Daniel Tenant, PhD
1. Chemically modified siRNAs/ASOs for therapeutic applications
RNA interference (RNAi) is a naturally occurring gene silencing mechanism that is mediated by RNA. Small interfering RNA (siRNA) is 21-22 nucleotides double-stranded RNA with two nucleotide 3’-overhangs. siRNAs have great potential in therapeutic applications. There are also some challenges associated with siRNA applications like nuclease susceptibility, unwanted binding to off-target due to partial complementarity, activation of unwanted immune responses, and in vivo delivery. The rational use of chemical modifications in sugar, nucleobase, and backbone of siRNAs can address most of these challenges.
Our objective is to synthesize chemically modified siRNAs and test their biochemical properties. Our primary goal is to carry out duplex stability study, serum stability study, and gene silencing studies using chemically modified siRNAs.
2. Design and development of RFP chromophore for selective detection of G-quadruplex
G-quadruplexes (G4) are non-canonical secondary nucleic acid structures formed by stacked guanine tetrads in G-rich DNA or RNA sequences. The guanine tetrad structures consist of four guanines arranged in a square planar arrangement through Hoogsteen hydrogen bonding. Depending upon the strand directionality, G-quadruplex can have various structural polymorphisms such as parallel, antiparallel, and hybrid structures with loop diversities. Several proto-oncogenes in the promoter region, including K-RAS, KIT, BCL-2, VEGF, and MYC, adopt parallel G-quadruplex structures and are overexpressed in cancers. G4 has emerged as a therapeutic target against anticancer, antiviral, and antibacterial because of their possible functions in DNA replication, telomere integrity, genome instability, and oncogene transcriptional control. Taking into account the potential therapeutic applications related to G4s, it has been of great importance to expand techniques to detect and visualize G4 DNA structures in live cells. We aim to develop a novel RFP-based chromophore that selectivity recognizes and stabilizes the specific G-quadruplex for potential therapeutic applications
3. Design and development of a modified G-quadruplex structure for protein binding
G-quadruplexes are non-classical secondary structures composed of G-tetrads and connected through Hoogsteen H-bonding. These structures are recognized for their potential in therapeutic applications due to their interaction with cellular proteins. The considerable thermal stability of the G-quadruplex provides them with moderate resistance against nucleases, which allows them a prolonged permanence in the bloodstream. Thus, the G-quadruplex be considered an interesting category of molecules with several potential applications in therapeutics. Notably, G-rich oligodeoxynucleotides (ODNs) that form G4s have shown antiproliferative effects, especially against cancer. For instance, AS1411 is a nucleolin protein binding aptamer that was evaluated in clinical trial 2, targeting nucleolin protein against cancer therapy. TBA is another G-quadruplex-forming aptamer known for binding with thrombin protein and acting as an anticoagulant. It also has antiproliferative properties. It has been reported that DNA aptamer T40214 inhibited the DNA-binding activity of Stat3. Also, it has been found that T40214 significantly suppressed the growth of breast, prostate, neck, head, and non-small cell lung cancer (NSCLC) tumors in nude mice xenograft models. Thus, exploring the interaction of G-quadruplex with specific proteins is very crucial for their therapeutic applications. Therefore, we aim to introduce chemical modification in the G-quadruplex, which can increase the binding affinity of the G-quadruplex while maintaining its native structure and stability.
4. Computational Studies on Nucleic Acid Systems
Our molecular modeling study investigates the structural properties of various chemically modified nucleic acid systems, including RNA and DNA homoduplexes as well as DNA:RNA heteroduplexes. We examine how different chemical modifications impact ribose sugar conformation and modulate duplex structure. Using MD simulations, we also explore interactions between chemically modified functional nucleic acids and various proteins. Additionally, our research utilizes Docking and MD simulation to analyze the binding modes of small-molecule ligands that selectively stabilize distinct G-quadruplex structures. Currently, we are also studying the binding interactions between different G-quadruplexes and protein systems.