Sequence specific hydrogen bond of DNA with denaturants affects its stability: Spectroscopic and simulation studies

https://doi.org/10.1016/j.bbagen.2020.129735Get rights and content

Highlights

  • Gdm+ of GdmCl and urea both intrudes into the groove region of DNA along with the interaction with its phosphate backbone.

  • Opposite effect of GdmCl and urea on the stability is a general property of B-DNA.

  • The extent of stabilization/destabilization of DNA in Gdm+ and urea depend on its sequence

  • Pattern of hydrogen bond is different for urea and GdmCl.

Abstract

Background

Several different small molecules have been used to target the DNA helix in order to treat the diseases caused by its mutation. Guanidinium(Gdm+) and urea based drugs have been used for the diseases related to central nervous system, also as the anti-inflammatory and chemotherapeutic agent. However, the role of Gdm+ and urea in the stabilization/destabilization of DNA is not well understood.

Methods

Spectroscopic techniques along with molecular dynamics (MD) simulation have been performed on different sequences of DNA in the presence of guanidinium chloride (GdmCl) and urea to decode the binding of denaturants with DNA and the role of hydrogen bond with the different regions of DNA in its stability/destability.

Results and conclusion

Our study reveals that, Gdm+ of GdmCl and urea both intrudes into the groove region of DNA along with the interaction with its phosphate backbone. However, interaction of Gdm+ and urea with the nucleobases in the groove region is different. Gdm+ forms the intra-strand hydrogen bond with the central region of the both sequences of DNA whereas inter-strand hydrogen bond along with water assisted hydrogen bond takes place in the case of urea. The intra-strand hydrogen bond formation capability of Gdm+ with the nucleobases in the minor groove of DNA decreases its groove width which probably causes the stabilization of B-DNA in GdmCl. In contrast, the propensity of the formation of inter-strand hydrogen bond of urea with the nucleobases in the groove region of DNA without affecting the groove width destabilizes B-DNA as compared to GdmCl. This study depicts that the opposite effect of GdmCl and urea on the stability is a general property of B-DNA. However, the extent of stabilization/destabilization of DNA in Gdm+ and urea depend on its sequence probably due to the difference in the intra/inter-strand hydrogen bonding with different bases present in both the sequences of DNA.

General significance

The information obtained from this study will be useful for the designing of Gdm+ based drug molecule which can target the DNA more specifically and selectively.

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.

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