Abstract achieved by measuring and comparing ctDNA viscosity

AbstractThe characterization of the binding interactionbetween DNA and small molecules has alwaysbeen a point of interest and extensively researched over the years. The primeinterest in these studies mainly lies in the fact that the DNA-binder agents aresupposed to have the potential to act as drugs. The presentexperimental study attempts to investigate the binding interaction between twotypes of acridone derivative, 8-choloro acridone(CA) and nitrile cyanide acridone (NCA), and calf thymus DNA (ctDNA). To performthis study, several spectroscopictechniques were employed. The obtained result revealed that CA and NCA can bindto ctDNA by a quenching constant of 2.

949×103 and 7.063×103M-1 respectively.Further analysis pointed out that the interaction between CA and ctDNA wascontrolled by a dynamic quenching mechanism while the dominant quenchingprocess in ctDNA-NCA interaction wasstatic. Calculating and analyzing the thermodynamic parameters allows anestimation of the forces that drives the complex formation. In present study, thethermodynamic properties of the bindingprocess indicated that the resulting ctDNA-CA and ctDNA-NCAcomplexes were stabilized by hydrophobic and van der Waals intercationsrespectively.

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The fluorescencedisplacement experiments were conducted by using ethidium bromide (EB) andacridone orang (AO) as fluorescence probes and showed that CA and NCA competeagainst intercalator probes and thereby they probably interact with ctDNAthrough intercalation. Given that the melting temperature ofctDNA went up by 6-8 C in the presence of CA and NCA, it can be thus inferredthat these two compound probably intercalated into ctDNA and brought greaterstability in ctDNA. A closer insight into the modes of binding was achieved by measuring and comparing ctDNAviscosity in the absence and presence of CA and NCA. The results displayed thatthe presence of CA resulted in a decrease in the relative viscosity of ctDNAwhich is a characteristic of non-classical intercalation bindings. In contrast,the ctDNA relative viscosity increased through binding on NCA which reflect thefact that NCA bound ctDNA intercalately. The effect of ionic strength on the interactions indicated that thectDNA-NCA and ctDNA-CA interactions were relatively dependent and independenton the salt concentration respectively.

Thesefindings allowed us to deduce that electrostatic interactions moderatelycontributed in the interaction between ctDNA and NCA. However, these external bindinginteractions are much weaker than intercalation. The CD spectra of ctDNAwere collected in the presence and absence of CA and NCA. The minor induced conformationalchanges in ctDNA through the interactions with CA and NCA were reflected in thegentle changes of ctDNA spectra upon incremental addition of CA and NCA.Moreover, it was shown that CA has higher affinity to ss ctDNA whereas NCA tends to bind native ctDNA more strongly. All things considered,our investigation and results implied that CA and NCA interacted with ctDNA viaintercalation mode. Moreover, the interaction between NCA and ctDNA is muchstronger compared to the ctDNA-CA binding interaction.