Unraveling DNA Triplex Assembly: Mass Spectrometric Investigation of Modified Triplex Forming Oligonucleotides for Enhanced Gene Targeting

Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors inc...

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Bibliographic Details
Published in:	Journal of the American Society for Mass Spectrometry Vol. 35; no. 9; pp. 2056 - 2063
Main Authors:	Chandrasegaran, Sarveenah, Klose, Jack W., Pukala, Tara L.
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 04-09-2024
Subjects:	Base Sequence DNA - chemistry DNA - genetics DNA, Bacterial - chemistry DNA, Bacterial - genetics Gene Targeting - methods Mass Spectrometry - methods Nucleic Acid Conformation Oligonucleotides - chemistry Pseudomonas aeruginosa - genetics
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Summary:	Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors including the length and composition of bases in the sequence. Here, different DNA base modifications have been explored, primarily using native mass spectrometry, in efforts to enable stronger binding between the triplex forming oligonucleotide (TFO) and duplex target sites. These modifications can also be used to overcome pyrimidine interruptions in the duplex sequence in promoter regions of genomes, to expand triplex target sequences for antigene therapies. Using model sequences with a single pyrimidine interruption, triplex forming oligonucleotides containing locked nucleic acid base modifications were shown to have a higher triplex binding propensity than DNA-only and dSpacer-containing TFOs. However, the triplex forming ability of these systems was limited by the competitive formation of multiple higher order assemblies. Triplex forming sequences that correspond to specific gene targets from the Pseudomonas aeruginosa genome were also investigated, with LNA-containing TFOs the only variant able to form triplex using these sequences. This work indicates the advantages of utilizing synthetically modified TFOs to form triplex assemblies in vivo for potential therapeutic applications and highlights the advantages of native mass spectrometry for the study of their formation.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1044-0305 1879-1123 1879-1123
DOI:	10.1021/jasms.4c00070