Sequence specific hydrogen bond of DNA with denaturants affects its stability: Spectroscopic and simulation studies
Graphical abstract
Introduction
The integrity and stability of DNA is an important aspect of the cell survival as DNA controls important biological processes such as replication and transcription of living cell owing to its capability to carry the hereditary information. Subtle transformation in the genetic information capability of DNA may affect its replication which can lead to several lethal diseases such as cancer, Huntington's disease, cystic fibrosis, muscular dystrophy, Down's syndrome, multifactorial disorders [[1], [2], [3], [4]]. Several different small molecules have been used to target the DNA helix in order to treat the diseases caused by its mutation [5,6]. The stabilization/destabilization of DNA by the interaction with small molecules occurs either by non-specific interaction with the charged sugar backbone or via groove binding as intercalation mode [[7], [8], [9], [10]].
Several different drug molecules recognize different regions of DNA such as backbone or groove region via the covalent or noncovalent interactions [11,12]. Drug induced destabilization of DNA helix has been suggested as a novel antitumor mechanism of action; however, the DNA destabilizing compounds are relatively rare. Indeed, it has been proposed that the original binding mode of DNA-stabilizing compounds may be different from the destabilizing ones [9,13]. Hence, the binding of small molecules with different region of DNA is important to understand to decode the stability/instability of DNA caused by these molecules in the prospect of drug designing.
Guanidinium based drug molecules are important class of molecule which have been extensively used for the cardiovascular, central nervous system, histamines and diabetic diseases [14,15]. Indeed, guanidinium chloride (GdmCl) along with urea is one of the important denaturant which have been extensively used to understand the structure of proteins as both destabilize the structure of proteins significantly [[16], [17], [18]]. However, there are few studies in which the effect of these denaturing agents on the stability and structure of DNA has been investigated [[19], [20], [21], [22], [23]]. It has been proposed that the effect of urea and GdmCl on the stability of DNA is opposite as GdmCl stabilize whereas urea destabilize DNA. Apart from that the denaturing effect of GdmCl and urea was studied on the nanostructure DNA origami and it was shown that both GdmCl and urea denature DNA origami structure, however, the denaturation capability of GdmCl is less than urea. [24,25] However, these experiments was performed in polymeric DNA and nanostructure whose sequences were not known and defined [26].
In this manuscript, we have studied the effect of GdmCl and urea on three different types of sequences (out of which two are AT rich whereas other is GC rich) using the spectroscopic and molecular dynamics simulation methods. It has been found that the propensity of the formation of distinctly different type of the hydrogen bond of GdmCl and urea with the nucleobases present in the groove region of the sequences of DNA result to the opposite effect on its stability. Interestingly, the stabilization or destabilization effect shown by GdmCl and urea on duplex DNA is its general property. However, the extent of stabilization/destabilization by denaturants depends slightly on the sequence of bases of DNA due to the formation of the different type of hydrogen bond between nucleobases and denaturants.
Section snippets
Materials and methods
High pressure liquid chromatography (HPLC) graded d(CGCAAAAAAGCG)2 (seq1), d(CGCATATATGCG)2 (seq2) and d(CGCGCGCGCGCG)2 (seq3) DNAs were procured from Integrated DNA Technologies (IDT). Stock solution of DNA was prepared by adding the required volume of Tris-HCl buffer of pH 7.2. Duplex DNA was formed by heating the stock up to 93 °C and then slowly cooling the solution to room temperature (25 °C). 4′, 6-diamino-2-phenylindole (DAPI, ≥98%, molecular biology grade) was purchased from Sigma
Results and discussion
The CD spectra of seq1 and seq2 DNA show the maximum positive ellipticity in the range of 270–280 nm and negative ellipticity ~250 nm along with zero point crossing at ~260 nm which is characteristic feature of the B form of DNA (Fig. S1a) [44]. The positive feature represents the base stacking and negative band depicts the helicity of B-DNA [45]. With the addition of denaturants, the spectral feature of CD for both the sequences of DNA does not change appreciably compared to buffer condition
Conclusion
The role of the hydrogen bonding of denaturants, GdmCl and urea on the stability of the duplex DNA of AT and GC rich sequences has been investigated using the spectroscopic and MD simulation studies. Spectroscopic studies indicate that GdmCl and urea both intrude into the groove region of DNA without perturbing its canonical structure. However, the mode of interaction of GdmCl and urea with DNA is different as GdmCl stabilize the DNA whereas urea destabilizes it comparatively to GdmCl and
Credit author statement
S.S., P.C.S. planned the experiment, S.S. performed the experiment, S.S. and P.C.S. analyzed the data and wrote the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
SS wants to thank DST-Inspire for the fellowship and CRAY facility of IACS for MD simulation.
References (54)
- et al.
Effects of replication and transcription on DNA structure-related genetic instability
Genes (Basel)
(2017) - et al.
Design, synthesis, and biological evaluation of substituted-N-(thieno[2,3-b]pyridin-3-yl)-guanidines, N-(1H-pyrrolo[2,3-b]pyridin-3-yl)-guanidines, and N-(1H-indol-3-yl)-guanidines
Bioorg. Med. Chem.
(2007) - et al.
Urea and guanidinium chloride denature protein L in different ways in molecular dynamics simulations
Biophys. J.
(2008) - et al.
Preferential binding of urea to single-stranded DNA structures: a molecular dynamics study
Biophys. J.
(2018) - et al.
Calorimetric measurements of the transition enthalpy of DNA in aqueous urea solutions
Biochim. Biophys. Acta (BBA) - Nucl. Acids Protein Synth.
(1977) - et al.
Alteration of the groove width of DNA induced by the multimodal hydrogen bonding of denaturants with DNA bases in its grooves affects their stability
BBA-Gen. Subj.
(2020) - et al.
Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides
Biochem. Biophys. Res. Commun.
(1990) - et al.
AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules
Comput. Phys. Commun.
(1995) - et al.
Refinement of the AMBER force field for nucleic acids: improving the description of α/γ conformers
Biophys. J.
(2007) - et al.
VMD: visual molecular dynamics
J. Mol. Graph.
(1996)