements that are crucial to the biology of our species.” Who knew Shakespeare’s Macbeth would be thus quoted!

It has been remarked by Richard Robinson that “most dark matter transcripts are not signals emerging from a hidden universe within the genome, but instead simply the noise emitted by a busy [sequencing] machine.” This view struck me as being extreme and possibly misleading, so I asked Piero Carninci, a leading expert in lncRNAs, for his opinion.   He share’s it here:

PieroCarninci

Piero Carninci, Leader of the Functional Genomics Technology Team (RIKEN, Japan)

“The rare non-coding transcripts are indeed truly transcribed, but in a much more tissue and cell specific manner, so they may appear as “background” transcription if the right cell is not selected. Therefore I do not think they are just ‘noise’ because of their specific expression pattern. Moreover, when looking at RNAs deriving by fractionating specific cell compartment (nuclei, chromatin, etc.) we see that these non-coding transcripts show very strong, specific compartment expression specificity. We are further characterizing the function of several of them and we see an important role in transcriptional control for several of them.  Although we cannot claim that all of them are functional, we cannot state they are ‘noise’. Experiments will tell their ultimate functions.”    

Readers interested in getting a sense of the extensive experimentation employed to unambiguously knockdown (or overexpress) a lncRNA and thus assess its biological function are directed to an investigation of a lncRNA called—would you believe—HOTAIR. This lncRNA reprograms chromatin and, in a brief review by Hung & Chang, is said to be overexpressed in ~25% of human breast cancers, drives metastasis in a mouse model, and is a prognostic marker for death and metastasis in human breast cancer. Association of HOTAIR and, moreover, a list of 166 other disease-related lncRNAs definitely makes for a “hot topic” (pun intended).

In wrapping up this blog, I wanted to add yet “more fuel to the fire” for ncRNAs that, until recently, have been linear molecules—long or short—by paraphrasing from an In Focus News article in Nature earlier this year entitled Circular RNAs throw genetics for a loop by Heidi Ledford. Basically, nonconventional sequencing of RNA led Memczak et al. to the discovery of thousands of well-expressed, stable, circular RNAs (circRNAs) that often show developmental-stage-specific expression. Tellingly, a ~1,500 (!) nucleotide-unit circRNA called CDR1as was found to have ~70 hybridization sites for a miRNA called miR-7 and, with other evidence, was shown to function as a molecular “sponge” that can sequester miR-7 and thus suppress activity of miR-7.

Molecular rendition of circRNA sequestering miRNA (Merlinnz Blog)

Molecular rendition of circRNA sequestering miRNA (Merlinnz Blog)

Metaphorical rendition of circRNA “sponge” for miRNA (EpiBeat)

Metaphorical rendition of circRNA “sponge” for miRNA (EpiBeat)

 

 

 

 

 

 

 

Ledford notes that circular RNAs have been hypothesized to also function by binding to viral microRNAs and RNA binding proteins, which suggests that circRNAs are a new class of regulatory RNA molecules. She quotes one researcher as saying that “[i]t’s yet another terrific example of an important RNA that has flown under the radar.” Another researcher is said to hypothesize that “[t]hey are so abundant, there are probably a multitude of functional roles.” Ledford concludes by asking Nobel Laureate Phillip A. Sharp what other shapes might RNAs take? To which Sharp responds: “I can’t think of another form we might have missed…[b]ut you know somebody will find one.”

Hopefully, by now I’ve grabbed your attention and you’d like to find out more.  Please check an lncRNA expert review by Piero Carninci and a continually updated database (lncRNAdb) containing a comprehensive list of lncRNAs that have been shown to have, or to be associated with, biological functions in eukaryotes. There is also a comprehensive analysis of human lncRNA gene structure, evolution and expression as of 2012 available in the GENCODE v7 catalog.

What do you think?

As always your comments are welcome.

Postscript

Henry Harris (University of Oxford) in the Correspondence section of Nature published online May 8, 2013 wrote the following in what was entitled History:  Non-coding RNA foreseen 48 years ago. 

The recent enthusiasm for studying non-coding RNAs (Nature 496, 127–129; 2013) brings to mind a largely forgotten review article that I wrote almost half a century ago in Evolving Genes and Proteins (V. Bryson and H. J. Vogel (eds) 469; Academic Press, 1965). This review reached a conclusion that was judged to be profoundly heretical at the time.

The article summarized years of work on the turnover of nuclear RNA, carried out during a period when pulse-labelled RNA was almost universally misdiagnosed as messenger RNA. It concluded: “Only a small proportion of the RNA made in the nucleus of animal and higher plant cells serves as a template for the synthesis of protein. This RNA is characterised by its ability to assume a form which protects it from intracellular degradation. Most of the nuclear RNA, however, is made on parts of the DNA which do not contain information for the synthesis of specific proteins. This RNA does not assume the configuration necessary for protection from degradation and is eliminated.”

Looking forward, not backwards, readers who wish to track future developments involving lncRNA can link to an lncRNA blog.

Also, there is a Keystone Symposium on February 27—Mar 4, 2014 called “Long Noncoding RNAs: Marching toward Mechanism” co-organized by Nobel Laureate Thomas R. Cech (1989; catalytic RNAs) and featuring a Keynote Address by Nobel Laureate Phillip A. Sharp,  (1993; split genes, i.e. RNA splicing).

Ripples from the 2013 TIDES Conference

Nucleic acids are getting small, SMARTT, long and weird

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

Jerry Zon’s standup & co-blogger Rick Hogrefe

Jerry Zon’s standup & co-blogger Rick Hogrefe

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

Getting Small:  Spherical Nucleic Acids Nanoparticle Delivery

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

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

chadmirkin

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

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

SNA

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

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

flaredetection

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

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

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

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

Getting SMARTT:  Block-Polymer Delivery

Dr. Mary Prieve, PhaseRx

maryprieve

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

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

SMARTT

SMARTT Polymer Technology (courtesy of PhaseRx)

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

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

Getting Long:  Targeting Long Non-Coding RNA

Dr. Jim Barsoum, CSO, RaNA Therapeutics

jimbarsoum

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

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

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

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

RaNA

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

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

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

Getting Weird:  Unnatural Base Pairs

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

ichirohirao

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

unnaturalbasepairs

Taken from Hirao and coworkers NAR 2012.

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

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

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