Development of new single-molecule techniques

As molecular systems of interest become more complex, conventional single-molecule technique is insufficient to investigate the complex biomolecular dynamics, and there is an ever increasing demand for more advanced techniques.

Hence, we aim to develop several single-molecule techniques with increased information contents, a high sensitivity, and  expanded scope.

This new method will allow us to solve many biologically important problems.

Various molecular machines in biology

By using our cutting-edge single-molecule techniques, we aim to reveal mechanisms underlying operations of various biologically important molecular machines as listed below.

Target recognition and cleavage mechanisms of type II topoisomerases

The double helical nature of DNA imposes intrinsic topological problems during DNA transactions such as replication, transcription, and repair. Type II topoisomerases are essential enzymes that resolve these problems. Decades of research have established that these ATP-dependent molecular machines operate by transporting a DNA duplex (the transport or T-segment) through a transient break in another DNA duplex (the gate or G-segment). Therefore, the DNA cleavage/ligation and opening/closing reactions of the enzyme are central to its catalytic function. However, we still have a poor understanding as to how this potentially dangerous, multi-step process is regulated, and integrated into a well-coordinated catalytic cycle. 

Target searching, recognition, and cleavage mechanisms of CRISPR-Cas DNA endonucleases

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) DNA endonuclease is a key enzyme of an adaptive immune response in bacteria and archaea. As part of its cellular functions, the enzyme recognizes and cleaves target dsDNA with a guide RNAs. This RNA-guided nuclease activity of Cas endonucleases has been harnessed for genome editing, but the regulation mechanism is not well understood.

Constructing next-generation type V CRISPR-Cas tools from structural dynamics

Class II Type V CRISPR-Cas system has emerged as attractive molecular scissors alternative to Cas9 owing to its unique features including fewer off-target effect, and alternative PAM sequence, pre-crRNA processing activity, and smaller gene size. Despite these advantages, Type V CRISPR-Cas nuclease has not been harnessed as recently reported next-generation genome editing tools: base and prime editors, because it does not have complete nickase variants unlike Cas9. The development of a novel strategy for type V CRISPR-Cas nuclease engineering including the complete nickase, requires a thorough understanding of the mechanism that govern the generation of complete double-stranded DNA breaks by the single catalytic site. To achieve this goal, we have used cutting-edge single-molecule fluorescence techniques. This would improve our ability to develop a rational design for more potently engineered type V CRISPR-Cas nuclease including the nickase form, extending the range of applications of type V CRISPR-Cas genetic scissor in the future.