At pH 6

At pH 6.0, the amplitude of the BLI transmission was similar to that observed in the work of Christetal.(26), that is, close to 9 nm in the presence of 500 nM iMab. conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to NOTCH1 blocks of 23 cytosines in single-stranded DNA, and call for more careful interpretation of results acquired with this antibody. == Graphical Abstract == == Graphical Abstract. == == Intro == i-Motifs (iMs, or i-DNA) are non-canonical, four-stranded secondary structures created by cytosine-rich DNA sequences upon mutual intercalation of hemi-protonated cytosinecytosine foundation pairs (CH+C), mainly at relatively low pH; temperature, ionic strength and the nature of metallic cations may also influence the stability of these constructions (17). iM constructions are known for more than three decades and have been well-characterizedin vitro(1,5,811); however, the debate concerning the presence, persistence, and possible functions of these constructions in cells is still relevant. It has been suggested that iMs play practical tasks as transcription regulators (3,12,13) and, consequently, could symbolize potential drug focuses on for a number of pathologies, including malignancy and neurodegenerative diseases (1316). The controversy regarding the presence of iMs in cells is mainly due to the strong pH dependence of their stability that, with a few exceptions (17,18), PF-3644022 may preclude their persistence at physiological pH and temp (19). To explain this controversy, the living of iM-stabilizing factors present in cells, such as molecular crowding or specific protein partners, has been postulated (2022). However, a very recent in-cell NMR study convincingly shown that iM-forming sequences within vitrotransition pH (pHT) < 7 are mainly unfolded in living HeLa cells, and that iM formation issues only a tiny portion (<1%) of putative iM-forming sequences (23). The molecular toolset to detect the presence of iMs in cells or to interrogate their functions is sparse. Therefore, despite a massive research effort, nobona fide(i.e., truly specific and strongly stabilizing) small-molecule iM ligand has been identified to date (24,25), which is inside a stark contrast to the plethora of highly specific, biologically active G-quadruplex (G4) ligands. To address this space, in 2018 the teams of D. Christ and M. Dinger raised an antibody (iMab) against the iM structure formed from the 22-mer C-rich DNA strand representing human being telomeric sequence (CCC(TAACCC)3T, or hTeloC; hTeloC-22 with this work) (26). The initial characterization of iMab shown high affinity for hTeloC-22 (KD= 59 nM at pH 6.0, as per biolayer interferometry (BLI) analysis) and excellent selectivity, while no binding to the mutated sequence unable to form an iM structure (hTeloC-mut), double-stranded and hairpin DNA, or G4 constructions was observed by using ELISA and BLI assays. At the same time, reduced yet non-negligible binding of iMab to the hTeloC strand was observed at pH 7.0 and even 8.0, which is rather surprising considering the transition pH of this sequence (pHT= 6.11 for 24-mer hTeloC at 20 C, in the presence of 110 mM Na+) (27) and the fact that iM single-strand transitions are highly cooperative, meaning that no significant portion of this sequence could be present in the iM form at pH 7.0 or, even less likely, at PF-3644022 pH 8.0 (5). Despite this inconsistency, immunostaining PF-3644022 of human being cells with iMab clearly evidenced the build up of iMab foci in the nuclei, which was interpreted as the presence of iM constructions in cells (26). Since this seminal work, iMab was commercialized and rapidly found several applications like a molecular biology tool to study the formation and dynamics of iM constructions in cells by immunofluorescence (28) and to obtain genome-wide maps of iM-forming sequences. Along these lines, using DNA immunoprecipitation and sequencing, Christet al.recognized in the human genome over 650 000 sequences that were identified by iMab and significantly enriched in cytosine; a biophysical validation of a subset of these sequences confirmed that most (but not all) were able to form iM structuresin vitro, at pH 6.0 and 25 C (29). At the same time, Richteret al., using Slice&Tag technique, found that most iMab peaks (8594%) were located in promoter regions of genes and significantly co-localized with open-chromatin markers, which was consistent with the putative implication of iMs in transcription rules (22). A biophysical validation confirmed that a.