Strategies for Characterization of Enzymatic Nucleic Acids

Abstract

Practical application of enzymatic nucleic acids has received more attention in recent years. Understanding the mechanism of catalysis and availability of information on potentials and limitations of these enzymes expands their application scope. A general approach for characterization of functional macromolecules including enzymatic nucleic acids is to perturb a specific set of condition and to follow the perturbation effect by biophysical and biochemical methods. This chapter reviews several perturbation strategies for functional nucleic acids, including deletion, mutation, and modifications of backbone and nucleobases, and consequent kinetic analysis, spectroscopic investigations, and probing assays. In addition to single point mutation and modifications, different combinatorial approaches for perturbation interference analysis provide reliable high amounts of data in a time-effective manner. The chapter compares various combinatorial perturbation interference analysis methods, that is, combinatorial mutation interference analysis (CoMA), nucleotide analogue interference mapping for RNA and DNA (NAIM and dNAIM), chemical and enzymatic combinatorial nucleoside deletion scanning (NDS), and dimethyl sulfate interference (DMSi).

Calibration of non-amplified nucleic acid quantitative fluorometer

“…When combined with stranded DNA, fluorescent dyes can increase the signal by tens to hundreds of times compared to single-strand DNA or RNA. [8] Therefore, by measuring the fluorescence intensity in the sample under excitation light, the concentration of double stranded DNA in the sample can be determined. The relationship between fluorescence and the concentration of dsDNA can be demonstrated in Figure 1.…”