Search for the Structural Basis of Biomacromolecular Function and Activity

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

My lab has made progress on several fronts. First, we have developed a novel algorithm and a method using AFM to study RNA conformational dynamics in solution. Briefly, we are now able to directly visualize individual RNA conformers in solution and determine the structures of individual RNA molecules; compute the total conformational space of RNA in solution. RNA molecules are highly dynamic and conformational-heterogeneous. This development is significant because it makes it possible to characterize individual molecules of heterogeneous conformations, such as RNA in solution, as opposed to an ensemble of molecules of homogeneous conformation. We have tested, bench-marked and applied our new approach and method in studying the RNA structural dynamics and conformational space in a number of important RNA molecules in solution. These include the HIV packaging signal RNA, Rev response element (RRE) RNA, the T-box riboswitch with/without tRNA ligand, cobalamine riboswitch RNA w/wo ligand, the 3' and 5'-UTR RNA of the COVID-19 and the RNaseP RNA (both the full-length and core particle). Three significant manuscripts are either under review or to be submitted. Second, we have demonstrated the feasibility of using RNA devices to control and regulate the PD-1 gene expression in mouse EL4 cells. The PD-1 gene is one of the critical genes for cancer immunotherapy. Thus this project is potentially translational. The basic idea is to use externally controllable RNA devices that are responsive to ligand bindings. We purposefully choose an FDA-approved ligand. The devices are engineered in a chromosome of T-cells using the CRISPR/Cas 9 technique. Built on the progress in the last year, now we have established the procedure and protocol to quantify the PD-1 expression at various ligand concentrations using both Western and qPCR methods. We have also obtained information on the kinetic characteristics of some of the RNA devices in cell. We are currently performing high-throughput screenings using lenti-libraries with the aim to identify the best RNA devices that are both of high efficiency and ideal kinetic characteristics in mammalian cells. Furthermore, we have crystallized one of the RNA devices in both presence and absence of ligand and thus opened the door for high-resolution structure determination. It is noteworthy to mention that the structure of any RNA devices has never been determined before. The high-resolution structure of an RNA may lead us to a better understanding of the ligand-triggered conformation changes at the atomic level and stimulate new designs of more efficient RNA devices. Lastly, we have made significant progress in improving the PLOR technology (Liu et al., Nat. 2015) using high-capacity DNA template attachedbeads. Our aim is to be able to synthesize kilo-base long mRNA with selectively-labeled or modified residues placed at desired positions. One of the applications of the improved PLOR could be manufacturing mRNA selectively labeled with modified pseudo-uridines, as opposed to the current uniform labelings such as mRNAs in the COVID-19 vaccines by Pfizer or Moderna.