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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Studying Telomeres in Space

  • Telomeres are DNA Biomarkers for Your Biological “Age”
  • Telomere Shortening Due to Stress was Expected During Spaceflight, but Exactly the Opposite has Been Found
  • Raising New Questions to Answer

After you read this blog about studying telomeres in space, I think you will agree with my opinion that scientific advances can sometimes occur amazingly fast. Telomeres (which are peculiar DNA structures that I’ll explain below) went from esoteric Nobel Prize subject matter in 2009 to the focus of spaceflight science in just six short years. Now, telomeres are being investigated by PCR on the International Space Station (ISS)! With a wink and a nod to Star Trek, this is indeed “warp speed” progress!

Taken from keywordsuggest.com

What are telomeres?

A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. For vertebrates like us, the repetitive sequence of nucleotides in telomeres is TTAGGG, with the reverse complementary DNA strand being AATCCC, as depicted below. This sequence of TTAGGG is repeated ~2,500 times in humans.

During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, causing the end of the chromosome to be shortened in each replication. The telomeres are thus disposable “buffers” at the ends of chromosomes which are truncated during cell division, as depicted below.

Taken from weeklyglobalresearch.wordpress.com

Telomers, however, are replenished by an enzyme named telomerase. This peculiar enzyme has an embedded RNA template and incorporates DNA nucleotides, as depicted below, and is therefore a special kind of reverse transcriptase. In people, it has been found that telomeres shorten with age in all replicating somatic cells that have been examined. In fact, average telomere length declines from about 11 kilobases at birth to less than 4 kilobases in old age, with the average rate of decline being greater in men than in women. Thus, telomere length can serve as a biomarker of a cell’s biological (versus chronological) “age” or potential for further cell division.

Taken from 2014hs.igem.org

‘Houston, we have a problem’

This now famous phrase, which was used in the past tense by the crew of the Apollo 13 moon flight to report a major technical problem back to their Houston base, echoed in mind when I learned that space flight might lead to telomere shortening. Yikes! This molecular-level change could indeed be a serious problem, and was first suggested by findings from laboratory microgravity simulations reported in 2008 by Chinese researchers. Since it was known that space flight leads to bone loss, they cultured bone stem cells (BSCs) under simulated microgravity in a rotary cell culture system.

This led to significantly decreased activity of telomerase. It was postulated that reduced bone formation in space flight may partly be due to the altered potential differentiation of BSCs associated with telomerase activity, which plays a key role in regulating the lifespan of cell proliferation and differentiation. Additionally, telomerase activation or telomerase replacement may act as a potential countermeasure for microgravity-induced bone loss.

Taken from energeticnutrition.com

If you’re thinking that these “potential countermeasures” are fanciful, you’d better think again. I recently came across a patent that was published last year on methods and compositions for increasing telomerase activity in cells, including pharmaceutical formulations. Moreover, there are now various commercially available supplements claiming to promote telomerase activity, such as that picture below. I hasten to add that I do not advocate use of any such supplement, and that interested readers should consult their primary care physician or certified nutritionist.

Twins and telomeres

Although identical twins are almost the same genetically, differences in environment, diet and other outside factors can affect their health in different ways. Consequently, identical twins have been enrolled in various studies that require deciphering effects due to “nature vs. nurture” (i.e. intrinsic genetics vs. external factors). Part of the Twins Study supported by NASA was aimed at examining the effects of space travel on one of a pair of twins: astronaut Scott Kelly, who stayed on the ISS for one year, while his twin brother, Mark, remained on Earth. In brief, Prof. Susan Bailey at Colorado State University is exploring differences between the twins’ telomeres to determine if telomeres respond differently to spaceflight and then how such changes relate to the various medical endpoints studied by other Twins Study investigators.

Scott Kelly (left) and his identical twin brother Mark in 2015 prior to Scott’s one-year mission to the ISS. Taken from space.com

Preliminary research results for this part of the NASA Twins Study (reported at NASA’s annual Investigators’ Workshop earlier this year) were a quite surprising because they were opposite of what was expected, thus raising more questions than providing answers. It had been theorized that exposure to microgravity and stress during prolonged spaceflight would shorten telomeres, but instead Bailey’s team found telomeres in Scott’s white blood cells increased in length while in space! This finding was rationalized as being due to increased exercise and reduced caloric intake during the space mission. Upon his return to Earth, however, these telomeres began to shorten again.

This is yet another example of a biomedical phenomenon being far more complex that first theorized, and one that becomes less understood as more and more data are obtained. We’ll all have to patiently stay tuned for how this telomeres-in-space story evolves. The good news, as I’ll explore in the next and final section of this blog, is that there are exciting plans to use PCR to measure telomere length extraterrestrially! This is very “far out” science—pun intended.

Studying telomeres in space by PCR

Taken from geeky-gadgets.com

The aforementioned Twins Study involved taking blood samples from an astronaut during spaceflight for lab analysis upon return to Earth. To obtain much more data, and to do so in real time while in space, NASA launched the Genes in Space-2 mission in April 2017. The goal is to determine whether astronauts aboard the ISS can analyze telomeres by PCR reactions in a small thermal cycling device (miniPCR system) and thus measure and monitor telomere changes during spaceflight.

In addition to testing the miniPCR system, the Genes in Space-2 mission has a secondary goal to test the feasibility of techniques used to measure telomere length. Currently, Single Telomere Length Analysis (STELA) is the only suitable technique for use on the ISS due to technical requirements. The Genes in Space-2 mission will also be testing the feasibility of a loop-mediated isothermal amplification (LAMP) colorimetric assay for detection of amplification aboard the ISS. Please stay tuned for updates on the outcome of these very important feasibility experiments.

Scheme for STELA procedure. Taken from Xing et al. (2009)

Julian Rubenfein. Taken from nydailynews.com

As a side note, the Genes in Space competition for 2017 selected this experiment on telomere amplification in microgravity from 375 submissions by nearly 850 students in grades 7 to 12 from across the US. This telomere experiment was proposed by 17-year old Julian Rubinfien from Stuyvesant High School in New York City, who is pictured below. I encourage you to read this interesting, although lengthy interview about his background and the experimental rationale. What’s even more interesting is this short video of Julian at the launch and his comments—very impressive!

I strongly encourage you to read more about all the award-winning experiments in this exciting round of competition among young, highly motivated, advanced students, who I’m sure will be successful in whatever they do in the future.

Your thoughts or comments here are welcomed.

Postscript

Profs. Elizabeth Blackburn and Carol Greiner—who received a Nobel Prize in 2009 for seminal work on telomeres—co-founded Telome Health Inc. (THI) in 2010 to leverage the predictive power of telomere-length assays to help assess health status, disease and mortality risk, and response to specific therapies. THI subsequently announced TeloTest™ as a diagnostic test that measures average telomere length by qPCR. TeloTest™ was the first saliva-based telomere test available on the market, and is currently offered by a company named TeloYears.

The clinical utility of testing telomere length in a saliva-based test was recently reported from an independent, large clinical study sponsored jointly by Kaiser Permanente, University of California, San Francisco (UCSF), and National Institutes of Health. In the study, the average telomere length of 100,000 Kaiser patients was measured and analyzed relative to other health domains and clinical outcomes.

My recently obtained TeloTest™ results from TeloYears indicated that my biological age is 4 years older than my chronological age. Naturally, I was hoping to learn that my telomere-based age would be less than my actual age. Alas, the results are what they are, so I’ll be following diet, exercise, sleep, and stress-management recommendations you can read about at TeloYears Learning Center.

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Is it time for “GQnomics”?

An Interview with Dr. Shankar Balasubramanian about DNA G-Quadruplexes (GQs) Quantitatively Visualized in Human Cells for the First Time

My previous post featured Watson & Crick’s revolutionary publication of DNA’s double-helix structure in Nature in April 1953 resulting from their work in Cambridge. Now I’m fast-forwarding 60 years to January 2013 to highlight yet another landmark publication on DNA structure, again in Nature (Chemistry) and again based on work in Cambridge, but this time from a team led by Prof. Shankar Balasubramanian, who is The Herchel Smith Professor of Medicinal Chemistry at Trinity College, University of Cambridge.

balasubramanianThe significance of Prof. Balasubramanian’s publication entitled “Quantitative visualization of DNA G-quadruplexes in human cells” coauthored with Giulia Biffi, David Tannahill and John McCafferty is evidenced by the fact that there were over 82,000 page views within 3 weeks of posting, as well as interviews with several news agencies, including the BBC! I’ve been very interested in GQs for quite some time so I reached out to Prof. Balasubramanian and he kindly agreed to my request for a telephone interview, which took place in March of this year. I’ve included some of my favorite questions from that interview below. For those of you who may be new to this area of research, I’ve included some background information on GQs as well as a postscript following the interview questions.

 

JZ: What inspired or led you to begin investigating cellular G-quadruplexes?

SB: My lab was actually working on the mechanism of human telomerase in the late 1990s and noted some interesting papers postulating the potential importance of G-quadruplex-folded (telomere) structure and telomerase function. This set me thinking about whether such structure might be ‘real’ in biological systems and how we might go about investigating them.

 

JZ: What has been the most challenging experimental aspect of this work?

SB: It is a culmination of much work done using molecular design and synthetic chemistry, antibody engineering, molecular biophysics and cell/molecular biology.  The biggest challenge was to figure out how to usefully integrate and harness all of these sub-disciplines in the project.

 

JZ: Was there a “most memorable moment” for you during this research?

SB: Seeing is believing. Observing the first images of G-quadruplex hotspots in cellular genomic DNA was an exciting moment.

 

JZ: What was a “big surprise” during this research?

SB: Seeing the levels of G-quadruplex signal go up on addition of a G-quadruplex-stabilising small molecule. Whilst this was in accord with the hypothesis that G-quadruplexes can serve as targets, I hadn’t expected such a visually clean outcome from the experiment.

 

JZ: How will it be possible to target G-quadruplexes in specific DNA promoters?

SB: To target G-quadruplexes associated with particular genes one must consider the molecular structure of that G-quadruplex, which can be done biophysically, and additionally how the functional state of that part of the genome (e.g. is it transcriptionally active) makes it more vulnerable towards small molecule ligands. It is important to consider a G-quadruplex (or a cluster of G-quadruplexes) within the architectural context of the genome and chromatin.

 

JZ: Any guesses about the DNA G-quadruplex epitope for the B4G antibody?

SB: We don’t, as yet, have high-resolution structural information on a protein-DNA complex. Given the antibody recognizes a wide variety of G-quadruplexes we have looked at, it would appear that it recognizes common molecular features such as those of the stacked G-tetrad core, rather than the hypervariable loops.

 

JZ: Is the BG4 antibody available to other research groups now or in the future?

SB: The plasmid will be made available to others for research purposes via a standard MTA with our university.

 

JZ: Do you foresee possibly visualizing DNA G-quadruplexes in living cells in real-time?

SB: We and others are taking steps towards enabling this through the design and development of new molecular probes and I expect we will soon see how it can be done.

 

JZ: What is currently most controversial about DNA G-quadruplexes?

SB: I would say whether they have biological ‘function’. This will of course also depend on one’s strict definition of function and what constitutes evidence of biological function. I consider there is already good experimental support that there are biological consequences of G-quadruplex formation and/or stabilization.

 

JZ: You have a historical connection with inventing what is now Illumina’s sequencing-by-synthesis—have you applied this technology in your G-quadruplex research?

SB: Indeed we have.  Last year we published a paper in which we used Solexa/Illumina sequencing to map the genomic sites of double strand breaks induced by a G-quadruplex stabilizing ligand. Gratifyingly these sites were mapped to regions richly populated by predicted G-quadruplexes.  More recently, we have been mapping G-quadruplex structures using an antibody to perform DNA immunoprecipitation followed by deep sequencing.

 

JZ: Do you think it’s appropriate to refer to this field as “GQnomics”?

SB: I’m not one for expanding the ‘omics’ lexicon.  However, I do think it is time for biologists and genome folk to consider how this non-Watson-Crick structural motif might play a role within their systems or questions under investigation.

Additional comments by Prof. Balasubramanian and photos are available in his interview with BBC News.

 

Background

DNA guanosine (G)-quadruplexes (GQs) of the type shown below have been extensively studied from a chemistry perspective for many years. However, investigations aimed at convincingly demonstrating that DNA GQs are present and have functional roles in cells are experimentally challenging and not without controversy. Obtaining data and interpretation is difficult in part due to the large number of possible structural motifs that can exist and be involved in complex dynamic equilibria.

GQ Structure Formation

Figure 1:  GQ Structural Formation

Prof. Balasubramanian’s publication on quantitative visualization of GQs in cells is the most recent contribution in a long line of his reports systematically elucidating the identity and function of this structural element. His early bioinformatics analysis of the human genome with Julian L. Huppert involved “putative quadruplex sequences” (PQS) defined and located by proposed working rules and search algorithms. They concluded that, in principle, as many as 370,000 PQS could exist simultaneously but that of these a smaller number would likely exist in a dynamic equilibrium with other structural forms of DNA. Importantly, it was found that PQS were present in a number of G-rich promoters throughout the genome and led to proposing that promoter GQs are directly involved in the regulation of gene expression.

But can putative GQs be detected and studied in cells? That critical question was answered in the affirmative by Prof. Balasubramanian and his coworkers in their publication noted above. Quantitative visualization in fixed cells was achieved using immunofluorescence microscopy enabled by a DNA GQ-specific single-chain antibody (BG4) that they obtained from a phage display library of more than 2 × 1010 different antibody clones. Importantly, various BG4-enabled visualization experiments established that GQ structures are present largely outside telomeres (Figure 2). Moreover, quantitative visualization results obtained with synchronized cell demonstrated that GQ formation in DNA is modulated during cell-cycle progression—maximum BG4 foci per nucleus at S (synthesis) phase—and that these structures can be stabilized by a known GQ-binding small-molecule, pyridostatin (PDS). The authors state that it was “anticipated that G-quadruplex formation most probably occurs during DNA replication, because the associated mechanisms necessitate that duplex strands become separated at replication forks, where single-stranded DNA may fold more easily into secondary structures.” Stabilization of GQs by PDS was said to allow for possible use of stabilizing ligands to target cellular GQs and intervene with their function.

Immunofluorescence for BG4

 

Figure 2:  Immunofluorescence for BG4 showing absence of large co-localization between telomeric TRF2 proteins (green) and G-quadruplex (red) in U2OS (osteosarcoma) cells. This suggests that endogenous G-quadruplex structures are present largely outside telomeres. Nuclei are counter stained with DAPI (blue). Scale bar, 20 µm.

Shifting molecular gears, so to speak, I encourage you to read a recent review coauthored by Prof. Balasubramanian on the occurrence and role of GQ structures in 5’ untranslated regions (5’-UTRs) of mRNA with regard to translational regulation and targeting. This emerging science is a fascinating and relatively new aspect of increasingly complex regulatory mechanisms involving RNA, some of which will be highlighted in a future post.

After researching the literature in this post, I have a far greater appreciation for the breadth and significance of molecular and cellular biology ascribed to GQ structures. So much so that at least for me there is a fascinating field of “GQnomics” in addition to genomics!

Comments about this post are welcomed!

 

Postscript

This and future occasional Postscripts are a way to share information found when researching topics and technical details for my posts that I think are interesting or in some way noteworthy.

For example, I learned that identification of GQs had its origins in finding surprisingly stable aggregates of short synthetic poly(dG) oligonucleotides described in 1962 by a team led by H. G. Khorana, who incidentally 6 years later was awarded a Nobel Prize with Robert W. Holley and Marshall W. Nirenberg for their interpretation of the genetic code and its function in protein synthesis. Only a few months after the poly(dG)-aggregation report by Khorana and coworkers in 1962, Gellert et al. proposed that this phenomenon was due to planar, stacked arrays of four H-bonded Gs as shown above. Coincidentally, 1962 was also the year when Watson & Crick were awarded their Nobel Prize.

Who knew then that GQ structures would be found as telomeres that maintain chromosome stability in all living cells, and become a key part of the science for which a Nobel Prize in 2009 was awarded to Profs. Elizabeth H. Blackburn, Carol W. Greiner and Jack W. Szostak—a video interview of these Nobel Laureates is well worth visiting to hear how these scientists came to investigate telomeres and co-discover telomerase, which is a ribonucleoprotein enzyme that adds DNA sequence repeats (“TTAGGG” in all vertebrates) to the 3′ end of DNA strands in the telomere regions. About 35 minutes into this 2009 video interview they are asked about potential therapeutic benefits of studying telomerase and telomeres. Profs. Blackburn and Greiner, who opine in response, co-founded Telome Health Inc. (THINC) one year later as a company that is “dedicated to leveraging the predictive power of telomere and telomerase assays (‘telome measures’) to assess health status, disease risk, and responses to specific therapies.”

One example of telomere-related therapy is a 13-mer oligonucleotide having a thio-phosphoramidate backbone named Imetelstat (GRN163L) that is in clinical trials by Geron Corp. Another is a phosphorothioate oligodeoxynucleotide called Cantide, which is antisense to human telomerase reverse transcriptase (hTERT; a catalytic subunit of telomerase) and has been recently reported to have promising anticancer activity in a nude mouse model of human primary hepatic lymphoma. Notwithstanding all of the above positive roles for GQs in biology and therapeutics, avoiding “runs of G” is well-known in design of PCR primers. This extends to antisense sequence selection, as exemplified by my investigations of a 15-mer having a run of 4 Gs that was seriously complicated by reversible intermolecular GQ formation evidenced by size-exclusion chromatography.

Comments about these Postscripts are also welcomed!