DNA ve RNA Oligonükleotitlerinin Elektrospray İyonizasyon Verimliliğini ve Sıvı Kromatografisi-Kütle Spektrometrisini Geliştirmek İçin Enzimatik Ligasyon Stratejisi

Özet (abstract)

Abstract Mass spectrometry (MS) is a powerful technique for characterizing modified RNA as it directly maps and quantifies all mass-altering modifications simultaneously. However, the physicochemical properties of RNA result in poor ionization efficiencies during electrospray ionization, presenting a major barrier to sensitive MS measurements necessary for low-abundance RNA samples and RNAs with low modification stoichiometries. Here, we report a ligation-based approach to increase ionization efficiencies of RNA oligonucleotides. We show that short (∼5 nt), chemically modified DNA oligonucleotides can be enzymatically ligated to RNA to serve as MS signal enhancers. Among a series of signal enhancers appended with various alkyl and alkylimidazolium functional groups, we found that decyl-functionalized derivatives improved MS sensitivity by ∼15-fold compared to the unlabeled oligonucleotide. When ligated to RNA standards, the decyl-modified signal enhancer increased MS signals by an average of 2.1-fold with the additional benefit of improved retention during liquid chromatography (LC) separations without ion-pairing agents. To apply the ligation-based approach to RNase T1 digests of longer RNAs, a multi-step enzymatic approach was optimized to maximize ligation efficiencies. We then ligated signal enhancers to a yeast transfer RNA (tRNA) digest and observed increased MS signals for numerous sequence-informative digestion products. Importantly, the sequences of RNA oligonucleotides ligated to signal enhancers were readily determined by tandem mass spectrometry with collision-induced dissociation. This ligation-based strategy for enhancing LC-MS/MS characterization of RNA creates opportunities to measure low-abundance RNA samples and their modifications.

Yazarların özeti; kaynağından alınmıştır. Analytical Chemistry, 2026 · DOI ↗