Decoding Complexity in Biomolecular Recognition of Dna I-Motifs

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 10,976 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 expressionin 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.

DNA i-motif levels are overwhelmingly depleted in living human cells: insights from in-cell NMR

“…Using the hRAD17 model as a paradigm, we showed that iMFPS displaying occur in cells as a mix of folded (iM) and unfolded states; we showed that the populations of folded (iM) and unfolded states differed between the M and early S phases of the cell cycle. Consistent with the existing literature, 14,17,41,44 we observed that our iMFSP models introduced into cells interacted with endogenous proteins.…”

“…It is essential to emphasize that all the scenarios outlined regard that iM formation, accounting for coil-to-iM and iM-to-coil transitions in chromatin context, is a consecutive (subordinate) step to competing processes related to strand separation (i.e., duplex-to-coil/coil-to-duplex transitions) and DNA-protein binding. 17,55 Evidence suggests that the chromatin microenvironment controls the duplex-to-coil/coil-to-duplex transitions in a double-stranded chromatin context; Selvam et al 24 observed that decreasing superhelicity in torsionally constrained dsDNA fragments in an acidic solution led to duplex destabilization and an increased formation of iM, consistent with earlier findings by Hurley’s group. 23 Later, the same research team demonstrated that the DNA duplex is also significantly destabilized under confined conditions marked by reduced water activity, 25 aligning with prior observations.…”

DNA i-motif levels are overwhelmingly depleted in living human cells: insights from in-cell NMR.

“…Consistent with the existing literature, 14,17,43,46 we observed that our iMFSP models introduced into cells interacted with endogenous proteins.…”

“…To con rm the protein binding to our constructs, we performed the shift-western blot 42 (WB) analysis of PAGE-resolved lysate prepared from HeLa cells transfected with hTel and hT121-6 (Fig. S7B), using antibodies against three promiscuous and previously described DNA C-rich binding proteins 14,16,17,43 , namely hnRNP K, hnRNP A1, and PCBP2. The WB con rmed the binding of all three proteins to hTel and hT121-6 (Fig.…”

“…Conversely, the potential presence of iM within living organisms, including humans, has been supported by several lines of evidence, including the discovery of iMFPS binding proteins [14][15][16][17] ; thermodynamic stabilization of iM by epigenetic modi cations in vitro 5,[18][19][20] and under conditions imitating a crowded cellular environment 8, 21,22 ; and the promotion of iM formation from double-stranded DNA under conditions emulating mechanistic forces connected with DNA helix unwinding and nanocon nement in the chromatin context. [23][24][25][26] Several indirect ndings also suggested that iM formation may play a role in regulating replication and transcription.…”