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Synthesis of novel modified L-RNA for SELEX of therapeutic aptamers 

 

RNA aptamers, with a high affinity for a variety of molecular ligands, have been identified as promising therapeutic candidates using systematic evolution of ligands by exponential enrichment (SELEX). SELEX-based RNA aptamers have many advantages, such as low immunogenicity and fast in vitro selection process. Scientists have also applied the non-natural diastereomeric L-type RNA aptamer to target small molecule or disease-related RNA motifs. The inherent property of L-type RNA makes it unable to be recognized by native RNase, which greatly enhances stability in physiological conditions. Our lab is trying to introduce functional chemical modifications into the L-RNA through chemical synthesis, thereby enhancing the binding affinity, selectivity and bio-stability of the L-RNA aptamer, and eventually leading to a promising RNA drug candidate. The promising targets of L-RNA based aptamers include cancer related miRNA elements, tRNA fragment and cancer cells.

 

Mechanistic studies on RNA self-replication in origin of life 

 

According to RNA world hypothesis, the non-enzymatic RNA polymerization is a critical transitional stage between prebiotic chemistry of ribonucleotides and the evolution of ribozyme-catalyzed RNA replication. A detailed, structure-based understanding of how the activated nucleotide substrates react with RNA primer-template duplexes will inform on the mechanism of the replication. Recently, it has been revealed that the non-enzymatic RNA polymerization is potentially driven by a 5'-5'-linked, imidazolium-bridged dinucleotide intermediate, which is a highly reactive substrate formed by two activated monoribonucleotides. We are utilizing X-ray and neutron crystallography to mechanistically study the RNA enzyme-free polymerization and ligation, which are catalyzed by 2-aminoimidazolium-bridge or ribozymes. We also plan to structurally study the non-RNA genetic polymers in polymerization, and hope the result will help to reveal the structural superiority of RNA over its analogues in evolution.

Synthetic RNA nanostructures for high-throughput cryo-EM study and development of novel drug delivery tool

 

The rapid development of nucleic acid nanotechnology provides a novel perspective for drug delivery, tissue engineering, biosensing and much more. Our lab try to utilize RNA nanotechnology to develop a platform for high-through cryo-EM-based structure determination of small (MW <100 kDa) RNA structures, and for the efficient drug delivery tool with higher stability in vivo. Several short RNA fragments can be designed, programmed and manipulated to assemble into the well-defined 3D objects with precise architecture in the nanometer range, and, in principle, any functional moieties can be included as part of the nanostructure, including DNA, RNA, protein or small molecules. In this way, the motif-of-interest containing nanostructure can serve as scaffold with high contrast for cryo-EM imaging. On the other hand, the synthetic mirror-image synthetic RNA can also be applied to construct the L-type nanostructure. The intrinsic stability of L-type conformation will offer the delivery tool with dramatic enhanced stability.

Structural and functional study on tRNA fragment-YBX1 complex for anti-cancer drug design

 

tRNA fragments were first detected in the urine and serum of patients with cancer about 40 years ago, but they were originally considered as the product of random degradation of tRNAs. Recently, the studies on their biogenesis strongly indicated that these small RNAs are generated through specific fragmentation rather than random degradation of tRNA. Moreover, these cleavage products are functional and highly conserved. It is evident that tRNA fragments are uniquely regulating biological functions in different cancer cells. Some extensively investigated examples are the tRFs derived from tRNAGlu, tRNAAsp, tRNAGly, and tRNATyr, which were discovered to be upregulated under hypoxic conditions in breast cancer cell lines. These specific tRNA fragments were able to function to suppress the oncogenic transcripts in breast cancer cells, by associating with the oncogenic RNA-binding protein Y box binding protein 1 (YBX1). In this way, the stability of multiple oncogenic transcripts is reduced, leading to the degradation of oncogene transcripts and the inhibition of cancer cells proliferation. It has been observed that cancer cell proliferation under serum starvation, cell invasion and metastasis were dramatically reduced after introduction of tRNA fragment-like small RNA molecules into breast cancer cell lines, and the expression of tRNAGlu, tRNAAsp, tRNAGly, and tRNATyrmolecules are at significantly different levels in metastatic versus non-metastatic cancer cells. These results clearly demonstrate the cancer-suppressive ability of tRNA fragments. 

Due to the unique mode of post-transcriptional regulation, both tRNA fragments and YBX1 protein have become potential biomarkers and novel therapeutic targets. However, the mechanistic understanding of how tRNA fragments bind to the YBX1 protein, especially at the molecular level, is still missing. Insights into the structural basis of binding between tRNA fragments and YBX1 will help determine how regulation of mRNA stability is achieved, and provide a molecular basis for designing potential novel RNA or small molecular drugs to target YBX1.

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