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Peer-reviewedCC BY 4.0Nucleic Acids Research

Sequence-specific targeting of GC-rich gene loci by parallel triplex-forming oligonucleotides containing a modified nucleobase

  • Ruolin Ma1
  • Michael Brazzill1
  • Dylan Justice1
  • Nicholas Buckham1
  • Cen Chen2,3
  • Shuichi Hoshika2,3
  • Steven A Benner2,3
  • David A Rusling1
  1. 1School of Medicine, Pharmacy and Biomedical Sciences, University of Portsmouth , Portsmouth PO1 2DT ,
  2. 2Foundation for Applied Molecular Evolution , Alachua, FL 32615 ,
  3. 3Firebird Biomolecular Sciences LLC , Alachua, FL 32615 ,
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Abstract

Targeting GC-rich gene loci is a major challenge owing to their high duplex stability, repetitive sequence composition, and propensity to adopt alternative DNA structures. Triplex-forming oligonucleotides (TFOs) provide a programmable strategy toward the recognition of GC-rich DNA, but their application is restricted by the limited recognition capabilities of natural nucleobases in a cellular setting. Here, we overcome this barrier using parallel-binding TFOs containing the synthetic nucleobase 6-amino-5-nitropyridin-2-one (Z), which enables pH-independent recognition of G-C base pairs. Using two structurally distinct regulatory elements within the MYC promoter, we show that Z-modified TFOs form stable, sequence-selective triplexes that repress promoter activity by 50%–80% in both episomal reporter assays and at endogenous gene loci. Notably, the greatest repression was observed at a GC-rich quadruplex-forming element that functions as a structural hub for transcription factor recruitment. To our knowledge, this represents the first demonstration that a simple nucleobase modification alone is sufficient to enable parallel-binding TFOs to repress expression of an endogenous gene, establishing a general strategy for targeting GC-rich regulatory elements through programmable DNA recognition.

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Cite

BibTeXRIS
BibTeX
@article{ma2026sequencespecific,
  title = {{Sequence-specific targeting of GC-rich gene loci by parallel triplex-forming oligonucleotides containing a modified nucleobase}},
  author = {Ruolin Ma and Michael Brazzill and Dylan Justice and Nicholas Buckham and Cen Chen and Shuichi Hoshika and Steven A Benner and David A Rusling},
  journal = {Nucleic Acids Research},
  year = {2026},
  month = oct,
  doi = {10.1093/nar/gkag998},
  url = {https://academic.oup.com/nar/article/doi/10.1093/nar/gkag998/8986556},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Sequence-specific targeting of GC-rich gene loci by parallel triplex-forming oligonucleotides containing a modified nucleobase
AU  - Ruolin Ma
AU  - Michael Brazzill
AU  - Dylan Justice
AU  - Nicholas Buckham
AU  - Cen Chen
AU  - Shuichi Hoshika
AU  - Steven A Benner
AU  - David A Rusling
PY  - 2026
DA  - 2026/10/01
JO  - Nucleic Acids Research
DO  - 10.1093/nar/gkag998
UR  - https://academic.oup.com/nar/article/doi/10.1093/nar/gkag998/8986556
AB  - Targeting GC-rich gene loci is a major challenge owing to their high duplex stability, repetitive sequence composition, and propensity to adopt alternative DNA structures. Triplex-forming oligonucleotides (TFOs) provide a programmable strategy toward the recognition of GC-rich DNA, but their application is restricted by the limited recognition capabilities of natural nucleobases in a cellular setting. Here, we overcome this barrier using parallel-binding TFOs containing the synthetic nucleobase 6-amino-5-nitropyridin-2-one (Z), which enables pH-independent recognition of G-C base pairs. Using two structurally distinct regulatory elements within the MYC promoter, we show that Z-modified TFOs form stable, sequence-selective triplexes that repress promoter activity by 50%–80% in both episomal reporter assays and at endogenous gene loci. Notably, the greatest repression was observed at a GC-rich quadruplex-forming element that functions as a structural hub for transcription factor recruitment. To our knowledge, this represents the first demonstration that a simple nucleobase modification alone is sufficient to enable parallel-binding TFOs to repress expression of an endogenous gene, establishing a general strategy for targeting GC-rich regulatory elements through programmable DNA recognition.
SN  - 0305-1048
SN  - 1362-4962
ER  -