Quantifying Double-stranded DNA Binding Properties Of G-quadruplex Ligands By Single Molecule Magnetic Tweezers

BackgroundThe unlimited proliferation of cancer cells requires fast replication and transcription of the genome,so the treatment of chemotherapy drugs targeting biomacromolecules nucleic acid is an important way of fighting cancer.Besides,double-stranded DNA,in recent years,small molecule ligands which target the genomic G-quadruplex have paid extensive attention.In the genome,a nucleic acid sequence that forms a G-quadruplex is enriched in the oncogene promoter region and the telomere region.Small molecules targeting G-quadruplex play a role in treating cancers by down-regulation of oncogene transcription and inhibition of telomerase expression.Most G-quadruplex ligands have a polyaromatic planar ring system,and the G-quadruplex structure and ligand are stabilized by interactions such as?-?stacking to hinder gene duplication and cause double-stranded DNA fragmentation.Some ligands to have a certain affinity with the double-stranded DNA,because of the planar ring structure of the G-quadruplex ligand and the positively charged group.It is still controversial whether the G-quadruplex ligand exerts anticancer effects by stabilizing G-quadruplex or intercalates double-stranded DNA.ObjectivesPreviously,it has been reported that ligands are able to interact with double-stranded DNA,but the sensitivity of traditional biochemical methods for detecting ligands/DNA binding is limited.In addition,the mode of ligand binding to double-stranded DNA and the number of the binding site in double-stranded DNA are unclear,which has an effect on the systematic understanding of the interaction between ligands and double-stranded DNA.Therefore,this study intends to detect the interaction of eight representative G-quadruplex ligands with long double-stranded DNA by using single-molecule magnet tweezers,which contributes to explaining the effect of ligands on double-stranded DNA and providing data support for highly selective G-quadruplex ligands.MethodsFirst,we used lambda-DNA as a template to amplify the 6618 bp double-stranded DNA with biotin and sulfhydryl groups at the 5'end respectively by PCR,purified and recovered the PCR product,and chemically modified the double-stranded DNA to the surface of the glass and the superparamagnetic sphere.In the meantime,the single-molecule mechanical study of double-stranded DNA was carried out.Secondly,we use the gradient magnetic force generated by the magnet to perform single-molecule double-strand DNA stretching.The tensile length change of double-stranded DNA was determined by recording the height of the magnetic sphere under different constant forces to study the force spectrum of the double-stranded DNA after stress.The elongation height of the DNA was recorded after adding to the ligand under different constant force.According to the elongation of the DNA,the possible binding mode of the ligand to the double-stranded DNA was determined,and the interaction between the ligand and the double-stranded DNA was obtained by Hill equation fitting.The dissociation constant(K_d),and calculate the amount of elongation of the DNA after the single molecule ligand is inserted into the DNA,and calculate the number of bases of the adjacent two ligand molecules that bind to the double-stranded DNA.ResultsWe found that the DNA elongation after the above four ligands binding with double-stranded DNA is greater than the DNA elongation after EB binding double-stranded DNA and the other ligands'are similar to EB's,when compared the force-bead height and concentration-elongation curves of acridine ligands BRACO-19?RHPS4,heterocyclic ligand CX-5461 and porphyrin TMPyP4 with the force-bead height curve of EB.In addition,we quantitatively analyzed the dissociation constant(K_d)ranging from 0.59?M to 2?M of the above four ligands to double-stranded DNA.The ligands BRACO-19,RHPS4,CX-5461,and TMPyP4 have strong intercalation binding with double-stranded DNA,which is consistent with previous reports.The distance of adjacent ligand molecules binding to double-stranded DNA ranged from 1.9 to 3.1 bp.In addition,the elongation of DNA is shorter than EB's after adding the porphyrin ligand NMM?quinoline ligands PDS?Phen-DC3 and quinazine ligand TrisQ,in which NMM is hardly bound to double-stranded DNA at low concentrations(1,4,8,80?M).And Phen-DC3 and TrisQ molecules can bind to double-stranded DNA more than adjacent three base pairs under a certain tension.We got the binding amount of eight G-quadruplex ligands to double-stranded DNA,in which NMM,PDS,Phen-DC3 and TrisQ have fewer bindings in double-stranded DNA,and other ligands'are close to EB's.ConclusionsIn summary,we found that ligands CX-5461,BRACO-19,RHPS4,and TMPyP4can strongly interact with double-stranded DNA;TrisQ,Phen-DC3,PDS and NMM are relatively weakly bound to double-stranded DNA,which shows better selectivity for G-quadruplex.