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bioRxiv · 10.64898/2026.07.30.741700

Sequence-Specific Targeting of GC-Rich Gene Loci by Parallel Triplex-Forming Oligonucleotides Containing a Modified Nucleobase

Rusling, D. A.·Ma, R.·Brazzill, M.·Buckham, N.·Justice, D.·Chen, C.·Hoshika, S.·Benner, S. A.
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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 towards 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. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/741700v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ee4ad6org.highwire.dtl.DTLVardef@173299eorg.highwire.dtl.DTLVardef@1b14d05org.highwire.dtl.DTLVardef@1379384_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Rusling, D. A., Ma, R., Brazzill, M., Buckham, N., Justice, D., Chen, C., Hoshika, S., Benner, S. A.. 2026-07-30. Sequence-Specific Targeting of GC-Rich Gene Loci by Parallel Triplex-Forming Oligonucleotides Containing a Modified Nucleobase. https://doi.org/10.64898/2026.07.30.741700

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