A high throughput approach for discovery of catalytic nucleic acids

Abstract

Enzymes are biologic polymers. Catalytic nucleic acids are extremely useful and hence there is constant need for improving and discovering of catalytic molecules for industrial, medical and biotechnological applications. In vitro evolution has established that single-stranded nucleic acids can display substrate dependent catalysis of specific biochemical reactions. These single stranded oligonucleotides are called aptamers. Mass production and qualitative detection of single-stranded deoxyribonucleic acids are essential for each round of successful in vitro evolutionary pathways for high affinity binding or catalytic aptamers. In this work we optimized the asymmetric polymerase chain reaction protocol for mass production and subsequently developed a new assay system for detection and quantification of single-stranded deoxyribonucleic acid on the native TBE gel. Further enzyme assay of asymmetric polymerase chain reaction product reflect the quality of single-stranded nucleic acids present in the bulk reaction. We would further translate the ePCR approach into the most powerful ultrahigh-throughput inexpensive droplet-based microfluidics system for discovery of new catalytic nucleic acid variants having stronger catalytic activity. Isolate variants can further be used in diagnostic assay upon modification of nucleotides for resistance to enzymatic and chemical degradation.