Nonselective Intercalation of G-Quadruplex-Targeting Ligands into Double-Stranded DNA Quantified by Single-Molecule Stretching

Abstract

Most G-quadruplex (G4)-targeting ligands reported so far contain planar heteroaromatic groups and can intercalate into adjacent base pairs of double-stranded DNA (dsDNA). However, quantitative data on the binding number γ (ligands/bp) of G4 ligands that intercalate into long dsDNA remain lacking, which are essential for understanding the selectivity of G4 ligands. Here, using a single-molecule stretching assay based on the lengthening of dsDNA, we analyzed the dissociation constants and the binding number of eight most commonly used G4 ligands that intercalate into dsDNA. We showed that five ligands (CX-5461, BRACO-19, RHPS4, TrisQ, and Phen-DC3) intercalate into dsDNA avidly (K d = 0.5−2.1 μM, saturated γ > 0.2 ligands/bp), which was similar to the typical dsDNA intercalator EB. Two bisquinolines, PDS and 360A, showed moderate intercalation ability (K d = 22.5 and 48.7 μM) and γ < 0.01 ligands/bp in the presence of 1 μM ligands. Porphyrin NMM showed no intercalative binding even at 200 μM. Molecular docking and molecular dynamics simulations were carried out to further evaluate the intercalative binding of these G4 ligands with dsDNA by calculating the binding energies and π−π stacking probability.

Unfolding rates of 1:1 and 2:1 complex of CX-5461 and c-<i>MYC</i> promoter G-quadruplexes revealed by single-molecule force spectroscopy

“…The Myc-20T hairpin construct was used because the CX-5461 intercalative binding into dsDNA handles can cause significant fluctuations in dsDNA extension (Liu et al 2023 ). The intercalative binding of CX-5461 to dsDNA handle disrupts the typical overstretching transition of dsDNA usually observed at ~65 pN (Backer et al 2019 ).…”

Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation

“…Our results showed that CX-5461 exhibits the strongest inhibitory effects on c- MYC transcription, which can be attributed to its multifaceted mechanisms of action, including strong c-MYC promoter G4s unfolding inhibition, the ability to intercalate into dsDNA, RNA polymerase I inhibition, chromatin remodeling . Notably, our recent findings indicate that CX-5461 also exhibits a strong affinity for double-stranded DNA ( K d = 0.5 μM) . The ability of CX-5461 to intercalate into dsDNA was similar to the typical dsDNA intercalator EB.…”

Force-Enhanced Sensitive Detection of New DNA-Interactive Agents from Microorganisms at the Single-Molecule Level

“…1B, where a 6618 bp dsDNA was tethered between a paramagnetic bead and a cover glass slide, serving as a sensor to detect the DNA interacting agents. [9,21] By monitoring the extension of dsDNA using magnetic tweezers and applying forces ranging from 1 pN to 75 pN, the extension changes induced by DNA binding small molecule can be measured. The elongation of dsDNA measured in the presence of different concentration of daunorubicin was shown in Fig.…”

“…[6][7] Understanding of genome structure and function of genes has also created new opportunities for DNA intercalators targeting cellular DNA or the associated proteins, including topoisomerase inhibitors, [8] and drug targeting non-canonical DNA structures like G-quadruplexes. [9][10] Given the escalating issue of drug resistance, there is a pressing need for novel DNA interacting agents. Additionally, considering the diverse clinical efficacy observed among drugs from various chemical families targeting the same molecule, novel DNA-binding agents hold promise for combating different types of tumors.…”