Deoxyribozyme-catalyzed labeling of RNA.

Site-specific covalent modification of RNA is important for enabling structure–function studies. For example, probes such as fluorescein are commonly used in fluorescence resonant energy transfer (FRET) investigations of RNA folding. Biotin is used for immobilization during single-molecule analysis, to enable RNA–protein cross-linking studies, and as a key element of selection schemes in vitro. The 5’ and 3’ termini of RNA may be derivatized, but many experiments instead demand internal modification, and no direct methods are known for site-specific modification within an arbitrary RNA sequence. Therefore, covalent modifications are typically introduced by enzymatic splint ligation in which a DNA template aligns oligoribonucleotide substrates that have modified nucleotides incorporated through solidphase synthesis. However, this approach often suffers from low yields and is unpredictable because identifying a highyielding ligation site in the target RNA can be difficult without directly testing several possibilities. Non-natural nucleotides have recently been used to transcribe modified RNA. Although this avoids the difficulties of splint ligation, extensive organic synthesis is required. As an alternative approach to RNA labeling, noncovalent Watson–Crick hybridization of a probe-labeled oligonucleotide has been used. However, this is invasive because long stretches of nucleotides must be inserted within the RNA, and duplex formation involving these inserted nucleotides must be tolerated. These limitations have led us to develop a general deoxyribozyme-based strategy for site-specific RNA modification. Deoxyribozymes are catalytic DNA molecules identified by in vitro selection, and our laboratory has reported several deoxyribozymes that ligate two RNA substrates. Herein, we have applied the 10DM24 deoxyribozyme in a new approach for site-specific internal RNA modification that we term deoxyribozyme-catalyzed labeling (DECAL). A single 5-aminoallylcytidine nucleotide is incorporated at the second position of a short “tagging RNA” by in vitro transcription (see the Supporting Information for all experimental procedures). The aminoallyl-modified transcript is coupled with the amine-reactive form of a desired biophysical probe to form the labeled tagging RNA (Scheme 1a). The tagging RNA is