Debranchase-resistant labeling of RNA using the 10DM24 deoxyribozyme and fluorescent modified nucleotides

Abstract

The 10DM24 deoxyribozyme can site-specifically label RNAs with fluorophore-GTP conjugates; however, the 2′,5′-branched RNA linkage is readily cleaved by debranchase. To prevent loss of labels upon cleavage, we synthesized phosphorothioate-modified, fluorescent GTP derivatives and elaborated conditions for their incorporation by 10DM24. RNAs labeled with fluorescent derivatives of Sp-GTPS were found to be resistant to debranchase.

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

“…Moreover, upon incubation with yeast debranching enzyme Dbr1, the phosphonate linkage in the FJ1 product was significantly more stable compared to the phosphodiester produced by the FH14 ribozyme with N 6 ‐biotin ATP. Under the in vitro conditions with highly active recombinant Dbr1 enzyme, the half‐life was 4–8‐fold increased (Figure b).…”

“…N 6 ‐modified ATP analogues would compete with cellular ATP and ATP‐dependent enzymatic processes, and result in ribozyme‐independent background labeling of RNA by polymerase incorporation . Moreover, the resulting 2′,5′‐phosphodiester linkage in the labeled RNA is easily cleaved by natural debranching enzymes . We sought to address these issues of lacking orthogonality and vulnerability to debranching enzymes by attachment of non‐natural nucleotide analogues.…”

“…Importantly, the resulting phosphonate linkages proved more stable towards yeast debranching enzyme (Dbr1) compared to the previously used natural phosphate linkages. Other chemical modification such as thiophosphates or thiophosphonates could further increase the half‐lives . In addition, it is worthwhile to continue searching for other ribozymes that can form bioorthogonal linkages completely resistant to enzymatic hydrolysis.…”

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

“…Other chemical modification such as thiophosphates or thiophosphonates could further increase the half-lives. [10] In addition, it is worthwhile to continue searching for other ribozymes that can form bioorthogonal linkages completely resistant to enzymatic hydrolysis. The currently available toolbox of orthogonal ribozymes allows the installation of FRET pairs on a single RNA, and to address multiple different RNAs in one experiment with different labels, simply by the corresponding design of the Watson-Crick binding arms of FH and FJ ribozymes.…”

“…[9] Moreover, the resulting 2',5'phosphodiester linkage in the labeled RNA is easily cleaved by natural debranching enzymes. [10] We sought to address these issues of lacking orthogonality and vulnerability to debranching enzymes by attachment of non-natural nucleotide analogues. Specifically, we considered tenofovir diphosphate as a ribozyme substrate.…”