Evolving New L-RNA Aptamers and Developing L-RNA aptamer-peptide Conjugates to Control G-quadruplex Structure-associated Gene Activity

G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that have been first detected few decades ago. Multiple lines of evidence suggested that these four-stranded nucleic acid structural motifs in DNA and RNA have crucial functional roles in a number of biological processes in cells, but challenges remain in selective targeting of functional G4 structures and precisely control the G4-associated biological events. To tackle this, the invention of novel G4 targeting tools— particularly with the capability to target specific G4 of interest, as well as the potential to localize and work in cells to control G4-associated gene activity — are two important aspects for further discovering new G4 biology and evolving original strategies for G4 targeting and gene regulation applications. Our group have an established track record of aptamer development and application, as well as targeting of G4 structures. Recently, we have developed the first series of G4 targeting L-RNA aptamers and applied them to target G4 of interest, of which the reported L-RNA aptamers exhibited exquisite binding and promising selectivity properties. In addition, we have designed and incorporated new functional modules to cyclize the aptamer to further improve its performance, such as binding affinity, selectivity, biostability, and demonstrated its application as G4 tool in cells.In this project, we will evolve new L-RNA aptamers with diverse properties to target G4, integrate the L-RNA aptamer with peptide to form L-RNA aptamer-peptide conjugate via different ligation methods to improve its nuclear permeability and localization ability, and study the effect of L-RNA aptamer-peptide conjugate in regulating G4-protein interaction and G4-associated gene activity, particularly in the nucleus. Our group have all the required apparatus and skillsets to complete the objectives in this proposal, and the novel approach and aptamer developed in this project, such as the aptamer selection method, the L-RNA aptamer sequence and L-RNA aptamer-peptide conjugate, will be patent-worthy.Specific G4 targets of interest include the G4s at the promoter of human c-kit and c-myc that are linked to cancers, and the G4 at the human telomere, which is related to telomere maintenance and genome instability. Other biological relevant G4s will also be investigated. Our data from pilot experiments are promising, and our group will continue to work meticulously together to ensure successful execution of this well-timed and exciting project. We expect the outcomes of this project will generate new and important insights into the understanding and manipulation of G4s’ roles in biology.