On the mechanism of RNA phosphodiester backbone cleavage in the absence of solvent

Abstract

Ribonucleic acid (RNA) modifications play an important role in the regulation of gene expression and the development of RNA-based therapeutics, but their identification, localization and relative quantitation by conventional biochemical methods can be quite challenging. As a promising alternative, mass spectrometry (MS) based approaches that involve RNA dissociation in ‘top-down’ strategies are currently being developed. For this purpose, it is essential to understand the dissociation mechanisms of unmodified and posttranscriptionally or synthetically modified RNA. Here, we have studied the effect of select nucleobase, ribose and backbone modifications on phosphodiester bond cleavage in collisionally activated dissociation (CAD) of positively and negatively charged RNA. 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.

Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

“…We instead attribute this phenomenon to charge redistribution within fragment ions by proton transfer according to Coulombic repulsion, [18] after backbone cleavage but before complex dissociation, that can weaken or strengthen individual interactions differently in different fragment ions,which in turn affects the competition between complementary c and y fragments for tat. In this scenario,anelectrostatic interaction of residue 6ofc 6 with tat can be sufficiently strong to compete with the interactions between the complementary fragment y 25 and tat, whereas in the c 7 , c 8 ,a nd c 9 ions,n egative charge will move away from residue 6t ot he terminal residues 7, 8, and 9, respectively, [18] thus weakening the electrostatic interaction of residue 6with tat. This does not exclude the possibility of tat interactions with residues 7, 8, and 9b ut suggests that they must be substantially weaker than that between tat and residue 6ofc 6 .…”

Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

“…[17] Thel ower-energy mechanism of phosphodiester backbone bond cleavage into c and y ions (Supporting Information, Figure S2), on the other hand, does not involve nucleobase loss. [18] Site-specific yields of c and y ions were generally higher on the 5'-side of guanosine (Figure 2a), which we have recently attributed to the hydrogen bonding between nucleobase and phosphodiester moieties at the cleavage site prior to dissociation. [18] Peptide binding somewhat reduces this effect (Supporting Information, Figure S3), consistent with the restricted conformational RNAf lexibility in the bound state.…”

“…We instead attribute this phenomenon to charge redistribution within fragment ions by proton transfer according to Coulombic repulsion, [18] after backbone cleavage but before complex dissociation, that can weaken or strengthen individual interactions differently in different fragment ions,which in turn affects the competition between complementary c and y fragments for tat. In this scenario,anelectrostatic interaction of residue 6ofc 6 with tat can be sufficiently strong to compete with the interactions between the complementary fragment y 25 and tat, whereas in the c 7 , c 8 ,a nd c 9 ions,n egative charge will move away from residue 6t ot he terminal residues 7, 8, and 9, respectively, [18] thus weakening the electrostatic interaction of residue 6with tat. This does not exclude the possibility of tat interactions with residues 7, 8, and 9b ut suggests that they must be substantially weaker than that between tat and residue 6ofc 6 .…”

Radical Transfer Dissociation for De Novo Characterization of Modified Ribonucleic Acids by Mass Spectrometry

“…Moreover, CAD of (M + Co III (NH 3 ) 6 Àn H) (nÀ3)À ions of RNA 2 (which lacks 2'-OH groups at positions 6-9, Table 1) produced virtually no c and y fragments from cleavage at sites 6-9 ( Figures S3 and S4), which agrees with the established nonradical mechanism for RNA dissociation into c and y fragments that involves the 2'-OH group. [4] The steep increase in the number of ions resulting from loss of (5 NH 3 + CNH 2 ) in CAD of (M + Co III (NH 3 ) 6 À9 H) 6À ions in the energy range 45-57 eV coincided with a steep increase in the number of d and w fragments. However, above 57 eV, the number of c and y fragments increased, whereas the number of d and w fragments decreased (Figure 2 A).…”