Single Molecule Bio-Spectroscopy: Deoxyribozyme Folding, Genetic Assay, and Observing Enzymatic Reactions

We have developed two novel single molecule spectroscopic techniques to monitor structures and dynamics of biological macromolecules; 3-color alternating laser excitation (ALEX) FRET and total internal reflection fluorescence (TIRF). With 3-colr ALEX FRET, we examined the folding of 8-17 deoxyribozyme, a member of synthetic enzymes composed of DNA whose catalytic functions include RNA/DNA cleavage, DNA ligation and modification. We found that the stem loop region of 8-17 deoxyribozyme played a critical role in obtaining the right structure and hence the proper chemical activity of the enzyme in Mg²⁺ -induced folding. Another application is single molecule detection of single nucleotide polymorphism (SNP), where the color pair identity of, and interprobe distance between, reference and probe DNAs differently labeled for each type of DNA bases help uniquely distinguish all 4 nucleobases at each SNP site.

In our TIRF study for the enzymatic reaction of 10-23 deoxyribozyme, which can cleave almost any RNA substrate under ion-, temperature-, and pH-dependent conditions, a particularly interesting case was demonstrated where the entire process of an enzymatic reaction was detected for the first time in real time at the single molecule level. We were also able to directly measure the RNA cleavage rate while varying temperature and Mg²⁺ ion concentration through real-time monitoring of binding between 10-23 deoxyribozyme and DNA substrate with dual RNA.