Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification.

Abstract

Several DNA/RNA sequencing strategies have been developed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In the reverse Sanger sequencing approach alpha-thiophosphate-containing NTPs are employed. Sequencing ladders are produced by the subsequent exonuclease cleavage, which is inhibited by the alpha-S-NTP at the 3' terminus. Here the reverse Sanger sequencing of RNA is described. The stability of RNA during the UV-MALDI process is higher relative to DNA, and RNA can be easily synthesized by transcription using bacteriophage RNA polymerase. alpha-S-rNTP was added to the reaction in a ratio of 1:3 to the native rNTPs and was incorporated statistically by the RNA polymerase. Four separate sequence ladders were produced, to avoid the problem of the only 1u mass difference between uridine and cytidine. However, it was shown that RNA transcription does not produce homogeneous transcripts. Therefore isolation of the full-length transcript is required to attain a non-ambiguous interpretation of cleavage spectra. This is achieved by the exclusive immobilization of the full-length transcript on a solid phase. The full-length transcripts were hybridized to magnetic beads, coated with short universal sequences, complementary to the in vitro RNA. After purification and isolation the RNA full-length transcript is cleaved by snake venom phosphodiesterase (SVP) and the obtained sequence ladder is analyzed by MALDI-MS.