De Novo Sequence Determination of Modified Oligonucleotides

Farand, Julie; Gosselin, Francis

doi:10.1021/ac802452p

Cited by 29 publications

(30 citation statements)

References 34 publications

(58 reference statements)

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“…Work on MS-based investigation of modified ONs has been published as well, e.g., on methylphosphonate or 2′-modified ONs [31][32][33][34][35][36]. Farand et al used a chemical and enzymatic digestion approach to degrade random mixtures of 2′-deoxy-, 2′-fluoro-, 2′-O-methyl-, and abasic oligoribonucleotides [32,33].…”

Section: Introductionmentioning

confidence: 99%

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Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

Nyakas

Stucki

Schürch

2011

J. Am. Soc. Mass Spectrom.

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Therapeutic approaches for treatment of various diseases aim at the interruption of transcription or translation. Modified oligonucleotides, such as 2′-O-methyl-and methylphosphonatederivatives, exhibit high resistance against cellular nucleases, thus rendering application for, e.g., antigene or antisense purposes possible. Other approaches are based on administration of cross-linking agents, such as cis-diamminedichloroplatinum(II) (cisplatin, DDP), which is still the most widely used anticancer drug worldwide. Due to the formation of 1,2-intrastrand cross links at adjacent guanines, replication of the double-strand is disturbed, thus resulting in significant cytotoxicity. Evidence for the gas-phase dissociation mechanism of platinated RNA is given, based on nano-electrospray ionization high-resolution multistage tandem mass spectrometry (MS n ). Confirmation was found by investigating the fragmentation pattern of platinated and unplatinated 2′-methoxy oligoribonucleotide hexamers and their corresponding methylphosphonate derivatives. Platinated 2′-methoxy oligoribonucleotides exhibit a similar gas-phase dissociation behavior as the corresponding DNA and RNA sequences, with the 3′-C-O bond adjacent to the vicinal guanines being cleaved preferentially, leading to w x -ion formation. By examination of the corresponding platinated methylphosphonate derivatives of the 2′-methoxy oligoribonucleotides, the key role of the negatively charged phosphate oxygen atoms in direct proximity to the guanines was proven. The significant alteration of fragmentation due to platination is demonstrated by comparison of the fragment ion patterns of unplatinated and platinated 2′-O-methyl-and 2′-O-methyl methylphosphonate oligoribonucleotides, and the results obtained by H/D exchange experiments.

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Section: Introductionmentioning

confidence: 99%

“…Farand et al used a chemical and enzymatic digestion approach to degrade random mixtures of 2′-deoxy-, 2′-fluoro-, 2′-O-methyl-, and abasic oligoribonucleotides [32,33]. Subsequent analysis using ESI tandem mass spectrometry allowed for de-novo sequence determination of the investigated oligonucleotides.…”

Section: Introductionmentioning

confidence: 99%

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

Nyakas

Stucki

Schürch

2011

J. Am. Soc. Mass Spectrom.

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“…The identities of the oligonucleotide-drug and different metabolites can be confirmed by the mass measurements [27,28]. Farand and coworkers have demonstrated that sequence confirmation and de novo sequencing of highly modified oligonucleotides can be achieved via mass spectrometry of chemically degraded oligonucleotides and tandem mass spectrometry analysis of the short fragments [29,30]. However, this method requires additional sample preparation steps and its efficacy on LNA-modified oligonucleotides has not been demonstrated.…”

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confidence: 99%

Ion trap collision-induced dissociation of locked nucleic acids

Huang

Kharlamova

McLuckey

2010

J. Am. Soc. Mass Spectrom.

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Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3=C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics. (J Am Soc Mass Spectrom 2010, 21, 144 -153)

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“…The determination of an unknown chemically modified siRNA by de novo sequencing has also been demonstrated [86]. A combination of chemical degradation and tandem mass spectrometry was first used to sequence the oligonucleotide followed by a nucleoside composition analysis by enzymatic digestion to crossreference the proposed de novo sequence.…”

Section: Sequencing Sirna By Mass Spectrometrymentioning

confidence: 99%