| Research in Ribonucleic acid (RNA) chemistry has grown dramatically with the recognition that RNA can act as enzymes as well as genetic carrier. Yet a lot remains to be known about how RNA utilizes its limited building blocks to manifest diverse structural motifs to support catalysis. The current work addresses two aspects of RNA catalysis: substrate binding and the link between folding and catalytic mechanisms. RNA binding motifs (aptamers) have been selected for flavin adenine dinucleotide (FAD), an essential redox cofactor, from a library of RNA molecules (∼ 1 x 1015 different species) using in vitro selection-amplification process. Distinct RNA architectures were obtained under two different selection conditions: a stem-loop structure and a three-way helical junction structure. Secondary structure of the first aptamer was investigated using comparative sequence analysis, mutational analysis and enzymatic foot-printing. The secondary structure of the second class of aptamers was obtained from comparative sequence analysis and modeling using MFOLD software. Although the first aptamer recognized the redox center of flavin, yet it was unable to differentiate between the two redox forms of FAD but the second aptamer was able to differentiate between FAD and FADH2 based on relative binding assays.; Mechanistic studies of a re-engineered RNA enzyme (ribozyme), hammerhead, were performed to understand its autocatalytic behavior in the presence of auxiliary loop elements. These RNA sequences enhance the ribozyme's cleavage activity in the range of 100 to 3000 min-1 in low concentrations of different divalents, where the ribozyme would otherwise show weak to no activity. These same sequences also confer thermal stability. These tertiary interactions help to populate the active conformation of the ribozyme. Even rearranging base-pairing sequences can alleviate the problem of inactive conformers as seen from kinetic studies of another natural hammerhead ribozyme from cave crickets. Kinetic analysis as a function of pH support a nucleobase-catalyzed acid-base mechanism in these stabilized ribozymes. The role played by magnesium is predominantly structural with evidence of at least one specific metal binding site from competition assays with cobalt hexammine, a structural analog of hexahydrated magnesium. |
