Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

Abstract

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.

Staphylococcus aureusCas9 is a multiple-turnover enzyme

“…The DNA did not have any other occurrences of either the target sequence or the PAMs (Table 1, DNA1) and was labeled with a 5’-FAM fluorophore on the PAM-containing strand (cleaved by the RuvC domain) and a 5’-ROX fluorophore on the non-PAM strand (cleaved by the HNH domain). Reaction aliquots were quenched with a final concentration of 50 mM EDTA, 1% SDS, and 0.1 units/μL of Proteinase K and analyzed by capillary electrophoresis (CE) (Greenough et al, 2016). The fraction of DNA cleaved was plotted versus time, and fit with a single exponential equation describing the observed rate (k obs ) and extent of DNA cleavage in the presence of a particular concentration of sgRNA (Fig.…”

Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

“…Reactions were quenched by addition of 8-fold molar excess of EDTA relative to the Mg 2+ present. Immediately following EDTA addition, DNA was purified by using a Monarch® PCR&DNA cleanup kit (NEB #T1030) before analyzing the DNA by capillary electrophoresis (30), PAGE, or next generation sequencing. DNA substrates used for digestion reactions were either generated by PCR using Q5® High-Fidelity 2X Hot Start Master Mix (NEB #M0494), oligonucleotides produced by IDT, or commercially available products such as M13mp18 single stranded DNA (NEB #N4040).…”

Recent Methods for Purification and Structure Determination of Oligonucleotides

“…The ratio of acrylamide to bisacrylamide affects the cross-linking frequency of the polyacrylamide mesh. For example, an increase in bisacrylamide concentration from 3.3% (acrylamide:bisacrylamide = 29:1 ( v / v )) to 5% (acrylamide:bisacrylamide = 19:1 ( v / v )) results in a decrease of the pore size, thus leading to a shift in the separation range toward smaller oligonucleotides [22]. A further increase in the concentration of bisacrylamide leads to an increase of the pore sizes because of non-uniform chain cross-linking.…”