Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring

Цветков, В. Б.; Turaev, Anton V.; Petrunina, Nataliia A.; Melnik, Denis M.; Khodarovich, Yu. M.; Pozmogova, Galina E.; Zatsepin, Timofei S.; Varizhuk, Anna M.; Aralov, Andrey V.

doi:10.1039/d1an00660f

Cited by 7 publications

(8 citation statements)

References 21 publications

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“…15,16 The identification and evaluation of i-DNA ligands are hampered by the lack of robust, uniform assays such as FRET-melting and FID assay that became routine in the field of G4 ligand. In fact, these methods have strong biases and provide untrustworthy results with i-DNA, as evidenced in recent publications, 17,18 prohibiting direct comparison of the ligands described by different groups. In this context, we have previously assembled two peptide-DNA conjugates that form i-motif structures, 19,20 with one of them, namely conjugate 2 (Fig.…”

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confidence: 99%

Assessment of presumed small-molecule ligands of telomeric i-DNA by biolayer interferometry (BLI)

et al. 2022

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confidence: 99%

Assessment of presumed small-molecule ligands of telomeric i-DNA by biolayer interferometry (BLI)

et al. 2022

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“…In the conditions of that study (50 mM Na cacodylate buffer, pH 5.8), the relative fluorescence variation at 520 nm (i.e., FAM emission) was less than 10%, and an equilibrium dissociation constant K D of around 490 nM was determined. However, it has been more recently shown that fluorophores typically used in the FRET assay may introduce strong biases into ligand interactions with i-DNA [ 29 , 30 ], namely by direct fluorophore–ligand interactions. Moreover, the authors have performed CD melting experiments and observed a very low effect of the ligand on thermal stability of bcl-2 i-DNA (∆ T m = 1 °C), which appears rather surprising considering the reported sub-micromolar affinity.…”

Section: Resultsmentioning

confidence: 99%

Novel Synthesis of IMC-48 and Affinity Evaluation with Different i-Motif DNA Sequences

et al. 2023

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During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity.

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“…Finally, the structure was optimized using Powell’s method. Calculations of the partial charges in the modified loops and 80 ns molecular dynamics (MD) simulations were performed according to published protocols …”

Section: Methodsmentioning

confidence: 99%

“…Calculations of the partial charges in the modified loops and 80 ns molecular dynamics (MD) simulations were performed according to published protocols. 42 Nuclease Hydrolysis. For endonuclease hydrolysis assays, S1 endonuclease (Thermo Fisher Scientific) was added to a 6 μM ODN solution in the S1 reaction buffer (50 mM sodium acetate, pH 4.5, 280 mM NaCl, and 45 mM ZnSO 4 ) to a final S1 concentration of 0.2 u/μL.…”

Section: Design and Synthesis Of Labeled Native And Modifiedmentioning

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Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules

et al. 2023

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DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau 1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.

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Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring

Abstract: The lack of HTS techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing effects of ligands...

Cited by 7 publications

References 21 publications

Assessment of presumed small-molecule ligands of telomeric i-DNA by biolayer interferometry (BLI)

Assessment of presumed small-molecule ligands of telomeric i-DNA by biolayer interferometry (BLI)

Novel Synthesis of IMC-48 and Affinity Evaluation with Different i-Motif DNA Sequences

Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules

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