Long-Range Ordered Water Correlations between A–T/C–G Nucleotides

Abstract

•Unzipping of dsDNA by atomic force microscopy-based single-molecule force spectroscopy•Weak long-range ordered water interactions between Watson-Crick base pairs•Special helical network structures of ordered water The interactions between complementary base pairs are intensively explored because of their unique impacts in hybridization processes. However, the interactions at long range, which also strongly influence the hybridization processes of nucleic acid, are still not clear. Moreover, it is also challenging to investigate these weak long-range interactions, which is restricted by experimental methods and instruments. Using atomic force microscopy and Raman spectroscopy, weak long-range interactions between A–T/C–G nucleotides were observed, which could be attributed to the ordered water chain interaction between the base pairs. Furthermore, theoretical calculations also were used to simulate the ideal structure of the ordered water, which might exist as a helical network structure. This ordered water structure between complementary base pairs could have a significant impact on DNA hybridization and other biological assembly processes. The interactions between complementary base pairs are crucial to the helical duplex structure of DNA. Although base-base interactions have been widely reported, the study of long-range interactions in the base-pairing processes is still a major challenge in this field. Here, we first study the long-range interactions between the base pairs by atomic force microscopy-based single-molecule force spectroscopy (SMFS). The SMFS results of A–T/C–G imply that there are weak long-range interactions between them, which have a range of 15–25 nm. Raman spectroscopy results imply that these weak interactions can be attributed to multiplex hydrogen bond interaction of ordered water structure between A–T/C–G nucleotides. In addition, the theoretical calculations deduce that the ideal structure of these water molecules exhibits a specific helical structure. Our findings might open up a new understanding of biological assembly processes and provide helpful strategies for bio-nanotechnology. The interactions between complementary base pairs are crucial to the helical duplex structure of DNA. Although base-base interactions have been widely reported, the study of long-range interactions in the base-pairing processes is still a major challenge in this field. Here, we first study the long-range interactions between the base pairs by atomic force microscopy-based single-molecule force spectroscopy (SMFS). The SMFS results of A–T/C–G imply that there are weak long-range interactions between them, which have a range of 15–25 nm. Raman spectroscopy results imply that these weak interactions can be attributed to multiplex hydrogen bond interaction of ordered water structure between A–T/C–G nucleotides. In addition, the theoretical calculations deduce that the ideal structure of these water molecules exhibits a specific helical structure. Our findings might open up a new understanding of biological assembly processes and provide helpful strategies for bio-nanotechnology. The discovery of the helical duplex structure of DNA by Watson and Crick in 19531Watson J.D. Crick F.H.C. A structure for deoxyribose nucleic acid.Nature. 1953; 171: 737-738Crossref PubMed Scopus (7778) Google Scholar brought forth a new era of molecular biology and stimulated the innovations in gene-engineering techniques such as DNA synthesis and DNA cloning.2Caruthers M.H. A brief review of DNA and RNA chemical synthesis.Biochem. Soc. Trans. 2011; 39: 575-580Crossref PubMed Scopus (38) Google Scholar,3Ma V.P. Salaita K. 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Direct observation of single-molecule stick-slip motion in polyamide single crystals.ACS Macro Lett. 2018; 7: 762-766Crossref Scopus (19) Google Scholar In this paper, we used AFM-based SMFS to explore the forces in long-range interactions during the unbinding of Watson-Crick base pairs. The SMFS force curves for A–T and C–G indicate that there are weak long-range (15–25 nm) interactions between the complementary base pairs. A combination of AFM-based SMFS and Raman spectroscopy indicated that these weak long-range interactions can be attributed to ordered water between the two complementary bases. The ideal structure obtained from theoretical calculations implies that the ordered water between complementary bases forms a helical network structure. Our findings indicate that DNA hybridization involves long-range interactions between Watson-Crick base pairs. This provides a new understanding of the biological assembly processes. In this experiment, AFM-based SMFS was used to investigate the interactions between A–T nucleotides, and the non-complementary strands (poly(dA)-poly(dC)) were also investigated as control. As shown in Figures 1A and 1B , the 5′ end of poly(dA) was chemically modified on an AFM tip with a poly(ethylene glycol) (PEG) linker, and the 3′ end of poly(dT) or poly(dC) was connected to the glass substrate with the same PEG linker. The modified AFM cantilever tip approached the substrate and stayed for about 2 s to allow the formation of a double-stranded DNA (dsDNA) helical structure (H1) between the cantilever and substrate in phosphate-buffered saline (PBS). During retraction of the cantilever tip, the dsDNA structure was unzipped, which enables evaluation of the mechanical stability of the base pairs and the long-range interactions between them. A typical force curve for unzipping of H1 in PBS is shown in Figure 1C. 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Because of the restrictions of the synthetic technique, dsDNA chains (H2) with an average G–C content, which were formed from chain A and chain B (Table S1), were used in these SMFS experiments. The unzipping force curve for H2, which was obtained in PBS, is shown in Figure 2C. A reproducible force plateau at about 24.5 ± 0.4 pN appeared in the force curve (Figure 2D; for details see Figure S3) due to the unzipping process of H229Carrion-Vazquez M. Oberhauser A.F. Fisher T.E. Marszalek P.E. Li H. Fernandez J.M. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.Prog. Biophys. Mol. Biol. 2000; 74: 63-91Crossref PubMed Scopus (360) Google Scholar,37Rief M. Clausen-Schaumann H. Gaub H.E. Sequence-dependent mechanics of single DNA molecules.Nat. Struct. Biol. 1999; 6: 346-349Crossref PubMed Scopus (677) Google Scholar,40Krautbauer R. Rief M. Gaub H.E. 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This can be attributed to their different H-bonded structures, i.e., two H bonds between A–T and three between C–G. A possibility of these interactions is that the long-range interactions between Watson-Crick base pairs arise from the effect of water, which is an indispensable solvent in biological systems.14Privalov P.L. Crane-Robinson C. Role of water in the formation of macromolecular structures.Eur. Biophys. J. 2017; 46: 203-224Crossref PubMed Scopus (38) Google Scholar,41Ball P. Water as an active constituent in cell biology.Chem. Rev. 2008; 108: 74-108Crossref PubMed Scopus (1534) Google Scholar,42Ouyang J.F. Bettens R.P. Modelling water: a lifetime enigma.Chimia (Aarau). 2015; 69: 104-111Crossref PubMed Scopus (38) Google Scholar Recently, Kurian et al. reported the potential presence of a water-mediated long-range correlation in DNA-enzyme interactions.43Kurian P. Capolupo A. Craddock T.J.A. Vitiello G. Water-mediated correlations in DNA-enzyme interactions.Phys. Lett. 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Raman spectra of water with DNA in different interaction states were obtained by using the following detection method. As shown in Figure 3A, two modified substrates were placed together and separated by micron particles (diameter ∼1 μm) at one end. The gap between the two surfaces increased continuously from 0 to 1,000 nm, and the interaction correlations between the different DNA molecules on the two surfaces changed with increasing gap distance between the two surfaces. The complementary DNA (cDNA) molecules should adopt a helical structure when the total length of the molecules is larger than the gap between the two surfaces. Otherwise, the DNA molecules on the two surfaces would show long-range interactions or free single-stranded states. As shown in Figure 3B, the laser beam was moved continuously between the middle of the two modified surfaces from 0 to 10 mm (Figure 3B; the gap distance increases from 0 to 500 nm), and spectra of the liquid water around the DNA molecules in different states were obtained and used to explore the changes in water in the system. For the A–T system, poly(dA) and poly(dT) were modified on a quartz slide and Si substrate, respectively. The changes in the interactions between them greatly depend on the molecular lengths. The molecules used are the same as those in SMFS, and the total length of the molecules is about 199.3 ± 2.2 nm for the A–T system (for details see Figure S6). Therefore, the gap distance was selected accordingly in the range of 0–500 nm, which corresponds to 0–300 nm for the inter-distance between DNA molecules. With the inter-distance increasing, the DNA molecules show three different states, i.e., a double-stranded state, a long-range interaction state, and a free single-stranded state (Figures 3C–3E). A detailed description of the C–G system is given in Supplemental Information. The Raman spectra of OH-stretching vibration of water that between poly(dA) and poly(dT) (Figure 3F)/chain A and chain B (Figure 3G) in three states were obtained after normalizing at 3,370 cm−1 (the isosbestic point of liquid water),49Walrafen G.E. Hokmabadi M.S. Yang W.H. Raman isosbestic points from liquid water.J. Chem. Phys. 1986; 85: 6964-6969Crossref Scopus (261) Google Scholar which reflects the broad range of H-bonding interactions in aqueous solution. After peak fitting, the spectra show three main peaks at 3,200, 3,400, and 3,600 cm−1, respectively (Figure S7).50Ninno A.D. Castellano A.C. Giudice E.D. The supramolecular structure of liquid water and quantum coherent processes in biology.J. Phys. Conf. 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We further carried out detailed Raman spectroscopic measurements by scanning the laser beam with a step width of 1 mm through the range of 0–10 mm, corresponding to DNA inter-distance from 0 to 300 nm. The relationship between the inter-distance of DNA chains and the average values of the area ratios of the peak at 3,200 cm−1 are shown in Figures 3H and 3I, respectively. After fitting, there exists a maximum peak around 50–100 nm for the A–T system and 30–80 nm for the C–G system, indicating a highest content of ordered water in this range. The range is a little larger than that obtained from SMFS, which might be attributed to the differences between the single molecular chain and multiple molecular chain, which is adopted in SMFS and Raman system, respectively. From the curves, it can also be observed that the amount of ordered water in the double-stranded state is greater than that of the free single-stranded state, since the DNA molecules should adopt a helical structure at the double-stranded state, in which some of the water molecules are oriented around the DNA backbone.51Hall J.P. Cook D. Morte S.R. McIntyre P. Buchner K. Beer H. Cardin D.J. Brazier J.A. Winter G. Kelly J.M. et al.X-ray crystal structure of rac-[Ru(phen)2dppz]2+ with d(ATGCAT)2 shows enantiomer orientations and water ordering.J. Am. Chem. Soc. 2013; 135: 12652-12659Crossref PubMed Scopus (68) Google Scholar,52McDermott M.L. Vanselous H. Corcelli S.A. Petersen P.B. DNA's chiral spine of hydration.ACS Cent. Sci. 2017; 3: 708-714Crossref PubMed Scopus (71) Google Scholar The control experiments of the non-complementary strands (A–C and T–C system, Figures S7 and S8) were also performed by using similar methods. The relationship between the inter-distance of DNA chains and the average values of the area ratios of the peak at 3,200 cm−1 are shown in Figures S9C and S9D. After fitting, however, compared with the A–T and C–G systems, there is no obvious change of ordered water around 0–100 nm in the A–C and T–C systems. This result indicates that there is no ordered water-mediated long-range interaction between non-complementary DNA nucleotides. The Raman results of complementary and non-complementary DNA strands provide evidence of the possibility that there exists ordered water with several tens of nanometers between the cDNA base pairs; however, the structure of the ordered water is still unclear. Inspired by Naserifar et al., whose molecular dynamics NVT (canonical ensemble) simulations suggested that water forms a dynamic multibranched polydisperse polymer network with ∼20 nm length,53Naserifar S. William A. Goddard I. Liquid water is a dynamic polydisperse branched polymer.Proc. Natl. Acad. Sci. U S A. 2019; 116: 1998-2003Crossref PubMed Scopus (27) Google Scholar we performed theoretical calculations to predict the possible structure of ordered water between two complementary base pairs. We performed density functional theory calculations by using the B3LYP functional with the 6-31G∗ basis set to optimize the related structures. All calculations were performed with the Gaussian 09 software package.54Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Scalmani G. Barone V. Mennucci B. Petersson G.A. et al.Gaussian 09, Revision E.01. Gaussian, Inc., Wallingford CT2013Google Scholar The calculations are programmed as follows. First, molecular models of A, T, C, and G nucleotides, and A–T and C–G base pairs, were optimized as shown on the left side of Figure 4. Based on the H-bonding characteristics of A–T/C–G, three water molecules as one layer were inserted and restrictedly optimized (the structures of A and T or C and G were fixed, and only the water molecules were optimized) to form one water layer between the A–T/C–G nucleotides. The DNA nucleotides were then fixed, and another two water layers were inserted to form three water layers to fit the requirement of ordered water arrangement by four H bonds formed with four neighboring water molecules.41Ball P. Water as an active constituent in cell biology.Chem. Rev. 2008; 108: 74-108Crossref PubMed Scopus (1534) Google Scholar By repeating the process to insert another water layer between the A–T/C–G nucleotides, DNA nucleotides with the different water layer structures were fully optimized again, as shown on the right side of Figure 4. Finally, water chains with a length of ∼20 nm including 228 water molecules between A–T/C–G nucleotides were also formed. By analyzing the molecular arrangement in the chains, it was found that the water molecules assembled in a helical manner as a molecular spring. The weak tensions of this molecular spring could act as a driving force for the long-distance recognition of DNA-hybridization processes. In the current study, long-range interactions between Watson-Crick base pairs (A–T and C–G) were systematically investigated by AFM-based SMFS, Raman spectroscopy, and theoretical calculations. The SMFS results indicate that there are weak long-range (15–25 nm) interactions between complementary base pairs, namely 2.3 ± 0.2 pN for A–T and 3.5 ± 0.2 pN for C–G. However, there are no apparent interactions between non-complementary base pairs (i.e., A–C and C–T) under the same conditions. Raman spectroscopy was used to detect the effect of water on the base-pairing process. The results indicate that weak long-range A–T (or C–G) interactions can be attributed to ordered water interactions between the pairs. In addition, the theoretical calculations imply a possible helical ordered water network structure between the complementary base pairs. All of these findings may provide a new understanding of long-distance recognition of DNA-hybridization processes. This long-range interaction could be affected by the salt concentration, pH, and temperature (for details see Figure S10) of the system, and we will explore this in future work.