Investigation of dynamical flexibility of D5SIC-DNAM inside DNA duplex in aqueous solution: a systematic classical MD approach

Abstract

Incorporation of artificial 3rd base pairs (unnatural base pairs, UBPs) has emerged as a fundamental technique in pursuit of expanding the genetic alphabet. 2,6-Dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN), a potential unnatural base pair (UBP) developed by Romesberg and colleagues, has been shown to have remarkable capability for replication within DNA. Crystal structures of a Taq polymerase/double-stranded DNA (ds-DNA) complex containing a DS-DN pair in the 3' terminus showed a parallelly stacked geometry for the pre-insertion, and an intercalated geometry for the post-insertion structure. Unconventional orientations of DS-DN inside a DNA duplex have inspired scientists to investigate the conformational orientations and structural properties of UBP-incorporated DNA. In recent years, computational simulations have been used to investigate the geometry of DS-DN within the DNA duplex; nevertheless, unresolved questions persist owing to inconclusive findings. In this work, we investigate the structural and dynamical properties of DS and DN inside a ds-DNA strand in aqueous solution considering both short and long DNA templates using polarizable, and non-polarizable classical MD simulations. Flexible conformational change of UBP with major populations of Watson-Crick-Franklin (WCF) and three distinct non-Watson-Crick-Franklin (nWCFP1, nWCFP2, nWCFO) conformations through intra and inter-strand flipping have been observed. Our results suggest that a dynamical conformational change leads to the production of diffierent conformational distribution for the systems. Simulations with a short ds-DNA duplex suggest nWCF (P1 and O) as the predominant structures, whereas long ds-DNA duplex simulations indicate almost equal populations of WCF, nWCFP1, nWCFO. DS-DN in the terminal position is found to be more flexible with occasional mispairing and fraying. Overall, these results suggest flexibility and dynamical conformational change of the UBP as well as indicate varied conformational distribution irrespective of starting orientation of the UBP and length og DNA strand.

Scheme 1

Schematic representation of the sequence of DS–DN incorporated DNA duplex of (A) MUD, (B) MUDL and (C) UUD. (D) depicts the 2D representations of DS and DN. E. Distance and angles associated with DNA.

Fig. 1

Optimized structures of (A) SYN and (B) ANTI conformers of DS–DN at ωB97x-D/6–311++g(d,p) level. NCIPLOT of (C) SYN and (D) ANTI represented the non-covalent interactions between DS and DN in optimized structures.

Fig. 2

Snapshot of different conformers i.e. (A) nWCFP1, (B) nWCFP2, (C) nWCFO, (D) WCF of UBP inside DNA duplex.

Fig. 3

Conformational change through intra- and inter-strand flipping observed during the MD simulations.

Fig. 4

(A) d_4–6 value: high value depicts nWCP conformers, low values depict WC and nWCO conformers, (B) DN–NB value: circles point out inter-strand flipping, (C) d_O–S values: low value generally indicates DS and DN are in same phase (SYN), high-value generally indicates DS and DN are in opposite phase (ANTI) for three replicates obtained from AMOEBA forcefield.

Fig. 5

RMSF values for all the systems obtained from AMBER simulation.

Fig. 6

Distribution and population of the conformers for all the replications for (A) MUD_SYN, (B) MUD_ANTI, (C) MUD_PAR and (D) MUDL. WC, nWCP1, nWCP2, nWCO are designated by Green, Purple, Brown and Pink respectively. Black pointer indicates inter-strand flipping with the change of SYN to ANTI and vice versa. Red pointer indicates inter-strand flipping with no conformational change. Blue pointer indicates intra-strand flipping.

Fig. 7

Snapshots of different geometries of UUD form of DNA duplex during simulations through intra and inter-strand flipping.