Secondary Structure Characterization of pH6DZl, a Fluorescence Signaling and RNA Cleaving DNA Enzyme

Abstract

<p> pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein we report on the sequence truncation and secondary structure characterization ofpH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three inter-helical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides. Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides. Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. A trans-acting DNA enzyme was also developed from the minimized version ofpH6DZl, which behaves as a true enzyme with a kcat value of~1 min"1 and generates a large fluorescence signal upon catalysis. This study should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors. </p>