Gas-phase Dissociation of homo-DNA Oligonucleotides

Abstract

Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS(3) of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series.

More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

“…The following highly charged homoDNA sequences (CL 100%) were investigated by MS/MS: ccggtt (H1) and ttccgg (H2) as well as the mixmer cacGT (HX) (lower case nucleobases indicate homoDNA-modified building blocks). The product ion spectra of H1 and H2 are both characterized by extensive NCOloss (Supplemental Figure 6), accompanied by additional base loss signals, which is in agreement with a recently published study [42]. In general, release of cyanate seems to be even more prevalent for homoDNA than for unmodified DNA.…”

Review of fragmentation of synthetic single‐stranded oligonucleotides by tandem mass spectrometry from 2014 to 2022

“…Furthermore, they observed that proton transfer is common, even at low‐charge states, which can involve, mostly, phosphate‐to‐phosphate but also, less commonly, phosphate‐to‐nucleobase transfer. This observation of proton transfer phosphate‐to‐nucleobase could confirm different mechanisms such as of Wang et al 49 or Stucki et al 53 where the proton is transferred from phosphate to a nucleobase.…”

“…Higher‐energy collision dissociation (HCD) was commercialised by Thermo Scientific but operates in the same manner as off‐resonance CID and is thus essentially a synonym. The first publications of CID (both off‐ and on‐resonance) of oligonucleotide ions (both DNA and RNA) 39 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 demonstrated that, in general, a‐B and w ions are obtained for DNA and c and y ions for RNA with reduced loss of base due to the presence of 2′‐OH, which stabilises the N ‐glycosidic bond on the nucleobase. 54 The formation of a‐B and w ions can follow several different pathways, as summarised by Wu and McLuckey 55 and Monn and Schürch.…”

DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

“…For succinctness, the oligonucleotide structure in Scheme 2 is abbreviated to three nucleotides and we only show the interaction of a purine at nucleobase position two (from the 5′ terminus) with the 5′ phosphate group, although purines at other positions can also follow the same mechanism. The first step in the formation of [phosphopurines] -may proceed via a mechanism similar to one recently proposed by Stucki et al in a study of homo-DNA oligonucleotides [47]. First, a lone pair of electrons from the ether oxygen (of the deoxyribose sugar) initiates cleavage of the purine base from the sugar backbone and subsequent nucleophilic attack of the 5′ phosphate group to form a phosphoamide tetrahedral transition species (Scheme 2, Step 1).…”