We investigate diverse aspects of microRNA -- from (1) the mechanisms of its biogenesis and regulation to (2) the mechanism of its action in RNA interference. We utilize the cutting edge technology of single-molecule fluorescence methods together with single molecule immunoprecipitation. 

 

Mechanism of pri-miRNA biogenesis

 

Since Drosha was first identified by prof. dr. Narry Kim, there have been many novel reports on Drosha. However, the lack of recombinant proteins of Drosha made it difficult to investigate its biochemical properties in depth. We seek to overcome this problem by innovatively borrowing the surface immobilization method from single-molecule approaches. The Drosha protein will be purified via immunoprecipitation and will be brought onto a surface with its cofactors bound. Biochemical experiments will be carried out by introducing substrates of interest while the reaction is being observed in real time via fluorescence at the single molecule resolution.

 

 

Processive translocation of Dicer protein

 

Human Dicer contains a DEAD/H box helicase domain. While Dicer is active even when ATP is not given or ATPase activity is blocked, there has been no good method of addressing the intriguing question as to why the helicase domain is necessary for Dicer, what it does and how it works if there is any function. Single-molecule methods are excellent in studying helicase functions of translocation and unwinding. We will use the single molecule approaches to investigate possible functions of the helicase domain of Dicer. 

 

 

Action of RISC mechanism 

 

Ago protein has received intensive attention from biotechnology companies as well as biologists due to its critical role of recruiting mature miRNAs and recognizing their target mRNAs. We will join the intense effort by introducing the unique advantage of single-molecule methods. 

 

Regulation of microRNA biogenesis by Lin28 and TUT4

 

The biochemical properties of Lin28 protein and its partner TUT4 are just beginning to be revealed. The role of Lin28 as a cofactor of uridylation activity will be studied by single-molecule FRET methods. 

After mastering single molecule methods, you may go outside and show pedestrians your excellent real-time results taken with nanometer resolution. They will say "Wow, what you are doing is gREEEat. Your data are AWEsome." You may ask them whether they are interested in working together. Then, nine out of ten, they will say, "Sorry but your technique won't be useful for my study because my protein is not available in the recombinant form, we don't have any crystal structure, we have no idea what's going on with my system at the molecular level and blah blah blah....."

 

The gap between conventional molecular biology and single molecule methods is huge. The reason is simple -- the single molecule methods are yet not practically useful and not accessible for general biologists. We want to build a solid bridge between the two fields by integrating the advantages of both methods of molecular biology and single molecule studies. The single molecule techniques need to be more readily adaptable to practical systems -- just like immunoprecipitation approaches. They should be useful even when the structural information is missing, for instance, by utilizing computational prediction algorithms. The approach should be more user-friendly and high-throughput as microarray and deep sequencing became available for everyone. The technique of SIMPlex is the basis of all the studies above.

Since the first human genome sequencing done with a laborious sequencing method, the "next-generation" sequencing techniques of Solexa, 454 and SOLiD have dramatically boosted up the sequencing speed. However, the next-generation techniques have intrinsic technical shortcomings that have limited their wide applications. To overcome them, single molecule-based sequencing techniques have been developed as the "third generation" approaches. However, due to different technical glitches, the new methods are still at their early stage with occupying only 1% of the sequencing market despite its great potential. We will develop a new single molecule approach to overcome the limitations of both the next generation and the recently developed single molecule sequencing techniques in collaboration with Seoul National University.