On the mechanism of RNA phosphodiester backbone cleavage in the absence of solvent.
Bottom Line: We found that CAD of RNA is a stepwise reaction that is facilitated by, but does not require, the presence of positive charge.Preferred backbone cleavage next to adenosine and guanosine in CAD of (M+nH)(n+) and (M-nH)(n-) ions, respectively, is based on hydrogen bonding between nucleobase and phosphodiester moieties.Moreover, CAD of RNA involves an intermediate that is sufficiently stable to survive extension of the RNA structure and intramolecular proton redistribution according to simple Coulombic repulsion prior to backbone cleavage into C: and Y: ions from phosphodiester bond cleavage.
Affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria.Show MeSH
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Mentions: Previous studies have shown that CAD of lowly charged (M−nH)n− ions of RNA (∼0.2 charges/nt, 8–76 nt) produces primarily (up to 98%) (23,24) c- and y-type fragments from phosphodiester bond cleavage (Scheme 1) (23,24,27,39,47), and that the number of a- and w-type fragments from alternative C3′–O bond cleavage increases with increasing (M−nH)n− ion net charge (24,39). In this study, we find that CAD of (M+nH)n+ ions of RNA also yields predominantly c and y, but virtually no a and w fragments, even at >0.2 charges/nt, as illustrated for RNA 2 (13 nt) with n = 4 (0.31 charges/nt) in Figure 2. Under conditions that gave ∼50% c and y fragments, using 44 and 72 eV (Figures 2 and 3A) for CAD of (M+4H)4+ and (M−4H)4− ions, respectively, the branching ratio between c, y and a, w fragments was ∼3:1 for negatively and ∼30:1 for positively charged RNA 2; similar behavior was observed for all other RNAs studied. Note that at these energies, 44 and 72 eV, the RNA ions dissociated to a similar extent, ∼75% for all (M+4H)4+ and ∼80% for all (M−4H)4− ions, respectively, such that the 10-fold difference in branching ratio is primarily a result of ion polarity rather than CAD energy.
Affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria.