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 elucidated the physical basis for the lattice phase transition triggered by ligand and discovered a fundamental phenomenon called molecular synchronization in crystal lattices. This project is fundamental to the visualization of molecular movies. The main technology and tool for this project is the X-ray free electron laser (XFEL), which is located in Meno Park, Ca. This part of the work has been published in four manuscripts, including a paper in Nat. Comm. within this FY. It is noteworthy to mention that my lab has established the research program using XFEL to study RNA structural dynamics, from sample prep, micron/nanocrystal characterization, data collection, data analysis in situ and post-data-acquistiion, computing, and interpretation. Thus we are in the position to take full advantage of this new technology, XFEL, to explore the structural dynamics and conformational space of RNA. Unfortunately, the travel ban due to the ongoing pandemic has prevented us from traveling to the site to perform experiments, preventing us from collecting data for the past 16 months and making any further progress challenging. As a result, I have made a conscientious and strategic change in our program. Instead of relying on the XFEL, the resource that we have no control over and is highly susceptible to interruption, given the current circumstance of non-travel, we were forced to adopt different technologies to carry out and fulfill our research goal of exploring RNA structural dynamics and conformational space. Second, 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 molecules in solution; 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). A number of manuscripts are at various stages of preparation for publication. Third, we are actively pursuing research of using riboswitch devices to control gene expression externally with the aim of a potential clinic application. Specifically, we aimed at control the expression of the pd-1 gene that 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. The devices are engineered in a chromosome of T-cells using the CRISPR/Cas 9 technique. In the past year, we have made meaningful progress in this endeavor. Briefly, we have established a cell culture and characterization room and procedures using our annually allocated resources; created the stable pd-1 knockout cell line for control; generated a stable cell line with a riboswitch device engineered in the 3' UTR of the pd-1; developed a software/program to randomly scramble sequences in key regions of riboswitch devices for high-throughput screening for efficient devices in mammalian cells. The progress is in addition to the previous work completed in the last FY during which we have designed about 40 different RNA devices that have near ideal kinetic characteristics for potential clinic applications.