Strand-Cleaving Deoxyribozymes Enable the Mid-Down Sequencing of mRNA by Mass Spectrometry with No Front-End...

We evaluated the possible application of RNA-cleaving deoxyribozymes to control the number and size of cleavage products obtained during the mid-down characterization of larger nucleic acids by mass spectrometry (MS). We assessed the structural determinants of substrate selectivity, as well as the effects of cofactor, additives, and environmental parameters on the cleavage activity of the histidine-dependent deoxyribozyme 3 (HD3). We designed dedicated sets of HD3 variants capable of cleaving RNA strands as large as the 758 nt enhanced green fluorescent protein (eGFP) mRNA. This substrate was dissected into only 10 oligonucleotides with lengths ranging from 46 to 120 nt, which compared favorably with the 87 unique products ranging from 3 to 14 nt to be expected instead from putative RNase T1 digestion. The complexity of such mixture was sufficiently limited to enable its comprehensive analysis by direct infusion nanospray-MS without front-end separation. Their unambiguous assignment was accomplished by matching experimental with predicted masses, which revealed the formation of mis-cleaved products and enabled the mass mapping of 100% of the initial substrate. Product identity was verified by collision-induced dissociation (CID), which provided individual sequence coverages ranging from 87 to 100%, with the lower figure obtained from a 120 nt mis-cleaved product. All sequence information combined accounted for 95% coverage of the 758 nt substrate, but without all the ambiguities engendered by classic nucleotide-specific endonucleases. This outcome demonstrated the feasibility of mid-down approaches based on deoxyribozymes and underscored their potential in the analysis of larger RNAs.