Our research group is interested in the chemical synthesis, biochemical properties, and molecular behaviour of nucleic acids and their analogues. We are particularly interested in developing new methods for the synthesis of nucleoside building blocks and RNA, including modified siRNA/miRNA, branched RNA and lariat RNA. We also devote an enormous effort to the study of nucleic acid structure.
More recently, our group reported the in situ synthesis of RNA on microarrays for the study and discovery of protein-RNA interactions that are relevant to important biological processes (e.g., RNAi, transcription, etc).
The main methods used in these investigations are solution and solid-phase synthesis; molecular biology techniques (gene silencing via RNAi, antisense, PCR, etc), high resolution NMR, UV and circular dichroism; and molecular modeling.
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This work has involved biochemical studies with novel chemically modified nucleic acids. Aspects of these studies include modifications that either (i) augment nuclease stability; (ii) improve catalytic cleavage of mRNA by RNase H or within the RNA-Induced Silencing Complex (RISC); or (iii) increase target hybridization accessibility.
Recent examples synthesized in our group include arabinonucleic acids (e.g., 2'-fluoro-ANA (FANA) and ANA), 2',5'-linked RNA, 2',4'-difluoro-RNA, and oxepane nucleic acids (oligonucleotides with a 7-membered carbohydrate ring). In collaboration, we are presently examining these compounds as anticancer oligonucleotides using antisense, anti-miRNA and siRNA compounds. We are also carrying out enzyme-mediated synthesis of novel 'genetic systems' and aptamers with therapeutic utility.
We have published several articles on sugar-modified nucleic acids that trigger the hydrolysis of mRNA by ribonucleases (RNaseH, Ago2). These are the predominant methods of activity of most antisense and siRNA oligonucleotides under clinical development. Our work on arabinose-based oligonucleotides (ANA and 2’F-ANA) has received significant attention by the nucleic acids community for several reasons: (i) These compounds represented the first examples of RNase H-competent oligomers that lacked the DNA sugar; (ii) our siRNAs are among a select few that allow full backbone modification without compromising potency; total modification is achieved in a non-sequence dependent fashion, and hence our siRNAs are readily applicable to most targets; and (iii) these findings provide a very important advancement in understanding the catalytic mechanism and substrate selectivity of RNase H (and Ago2), and underscores the potential of arabinose-based oligomers as gene-silencing agents. Delivered via aerosol, arabinose-based oligomers inhibit gene expression pathways linked to chronic obstructive pulmonary disease (Topigen). Our 2’F-ANA antisense and siRNA technologies have been recently licensed to AUM LifeTech and Paladin Labs, respectively.
The 3 major hurdles encountered in RNAi/antisense technologies are, as John Rossi attests, "Delivery, Delivery, and Delivery". We are addressing this issue through the development of "siRNA prodrug" conjugates, and integrating expertise in RNA targeting and antibody nanomicelle delivery. In collaboration with the Shoichet lab (University of Toronto), we have showed targeted gene downregulation using novel siRNA- and antisense-polymeric nanomicelles, demonstrating their capacity to achieve selective gene downregulation.
We are studying the structure of DNA/RNA hybrids, siRNA duplexes, and triple- and tetra-stranded DNA complexes. Interest in studying these structures, e.g., tetraplexes, has been renewed due to evidence suggesting biological roles in vivo (telomeres, DNA recombination), and because the formation and/or stabilization of DNA/RNA and RNA/RNA duplexes provides a basis for artificial control of gene expression through antisense and RNA interference.
Current efforts are also focused on understanding the recognition of (a) branched RNA (bRNA) by the lariat debranching enzyme and spliceosomal factors, (b) siRNA duplexes by the RNA-induced silencing complex (RISC), and RNA hairpins by the HIV-1 Reverse Transciptase. This research goals include gaining a detailed understanding of inter- and intramolecular interactions between various nucleic acid components or between nucleic acids and proteins as these key biochemical processes occur. We approach all of these studies through the use of chemically-modified oligonucleotides.
Our work on branched and dendritic oligonucleotides have been often cited in the context of “chiral dendrimers” and as one of the first dendrimers ever made from biological monomers (Hudson and Damha, JACS 1993). Our synthetic strategies are being implemented by other research groups to construct novel 3D DNA nanostructures. More recently, we are studying telomeric and centromeric DNA structures (G-quadruplexes, i-motifs) by 2'-fluorination of the sugar-phosphate DNA backbone (e.g., Nucleic Acids Res., 2007; JACS 2013). This approach will make it possible to study these structures at pH 7, where detailed structural information on, e.g., i-motifs, is lacking.
Synthetic branched RNAs (bRNA) serve as invaluable tools for probing mechanistic aspects of pre-mRNA splicing and debranching that occurs at the cellular level, and structural studies on the RNA Lariat Debranching Enzyme. Furthermore, our synthetic strategies are being implemented by other research groups to construct novel 3D DNA nanostructures.
Recently, the crystal structure of purified, recombinant Dbr1 from the amoeba Entamoeba histolytica (Eh) was determined, providing the first picture of an RNA lariat debranching enzyme in the absence of nucleic acids as well as in complex with several synthetic RNAs (Nucleic Acids Res., 2014 - Breakthrough Article).
Our work in this area was first published in 2004 and was featured on the cover of Nucleic Acids Research (2004). We made the exciting discovery that short hairpin RNAs interfere specifically with the function of the RNase H domain of HIV-1 Reverse Transcriptase (RT) at the low μM range, without affecting E. coli or human RNase H. Remarkably, the DNA polymerase activity, an intrinsic property of HIV RT, was not inhibited by these RNAs, a property not previously observed for any nucleic acid aptamer directed against RT RNase H. These findings bring into light a new class of nucleic acid aptamers that act exclusively upon HIV-1 RT RNase H in vitro.
More recently, we have been carrying out structure-guided synthesis of chemically modified branched RNAs (bRNAs), as a strategy for identifying inhibitors of the Lariat RNA Debranching enzyme (Dbr1) (J.Org.Chem. 2013). The knockdown of Dbr1 activity in yeast, primary rat neurons, and in a selected human neuronal cell line has been shown to offer protection against toxicity mediated by TDP-43, an RNA-binding protein implicated in amyotrophic lateral sclerosis (ALS) and the related disorder, frontotemporal lobar degeneration (FTLD). Hence, these endeavors may open up a novel therapeutic avenue for the treatment of neurodegenerative diseases associated with aging.
Our work in this area is aimed at finding ribonucleoside synthons that potentially benefit three critical aspects of RNA manufacturing: yield, cost and “green” methods of synthesis (CanJChem, 2007; JOC 2006). In collaboration with the Chan group (McGill), we have explored the conjugation of ionic tags to RNA, allowing easy isolation of the product from a reaction mixture through simple precipitations or extractions. We have demonstrated that RNA oligomers may be grown (via Ogilvie's TBDMSi chemistry) on dialkylimidazolium and tetraalkylphosphonium tagged soluble supports in high yields and purity, without the need for chromatography until the desired length polymer is obtained and deprotected. We believe that ionic tags will continue to be versatile and their applicability to biopolymer synthesis as soluble supports will remain a source of interest for some time to come. Our ion-tagged RNA synthesis methods have been licensed to AUM Life Tech.
In collaboration with researchers at the University of Wisconsin-Madison, we reported the first in situ synthesis of RNA on microarrays (“RNA chips”, JACS, 2009). These “RNA chips” will have immediate applications in the study (and discovery of) protein-RNA interactions that are relevant to important biological processes (e.g., RNAi, etc), as well as in the rapid screening of aptamers. Our RNA chip chemistries are being commercialized by ChemGenes Corporation.