Inclusion of methoxy groups inverts the thermodynamic stabilities of DNA–RNA hybrid duplexes: A molecular dynamics simulation study

Highlights

• 2′-O-methyl modifications drive the conformation of hybrid duplexes towards A-like.

• Hydrophobicity of methyl groups inverses thermodynamic stability of hybrids upon modification.

• Structural changes in hybrid prone to 2′-O-methyl modification depend on the base composition.

Abstract

Modified nucleic acids have found profound applications in nucleic acid based technologies such as antisense and antiviral therapies. Previous studies on chemically modified nucleic acids have suggested that modifications incorporated in furanose sugar especially at 2′-position attribute special properties to nucleic acids when compared to other modifications. 2′-O-methyl modification to deoxyribose sugars of DNA–RNA hybrids is one such modification that increases nucleic acid stability and has become an attractive class of compounds for potential antisense applications. It has been reported that modification of DNA strands with 2′-O-methyl group reverses the thermodynamic stability of DNA–RNA hybrid duplexes. Molecular dynamics simulations have been performed on two hybrid duplexes (DR and RD) which differ from each other and 2′-O-methyl modified counterparts to investigate the effect of 2′-O-methyl modification on their duplex stability. The results obtained suggest that the modification drives the conformations of both the hybrid duplexes towards A-RNA like conformation. The modified hybrid duplexes exhibit significantly contrasting dynamics and hydration patterns compared to respective parent duplexes. In line with the experimental results, the relative binding free energies suggest that the introduced modifications stabilize the less stable DR hybrid, but destabilize the more stable RD duplex. Binding free energy calculations suggest that the increased hydrophobicity is primarily responsible for the reversal of thermodynamic stability of hybrid duplexes. Free energy component analysis further provides insights into the stability of modified duplexes.

Introduction

Chemically modified nucleic acids (AOs) are attractive compounds in biomedical treatments involving nucleic acids because of their special features compared to pure DNA and RNA nucleic acids [1]. Regulation of gene expression by introducing chemically modified nucleic acids offer several advantages over traditional methods [2], [3]. The transfer of genetic information from DNA to protein can be blocked at several stages, including mRNA, ribosome and double stranded RNA (RNA interference) using AOs. In general, two mechanisms are accepted for the action of AOs in regulating gene expression: (a) binding of AOs to mRNA sequence to form a stable duplex, and (b) activation of ribonuclease H (RNase H) activity that involves cleavage of mRNA molecules [4], [5]. RNase H is an endonuclease enzyme that specifically identifies and cleaves the RNA strand of the DNA–RNA hybrid duplex without affecting the DNA strand. This recognition is non-sequence specific and can be exploited for biomedical purposes known as antisense and antiviral therapies [6], [7], [8]. This approach has been shown to be a potential way to block the transfer of genetic information. Since the first generation antiviral drugs are already in market, several studies have been carried out on the AOs [9]. A number of chemical modifications have been studied in great detail [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. Despite several available studies, search for suitable chemical modifications that can form stable duplexes with the mRNAs is an essential exercise.

Chemical modifications at 2′-position of the furanose sugar have been studied extensively [1], [10], [14], [15], [16]. Modifications at this position offer several advantages over other modifications. Their synthetic preparation is relatively easy compared to others and preorganizes the sugar conformation into C3′-endo similar to A-RNA duplex. These substitutions also modulate the duplex stability, affinity towards mRNA, nuclease resistance, uptake, hydrophobic interactions and hydration [1]. 2′-Methoxy modified nucleic acid is one such promising oligonucleotide that exhibits high affinities toward RNA targets [22]. It has been shown that the 2′-O-methyl modified nucleic acids are resistant to RNase H activity [23] and this modification shows a significant improvement in both nuclease activity and high affinity towards RNA target [24]. Previous studies have shown that the effect of 2′-O-alkyl and 2′-F modifications depends on the base composition present in duplexes [24], [25]. DNA–RNA hybrids are hybridized molecules between pure DNA and pure RNA duplexes and are formed as key intermediates in many important biological processes like DNA replication, reverse transcription, etc. [26], [27]. These are substrates for RNase H enzyme and are usually less stable than RNA duplexes [6], [7], [8]. They exhibit a conformation intermediate to A-form and B-form [8]. Previous studies have suggested that the hybrid properties depend on the deoxypyrimidine and GC content present in hybrids [28]. These molecules have extensively been studied by different experimental methods and observed that the hybrids with high deoxypyrimidine content are thermodynamically more stable than other hybrids and highly resistant to RNase H activity [28].

Duplex stability due to 2′-O-methyl modifications arise because of their ability to shift the conformational equilibrium of deoxyribose sugars toward C3′-endo (N-type) that results in A-RNA like conformation for the oligonucleotide and increases stacking interactions among nucleobases [1], [24], [25]. The resulting RNA-like conformation of oligonucleotides due to 2′-O-methyl modifications was confirmed by CD spectroscopy [24], [29], NMR spectroscopy [30], and X-ray studies [31]. Modelling studies have also supported the A-like conformation to 2′-O-methyl modified nucleic acids in line with the experimental studies [32], [33], [34]. In addition to A-like conformation, the 2′-O-methyl modified duplexes have under-winding helical structure suggested by recent studies [34]. Previous studies have shown that the 2′-O-methyl modified DNA–RNA hybrids display higher stability than their RNA counterparts that may arise due to the steric interaction between the 2-carbonyl groups of pyrimidines and the 2′-O-methyl substitutions, and enhanced base stacking [35], [36]. These putative hydrophobic interactions arise in modified DNA because of the consecutive 2′-O-methyl groups positioned on the surface of the minor groove [30]. Previous studies have suggested that inclusion of 2′-O-methyl modifications to the deoxyribose sugars of DNA–RNA hybrids reverses their thermodynamic stability [37]. This effect also depends on the AT/GC content present in the duplex and the modified duplexes display higher stability than RNA duplex. It has been suggested that there are two effects, conformational and hydrophobic, responsible for the total stabilization effect of the modified duplexes. The structural changes induced by 2′-substituent and the reasons for differential effects on the thermodynamic stability of hybrid duplexes with different base composition are yet to be explored. Molecular dynamics simulations can be efficiently used to understand the dynamic nature of biological macromolecules [38], [39], [40], [41], [42], [43]. The aim of the present study is to understand the impact of 2′-O-methyl modification on two different hybrid duplexes and the factors responsible for the reversal in thermodynamic stability of hybrid duplexes upon modification.