Day 1 :
Keynote Forum
Jonathan Perreault
INRS - Institut Armand-Frappier, Canada
Keynote: The versatility of nucleic acids in biology and their promise in medicine
Time : 10:50-11:30
Biography:
Jonathan Perreault has completed his PhD at the University of Sherbrooke in Canada and Post-doctoral studies at Yale University in the laboratory of Ronald Breaker. He joined INRS – Institut Armand-Frappier in 2011. His work on functional nucleic acids has been published in reputed journals such as Nucleic Acids Research and Nature. It encompasses bioinformatics, biochemistry, molecular biology and microbiology approaches aiming at discovering and elucidating ncRNAs
Abstract:
Since the double helix structure of DNA has been solved more than fifty years ago, the field of molecular biology has produced tremendous discoveries which impacted medicine on many levels. The study of DNA and RNA unveiled the code of all living things and helped understand all processes in biology, but nucleic acids were revealed as fascinating subjects on their own. For instance, the discoveries of ribozymes and riboswitches revealed the potential duality of RNA, which can bear information and function at the same time. Largely fuelled by technology advances, numerous discoveries regarding both DNA and RNA exposed the complexity and beauty of these macromolecules. In parallel, many of these discoveries led to the development of new drugs using or targeting nucleic acids, as well as approaches that hold great promises for the future of both diagnostics and therapeutics.
Keynote Forum
Anatoly D. Altstein
Institute of Gene Biology RAS, N.F. Gamaleya FSRC of Epidemiology and Microbiology, I.M.Sechenov First Moscow State Medical University, RF MH, Moscow, Russia
Keynote: Origin of biological nucleic acids and the fi rst genetic system (the progene hypothesis)
Time : 11:50-12:30
Biography:
Anatoly D. Altstein is a Virologist. He studied problems of viral carcinogenesis, development of viral vaccines, biotechnology and origin of genetic mechanisms. Now, he is a Main Scientist of Institute of Gene Biology and N F Gamaleya Federal Scientific Center of Epidemiology and Microbiology and also Professor of I.M.Sechenov 1st Moscow State Medical University. He has published more than 100 papers in international and Russian journals and has been serving as an Editorial Board Member of the “Molecular Genetics, Microbiology and Virology” journal (Moscow, Russia).
Abstract:
Biological nucleic acids perform some well-known fundamental functions like: template; coding of polypeptides; transport of activated specific amino acids; enzymatic (ribozymes); regulating (small RNAs) and structural (ribosomes, chromosomes, virions). The most accepted concept of the origin of nucleic acids and life is the RNA world hypothesis which is supported by many scientists. It is suggested that the first polynucleotides are synthesized spontaneously from mononucleotides on prebiotic Earth. According to the RNA world hypothesis, the first living beings (protoorganisms) consisted of RNA with both the template and the enzymatic functions, without any proteins; the translation process and the genetic code appeared later in evolution. There are very strong objections against the RNA world hypothesis: 1) The emergence of the first protoorganism is impossible without a processive (moving along template) polynucleotide polymerase; at present only distributive polymerases out of nucleotides were obtained; processive polymerases of such nature are unknown and apparently cannot exist in principle.2) Synthesis of long polynucleotides from racemic mixture of different prebiotic mononucleotides is impossible without stereospecific catalysts. 3) Within frames of the RNA world there is no clear understanding of mechanism of genetic code and translation arising. In order to overcome these obstacles and to explain how the first biological nucleic acid (the first gene)arises simultaneously with a specific protein (a processive polymerase) forming a bimolecular genetic system (BMGS), I have proposed an alternative hypothesis (the progene hypothesis) (Altstein, 2015). According to this hypothesis, BMGS emerges not from mononucleotides and monoamino acids, but from progenes, namely, trinucleotides aminoacylated on 3’-end by a nonrandomamino acid (NpNpNp~pX~Aa, where N – deoxyribo- or ribonucleoside, P –phosphate, X - a bifunctional agent, for example ribose, Aa - amino acid, ~ macroerge bond). The progenes are used as the only substrates for interconnected synthesis of a polynucleotide and a polypeptide. The growth of the system “polynucleotide – polypeptide” is controlled by the enzymatic properties of the growing polypeptide, and the BMGS emerges as an extremely rare event. The progene forming mechanism(NpNp+Np~pX~Aa) makes it possible to explain the emergence of the prebiotic physicochemical group genetic code, as well as the selection of organic compounds for the future genetic system from the racemic heterogeneous environment. The BMGS is reproduced on a progene basis via replicative transcription-translation (the first molecular genetic process) that is similar to its modern counterparts. Nothing is required for the emergence and reproduction of the BMGS except for progenes and conditions for their formation, including lipid vesicles and short oligonucleotides (2-6 nb).
- Track 1:Nucleic Acids | Track 2:RNA Interference | Track 3: Nucleic Acid Therapeutics
Location:
Session Introduction
Woojin An
University of Southern California, USA
Title: MacroH2A acts as a key regulator of osteolytic bone metastasis of cancer cells
Time : 12:30-13:00
Biography:
Woojin An has completed his PhD from Oregion State University and Post-doctoral studies from the Rockefeller University. He is an Associate Professor of University of Southern California Keck School of Medicine. He has published more than 50 research papers in reputed journals and has been serving as a reviewer for multiple journals and grant study sections.
Abstract:
Breast cancer tends to metastasize to bone, and more than half of all patients dying of breast cancer have evidence of osteolytic bone metastasis. Breast cancer-derived factors facilitate this bone metastatic process by generating a permissive microenvironment in the target organs for the engraftment of cancer cells as well as the establishment of metastatic foci. Histone modifications and variants play a crucial role in remodelling chromatin organization and modulating gene expression in various cancer cells including breast cancer cells. Altered expression of metastasis- associated genes by distinct combinations of histone modifications and variants is linked to multiple stages of breast cancer metastasis to bone. Yet, how this epigenetic resetting of gene expression in breast cancer cells contributes to osteolytic metastasis and bone lesions remains unclear. We show that histone variant macroH2A is capable of governing the early events of osteolytic lesion formation by inhibiting the expression of lysyl oxidase (LOX), which is a key factor for osteoclastogenesis. We demonstrate that macroH2A, in complex with EZH2 histone methyltransferase and Mybbp1a transcription repressor, occupies the promoter and coding regions of LOX gene, elevates the levels of histone H3 lysine 27 trimethylation, and thereby keeps the gene in a repressed state. Our data unravel an unexpected role for macroH2A in regulating the pathogenesis and progression of breast cancer bone metastasis and illustrate the power of combined biochemical and cellular approaches for mechanistic analyses.
Biography:
Yuhong Wang has completed her PhD in 2002 from the Johns Hopkins University and postdoctoral studies from Caltech and UPenn. She is an associate professor in University of Houston. She developes new biophysical tools, such as single molecule FRET and mechanomagnetic force spectroscopies. Her research interests are ribosome mechanism and non-invasive detection of microRNAs.
Abstract:
The ribosomal frameshifting is a rare but ubiquitous process. The putative "-1" frameshifting motif includes a slippery sequence, a spacer, and a secondary mRNA structure. We report a new force-based method to direct observation of “-1” and “-2” frameshiftings with single nucleotide resolution. We reveal that EF-G•GTP is indispensable to frameshifting. The biological relevance of the in vitro results is verified by protein translations in the cell. The mechanistic insights provided by our assay demonstrated the application of this method to study the ribosome system. The ribosome pre- and post-translocation complexes are tethered to the surface by biotinylated mRNA. The 3'-mRNA uncovered by the ribosome forms duplexes with DNA probes with known sequences. The probes are labeled with magnetic beads. Under external forces, the duplexes dissociate step-wisely according to the base pairs in the duplexes. The dissociation is detected by an atomic magnetometer and reflects the ribosome position with single nucleotide resolution. Three consecutive translocation steps were tracked to unambiguously identify the total of nine possible ribosome positions on the mRNA under in vitro conditions. Mechanistic studies were carried out by modifying the motif, introducing a secondary structure, and varying other experimental conditions. Meanwhile, in vivo and in vitro protein synthesis experiments were performed to demonstrate the biological significance of the frameshifting results.
Jonathan Perreault
INRS - Institut Armand-Frappier, Canada
Title: Design and selection of hammerhead ribozymes
Time : 14:30-15:00
Biography:
Jonathan Perreault has completed his PhD at the University of Sherbrooke in Canada and postdoctoral studies at Yale University in the laboratory of Ronald Breaker. He joined INRS – Institut Armand-Frappier in 2011. His work on functional nucleic acids has been published in reputed journals such as Nucleic Acids Research and Nature. It encompasses bioinformatics, biochemistry, molecular biology and microbiology approaches aiming at discovering and elucidating ncRNAs as well as developing applications.
Abstract:
Since the 1990s, hammerhead ribozymes have been studied in regards to gene therapy because of their simple RNA-cleaving catalytic property and the relative ease with which they could be designed to target mRNAs through sequence modifications permitting base complementarity with the RNA to be cleaved. Since then, the discovery of RNA interference and CRISPR have relegated ribozymes behind, while spurring a renewed interest in noncoding RNA-mediated gene therapy. However, meanwhile advances in design and major discoveries on hammerhead ribozymes, such as better activity when stem I and II interact, have opened new avenues. We have demonstrated the high efficiency of hammerhead ribozymez by using combinations that target the same mRNA. Moreover, the automated design software, RiboSoft, streamlines the use of ribozymes for gene knockdowns. Results of RNA targeting against PABPN1, a gene involved in hereditary diseases, and other RNAs will be shown. Ribozymes have many advantages over RNAi and CRISPR: they are inherently active and do not rely on any accessory protein or component, making them easily portable to any organism; since they do not require processing and rely on structure for their activity, other modules can be added for their function; and they can be assayed in vitro. In short, the revived interest in using RNA for gene therapy is likely to also help ribozymes make a come back by stimulating research for general problems such as gene delivery.
Eylon Yavin
The Hebrew University of Jerusalem, Israel
Title: RNA detection in living cancer cells by far-red emitting PNA-FIT probes
Time : 15:00-15:30
Biography:
Dr. Yavin completed his PhD at the Weizmann Institute of Science (Israel). He did his postoctoral work at the laboratory of Prof. Jacqueline Barton at Caltech (CA, USA). In 2006 he joined the School of Pharmacy at Jerusalem as a faculty member. Eylon is currently a senoir lecturer and has an active research lab in the field of Nucleic Acids. He has published more than 40 papers in reputed journals and has been recently elected as the president of The Medicinal Chemistry Section of the Israel Chemical Society.
Abstract:
To diagnose cancer early on has no doubt a huge impact on the success rate of anticancer therapy in almost all clinical settings. One promising approach is based on the detection of RNA biomarkers; particularly, mutated RNA that gives a distinct signature and allows a better choice of treatment. One attractive approach is based on the use of FIT-PNA (forced intercalation – peptide nucleic acid) probes. These PNA molecules hybridize to complementary RNA and gain fluorescence only after binding to their RNA target. We have previously shown that such FIT-PNAs with TO (thiazole orange) as a surrogate base, fluoresce in living pancreatic cancer cells (Panc-1) that express mutated kRAS mRNA (G to A single point mutation at codon 12) but not so in colon cancer cells (HT-29) that express wild-type kRAS mRNA. Herein we report on the design and synthesis of a new surrogate base (BisQ) with the unique feature of far-red emission. This surrogate base was introduced into PNAs that target the mutated kRAS oncogene. PNAs with a short cell penetrating peptide (CPP) consisting of 4 D-Lysines were shown to readily penetrate living cancer cells and fluoresce in the far-red region (ï¬max = 609 nm) exclusively in pancreatic cancer cells (Panc-1) that express the mutated form of kRAS but not in pancreatic cancer cells that are non-mutated (wild type) in kRAS (BxPC-3). We are currently developing new FIT-PNA probes targeting other RNA biomarkers and exploring new surrogate bases with unique spectral properties.
Thenmalarchelvi Rathinavelan
Indian Institute of Technology Hyderabad, India
Title: Mechanism of B-to-Z transition induced by A…A mismatch in a DNA duplex comprising of CAG trinucleotide repeat expansions
Time : 15:30-16:00
Biography:
Rathinavelan has completed her PhD from Department of Crystallography and Biophysics, University of Madras. Subsequently, she did her postdoctoral studies from Center for Bioinformatics/Department of Molecular Biosciences, The University of Kansas, US. Currently, she is working as an Assistant Professor in IIT Hyderabad and she has published more than 10 research papers in reputed journals.
Abstract:
Conformational polymorphism of DNA is a major causative factor behind several incurable trinucleotide repeat expansion disorders (TREDs) that arise from overexpansion of TREs located in coding/non-coding regions of specific genes. Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington’s disorder and spinocerebellar ataxias. Hairpin structure formed during CAG repeat overexpansion contains periodic occurrence of A…A mismatches and hijacks the mismatch repair proteins (MSH2-MSH3) through tighter binding. Although DNA hairpin stem structure generally embraces B-form with canonical base pairs, it is poorly understood in the context of periodic non-canonical A…A mismatch in a CAG overexpansion. Molecular dynamics simulations on a DNA hairpin stem containing A…A mismatches as in a CAG repeat overexpansion show that A…A dictates local Z-form irrespective of starting glycosyl conformation, in sharp contrast to canonical DNA duplex. B-to-Z transition occurs through ‘zipper mechanism’ facilitated by base extrusion, backbone and/or base flipping. Root cause for such B-to-Z transition is due to the mechanistic effect that originates from the pronounced non-isostericity exhibited by A…A mismatch with flanking canonical base pairs. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A...A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair.