ABSTRACT
A new experiment for the measurement of nJ(C,P) coupling constants along the phosphodiester backbone in RNA and DNA based on a quantitative-J HCP experiment is presented. In addition to coupling constants, in which a carbon atom couples to only one phosphorus atom, both the intraresidual 3J(C4'i,Pi) and the sequential 3J(C4'i,Pi+1) for the C4' resonances that couple to two phosphorus atoms can be obtained. Coupling constants obtained by this new method are compared to values obtained from the P-FIDS experiment. Together with 3J(H,P) coupling constants measured using the P-FIDS experiment, the backbone angles ß and ∈ can be determined.
ABSTRACT
Synthetic oligoribonucleotides up to 22 bases have been sequenced by observing different kinetics in exonuclease-induced phosphodiester bond hydrolysis and detecting the fragments by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Common mass spectrometric sequencing methods have disadvantages concerning read length, cost, and specialist instrumentation using RNA as the target molecule because uridine and cytidine have similar masses. Now we are in the position to distinguish U and C by different peak intensities. The method proposed has been verified using specific endonucleases and 13C-labeled nucleotides. This new nongel-based and nonlabeling sequencing strategy offers first RNA sequencing data using the advantages of fast and accurate MALDI-TOF-MS. Preparation steps and measurements are performed in less than 1 h.
