Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase

Abstract

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuinecognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.

RNA‐TAG Mediated Protein‐RNA Conjugation

“…A commercial BG-NHS ester was reacted with a commercially acquired preQ1-C6-NH2 probe precursor as previously described (Scheme S1). [21] The resulting small molecule was tested for TGT recognition by inserting it into the 17 nucleotide TAG2 RNA-TAG hairpin substrate, yielding the TAG2-BG modified RNA. The modification was detected via urea PAGE gel shift analysis, where an upward shift in the product band due to an increase in molecular weight was clearly visible (Figure 1).…”

“…[18,19] Previous work in our lab has established enzymatic nucleic acid modification strategies, RNA-and DNA-TAG (transglycosylation at (deoxy)guanosine). [20,21] These technologies rely on the tRNA guanine transglycosylase (TGT) enzyme from Escherichia coli (E. coli), which exchanges a specific guanine for derivatives of the 7-deazaguanine small molecule substrate, prequeuosine1 (preQ1), in nucleic acids containing a cognate hairpin loop. TGTs are tRNA modifying enzymes that are present across all three kingdoms of life, differing slightly in their small molecule and RNA substrate specificities.…”

“…[23][24][25][26][27][28] The more recently developed DNA-TAG technology employs the same enzyme to modify a set of DNA hairpin substrates and has been used to quickly and inexpensively generate fluorescence in situ hybridization (FISH) probe sets and a near-infrared (IR) fluorescent Northern blot (irNorthern) probe. [21] Both RNA-TAG and SNAP-tag technologies rely on enzymes which recognize specific small molecule substrates. We speculated that combining the function of these two enzymatic tools would enable a straightforward, fully enzymatic methodology to site specifically conjugate nucleic acids to proteins.…”