Decoding complexity in biomolecular recognition of DNA i-motifs with microarrays

Abstract DNA i-motifs (iMs) are non-canonical C-rich secondary structures implicated in numerous cellular processes. Though iMs exist throughout the genome, our understanding of iM recognition by proteins or small molecules is limited to a few examples. We designed a DNA microarray containing 10976...

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Bibliographic Details
Published in:Nucleic acids research 2023-12, Vol.51 (22), p.12020-12030
Main Authors: Yazdani, Kamyar, Seshadri, Srinath, Tillo, Desiree, Yang, Mo, Sibley, Christopher D, Vinson, Charles, Schneekloth, John S
Format: Article
Language:English
Subjects:
Chemical Biology and Nucleic Acid Chemistry
DNA - chemistry
G-Quadruplexes
Heterogeneous-Nuclear Ribonucleoprotein K
Humans
Mitoxantrone
Nucleotide Motifs
Oligonucleotide Array Sequence Analysis
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Summary:Abstract DNA i-motifs (iMs) are non-canonical C-rich secondary structures implicated in numerous cellular processes. Though iMs exist throughout the genome, our understanding of iM recognition by proteins or small molecules is limited to a few examples. We designed a DNA microarray containing 10976 genomic iM sequences to examine the binding profiles of four iM-binding proteins, mitoxantrone and the iMab antibody. iMab microarray screens demonstrated that pH 6.5, 5% BSA buffer was optimal, and fluorescence was correlated with iM C-tract length. hnRNP K broadly recognizes diverse iM sequences, favoring 3–5 cytosine repeats flanked by thymine-rich loops of 1–3 nucleotides. Array binding mirrored public ChIP-Seq datasets, in which 35% of well-bound array iMs are enriched in hnRNP K peaks. In contrast, other reported iM-binding proteins had weaker binding or preferred G-quadruplex (G4) sequences instead. Mitoxantrone broadly binds both shorter iMs and G4s, consistent with an intercalation mechanism. These results suggest that hnRNP K may play a role in iM-mediated regulation of gene expression in vivo, whereas hnRNP A1 and ASF/SF2 are possibly more selective in their binding preferences. This powerful approach represents the most comprehensive investigation of how biomolecules selectively recognize genomic iMs to date. Graphical Abstract Graphical Abstract
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkad981