Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry

Abstract

Oligoribonucleotides (RNA) and modified oligonucleotides were subjected to low-energy collision-induced dissociation in a hybrid quadrupole time-of-flight mass spectrometer to investigate their fragmentation pathways. Only very restricted data are available on gas-phase dissociation of oligoribonucleotides and their analogs and the fundamental mechanistic aspects still need to be defined to develop mass spectrometry-based protocols for sequence identification. Such methods are needed, because chemically modified oligonucleotides can not be submitted to standard sequencing protocols.In contrast to the dissociation of DNA, dissociation of RNA was found to be independent of nucleobase loss and it is characterized by cleavage of the 5=-POO bond, resulting in the formation of c-and their complementary y-type ions. To evaluate the influence of different 2=-substituents, several modified tetraribonucleotides were analyzed. Oligoribonucleotides incorporating a 2=-methoxy-ribose or a 2=-fluoro-ribose show fragmentation that does not exhibit any preferred dissociation pathway because all different types of fragment ions are generated with comparable abundance. To analyze the role of the nucleobases in the fragmentation of the phosphodiester backbone, an oligonucleotide lacking the nucleobase at one position has been studied. Experiments indicated that the dissociation mechanism of RNA is not influenced by the nucleobase, thus, supporting a mechanism where dissociation is initiated by formation of an intramolecular cyclic transition state with the 2=-hydroxyl proton bridged to the 5=-phosphate oxygen. A ntisense oligonucleotides are nucleic acids of about 12 to 20 nucleobases, which are designed to hybridize to a complementary messenger RNA (mRNA) sequence, thus, inhibiting gene expression [1][2][3]. Therefore, they are of great interest in human cancer therapy and for diagnostic applications. Besides the high binding specificity of antisense oligonucleotides to their target mRNA, affinity, bioavailability, and biostability are of foremost importance. However, a major drawback of the application of unmodified phosphodiester DNA or RNA oligonucleotides is that these structures are subjected to rapid nuclease degradation under physiological conditions. To improve these factors, oligonucleotide analogs, exhibiting chemical modifications of the phosphodiester backbone, of the ribose, and, to a limited extent, also of the nucleobases, are evaluated.Mechanisms of inhibition of gene expression by antisense oligonucleotides involve mRNA degrading enzymes such as RNase H, blockade of translation initiation, or modulation of splicing [4][5][6][7]. These mechanisms allow the application of new chemical modifications to increase the binding affinity and the nuclease resistance. Possible positions of the modifications are the phosphodiester backbone, the sugar unit and the nucleobases. Introduction of a phosphorothioate backbone increases nuclease resistance of antisense oligonucleotides and simultaneously serves as a very efficient su...

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

“…The methoxy group at the 2′-position does not provide a bridging oxygen for the formation of a cyclic structure, thus the generation of c-and y-ions should be inhibited according to the published mechanism [29]. As this stands in contrast to our experimental data, there must be an alternative dissociation pathway accessible for 2′-O-methyl oligoribonucleotides.…”

“…Since the ON is fully 2′-modified, the presence of uninterrupted series of c-and y-ions is remarkable, in the sense that the generally accepted mechanism for dissociation of unmodified RNA by Tromp and Schürch does not apply [28,29]. The mechanism assumes the 2′-hydroxyl substituent to induce cleavage of the 5′-P-O bond, which leads to cand y-fragment ions, whereas the c-ion rearranges to the corresponding 2′-3′-cyclic phosphate.…”

“…In contrast to DNA, RNA tends to form c-and y-ions under CID conditions [27][28][29]. This difference is caused by the influence of the 2′-OH group, which participates in a proton transfer reaction leading to a cyclic c-ion.…”

RNA fragmentation in MALDI mass spectrometry studied by H/D-exchange: Mechanisms of general applicability to nucleic acids

“…These authors suggested a 5=-POO cleavage mechanism involving the bridging of the 2=-OH proton to 5=-PO oxygen. In a recent study of the fragmentation of doubly deprotonated RNA tetramers, Tromp and Schürch [12] found that a 5=-POO bond was cleaved to a similar extend in absence of a 3=-adjacent nucleobase (replaced by hydrogen). They concluded that the nucleobase did not play a key role in 5=-POO cleavage.…”

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

“…This ion pair is formed in a two-step mechanism triggered by the loss of a nucleobase [9]. In RNA, the primary fragmentation products are c-and y-ions, which has been explained by an alternative fragmentation mechanism involving the 2'-OH group [10]. Moreover, some of the synthetic modifications introduced in antisense oligonucleotides are also known to alter the gas-phase dissociation mechanism.…”