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• W2890696136 abstract "•Joint X-ray/neutron structures of A-form DNA•Backbone phosphate of Ade7 is protonated at room temperature•Metal ion replaces proton on backbone phosphate at low temperature•Neutron structures show importance of structural analyses at different temperatures Nucleic acids can fold into well-defined 3D structures that help determine their function. Knowing precise nucleic acid structures can also be used for the design of nucleic acid-based therapeutics. However, locations of hydrogen atoms, which are key players of nucleic acid function, are normally not determined with X-ray crystallography. Accurate determination of hydrogen atom positions can provide indispensable information on protonation states, hydrogen bonding, and water architecture in nucleic acids. Here, we used neutron crystallography in combination with X-ray diffraction to obtain joint X-ray/neutron structures at both room and cryo temperatures of a self-complementary A-DNA oligonucleotide d[GTGG(CSe)CAC]2 containing 2′-SeCH3 modification on Cyt5 (CSe) at pH 5.6. We directly observed protonation of a backbone phosphate oxygen of Ade7 at room temperature. The proton is replaced with hydrated Mg2+ upon cooling the crystal to 100 K, indicating that metal binding is favored at low temperature, whereas proton binding is dominant at room temperature. Nucleic acids can fold into well-defined 3D structures that help determine their function. Knowing precise nucleic acid structures can also be used for the design of nucleic acid-based therapeutics. However, locations of hydrogen atoms, which are key players of nucleic acid function, are normally not determined with X-ray crystallography. Accurate determination of hydrogen atom positions can provide indispensable information on protonation states, hydrogen bonding, and water architecture in nucleic acids. Here, we used neutron crystallography in combination with X-ray diffraction to obtain joint X-ray/neutron structures at both room and cryo temperatures of a self-complementary A-DNA oligonucleotide d[GTGG(CSe)CAC]2 containing 2′-SeCH3 modification on Cyt5 (CSe) at pH 5.6. We directly observed protonation of a backbone phosphate oxygen of Ade7 at room temperature. The proton is replaced with hydrated Mg2+ upon cooling the crystal to 100 K, indicating that metal binding is favored at low temperature, whereas proton binding is dominant at room temperature. Nucleic acids encode life by storing and transferring genetic information in all living organisms including viruses. DNAs and RNAs of various length can fold into well-defined 3D structures, as many proteins do, with the information about their biological function implicitly woven into their sequences as well as structures (Leslie et al., 1980Leslie A.G. Arnott S. Chandrasekaran R. Ratliff R.L. Polymorphism of DNA double helices.J. Mol. Biol. 1980; 143: 49-72Crossref PubMed Scopus (365) Google Scholar, Hoogstraten and Sumita, 2007Hoogstraten C.G. Sumita M. Structure-function relationships in RNA and RNP enzymes: recent advances.Biopolymers. 2007; 87: 317-328Crossref PubMed Scopus (26) Google Scholar, Dickerson et al., 1982Dickerson R.E. Drew H.R. Conner B.N. Wing R.M. Fratini A.V. Kopka M.L. The anatomy of A-, B-, and Z-DNA.Science. 1982; 216: 475-485Crossref PubMed Scopus (541) Google Scholar). Knowing the precise 3-dimensional structures of nucleic acids can offer more information on the molecular mechanisms of various diseases, including genetic disorders, viral infections, and cancers (Park et al., 2011Park Y.J. Claus R. Weichenhan D. Plass C. Genome-wide epigenetic modifications in cancer.Prog. Drug Res. 2011; 67: 25-49PubMed Google Scholar). The structural knowledge can also be used in the development of nucleic acid-based therapeutics, such as antisense oligonucleotides and aptamers (Sharma et al., 2014aSharma V.K. Sharma R.K. Singh S.K. Antisense oligonucleotides: modifications and clinical trials.Med. Chem. Comm. 2014; 5: 1454-1471Crossref Google Scholar, Sharma et al., 2014bSharma V.K. Rungta P. Prasad A.K. Nucleic acid therapeutics: basic concepts and recent developments.RSC Adv. 2014; 4: 16618-16631Crossref Scopus (66) Google Scholar, Zhou and Rossi, 2014Zhou J. Rossi J. Cell-type-specific aptamer and aptamer-small interfering RNA conjugates for targeted human immunodeficiency virus type 1 therapy.J. Investig. Med. 2014; 62: 914-919Crossref PubMed Scopus (19) Google Scholar). Over the past several decades, the structure determination of biomacromolecules, including nucleic acids, has gone through several revolutionary changes due to advancements in X-ray crystallographic technologies, such as developments in synchrotron radiation, automation of crystal growth and data collection, and detectors. It is estimated that the number of functional nucleic acids (including non-coding RNAs) and nucleic acid-protein complexes is much larger than that of proteins. However, the rate of protein structure determination has dramatically outpaced that of nucleic acids, with >120,000 X-ray structures of proteins versus just ∼3,000 of nucleic acids deposited in the PDB (Berman et al., 2000Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. The Protein Data Bank.Nucl. Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27379) Google Scholar, www.rcsb.org). Such tremendous disparity in the number of available 3D structures of proteins and nucleic acids can be attributed to the differences in their natural ability to crystallize and to the difficulties in preparation of chemically and structurally homogeneous nucleic acid samples. The presence of negatively charged phosphate backbone on the surface, chemical and structural inhomogeneity, and the structural plasticity of nucleic acids make their crystal packing much more challenging (Dock-Bregeon et al., 1999Dock-Bregeon A.-C. Moras D. Giege R. Nucleic acids and their complexes.in: Ducruix A. Giege R. Crystallization of Nucleic Acids and Proteins—A Practical Approach. Oxford University Press, 1999: 209-243Google Scholar, Ke and Doudna, 2004Ke A. Doudna J.A. Crystallization of RNA and RNA-protein complexes.Methods. 2004; 34: 408-414Crossref PubMed Scopus (127) Google Scholar, Mooers, 2009Mooers B.H.M. Crystallographic studies of DNA and RNA.Methods. 2009; 47: 168-176Crossref PubMed Scopus (26) Google Scholar, Choi and Majima, 2011Choi J. Majima T. Conformational changes of non-B DNA.Chem. Soc. Rev. 2011; 40: 5893-5909Crossref PubMed Scopus (264) Google Scholar). X-ray crystallography has been the gold standard of structural biology. However, other techniques, such as nuclear magnetic resonance, cryoelectron microscopy, and neutron crystallography, are rapidly advancing. Neutrons have the advantage over X-rays in that they scatter off atomic nuclei instead of electron clouds, and the neutron scattering power of an atom does not depend on its atomic number. Thus, the lightest atom, hydrogen (H), can be directly observed, usually as its heavier isotope deuterium (D), in neutron structures at resolutions as low as 2.5–2.6 Å (Blakeley, 2009Blakeley M.P. Neutron macromolecular crystallography.Cryst. Rev. 2009; 15: 157-218Crossref Scopus (86) Google Scholar, Banco et al., 2016Banco M.T. Mishra V. Ostermann A. Schrader T.E. Evans G.B. Kovalevsky A. Ronning D.R. Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states.Proc. Natl. Acad. Sci. USA. 2016; 113: 13756-13761Crossref PubMed Scopus (22) Google Scholar, Gerlits et al., 2017aGerlits O.O. Coates L. Woods R.J. Kovalevsky A. Mannobiose binding induces changes in hydrogen bonding and protonation states of acidic residues in concanavalin A as revealed by neutron crystallography.Biochemistry. 2017; 56: 4747-4750Crossref PubMed Scopus (22) Google Scholar, Gerlits et al., 2017bGerlits O. Keen D.A. Blakeley M.P. Louis J.M. Weber I.T. Koavelsvky A. Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K.J. Med. Chem. 2017; 60: 2018-2025Crossref PubMed Scopus (21) Google Scholar). In contrast, observing H atoms in X-ray structures requires data collected to ultra-high resolutions of 1.0 Å or better, although even at such high resolutions most interesting and functionally important H atoms are often still not detected by X-ray crystallography (Gardberg et al., 2010Gardberg A.S. Del Castillo A.R. Weiss K.L. Meilleur F. Blakeley M.P. Myles D.A.A. Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 558-567Crossref PubMed Scopus (37) Google Scholar). In addition, neutrons used in neutron crystallographic experiments have wavelengths in the range of 1–5 Å; these “cold” neutrons do not cause direct radiation damage to macromolecular crystals, in contrast to X-rays with the same wavelengths, producing radiation damage-free biomacromolecular structures. Furthermore, the benign nature of cold neutrons allows neutron crystallographic data to be collected equally well at room and cryo temperatures, allowing functional observations to be made at room temperature. Herein we examine in detail the joint X-ray/neutron (XN) structures of the self-complementary A-DNA octamer d[GTGG(CSe)CAC]2 (1), exchanged with D2O, obtained at room temperature (1_RT structure at 2.0-Å resolution) and at 100 K (1_LT structure at 1.9-Å resolution). This study was enabled by selenium (Se) derivatization at the 2′-position of the ribose in Cyt5 nucleotide (CSe), making it possible to collect neutron crystallographic data from radically smaller crystals of ∼0.1 mm3 in volume than previously required sizes of several cubic millimeters. By introducing the 2′-SeCH3 group, crystallization of the oligonucleotide was enhanced, allowing us to obtain well-diffracting crystals suitable for neutron diffraction. Chemical modification of nucleic acids with Se was pioneered and developed in the lab of Zhen Huang (Lin et al., 2011Lin L. Sheng J. Huang Z. Nucleic acid X-ray crystallography via direct selenium derivatization.Chem. Soc. Rev. 2011; 40: 4591-4602Crossref PubMed Scopus (56) Google Scholar). It was demonstrated that introduction of Se to nucleobases and the ribose sugar ring significantly improved crystal growth and the diffraction quality of oligonucleotide crystals, and helped with phasing X-ray crystallographic data (Jiang et al., 2007Jiang J. Sheng J. Carrasco N. Huang Z. Selenium derivatization of nucleic acids for crystallography.Nucleic Acids Res. 2007; 35: 477-485Crossref PubMed Scopus (57) Google Scholar, Salon et al., 2008Salon J. Jiang J. Sheng J. Gerlits O.O. Huang Z. Derivatization of DNAs with selenium at 6-position of guanine for function and crystal structure studies.Nucleic Acids Res. 2008; 36: 7009-7018Crossref PubMed Scopus (55) Google Scholar, Salon et al., 2010Salon J. Sheng J. Gan J. Huang Z. Synthesis and crystal structure of 2′-Se-modified guanosine containing DNA.J. Org. Chem. 2010; 75: 637-641Crossref PubMed Scopus (27) Google Scholar). The most striking observation in the current neutron crystallographic study was the detection of the backbone protonation at RP oxygen of Ade7 phosphate in 1_RT, crystallized without organic polycations at a pH of 5.6. Moreover, when the crystal was flash-frozen in liquid nitrogen, this backbone phosphate protonation was replaced with magnesium (Mg2+) ion coordination in 1_LT, with the metal cation having proper octahedral coordination by five D2O water molecules and the RP oxygen (Figure 1). It appears that the metal coordination of a phosphate in the A-DNA is very labile, with most Mg(D2O)62+ ions being disordered in the crystal at room temperature. Conversely, metal coordination may correspond to the global energy minimum on the internal potential energy hypersurface of the A-DNA, while at room temperature the oligonucleotide adopts a configuration that corresponds to a local energy minimum whereby the backbone phosphate of Ade7 is protonated, similar to what was previously suggested in studies of proteins (Gerlits et al., 2017aGerlits O.O. Coates L. Woods R.J. Kovalevsky A. Mannobiose binding induces changes in hydrogen bonding and protonation states of acidic residues in concanavalin A as revealed by neutron crystallography.Biochemistry. 2017; 56: 4747-4750Crossref PubMed Scopus (22) Google Scholar, Gerlits et al., 2017bGerlits O. Keen D.A. Blakeley M.P. Louis J.M. Weber I.T. Koavelsvky A. Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K.J. Med. Chem. 2017; 60: 2018-2025Crossref PubMed Scopus (21) Google Scholar, Kovalevsky et al., 2018Kovalevsky A. Aggarwal M. Velazquez H. Cuneo M.J. Blakeley M.P. Weiss K.L. Smith J.C. Fisher S.Z. McKenna R. “To be or not to be” protonated: atomic details of human carbonic anhydrase-clinical drug complexes by neutron crystallography and simulation.Structure. 2018; 26: 383-390Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The octameric self-complementary oligonucleotide 1 crystallizes in a tetragonal unit cell (P43212), with one DNA oligomer in the asymmetric unit and the double helix generated through the crystallographic 2-fold axis, similar to the PDB-deposited structure PDB: 4FP6 (our unpublished data). Crystals of 1 suitable for X-ray and neutron crystallography grew in organic polycation-free conditions, containing Mg2+ as the sole source of the DNA counter ion. The neutron structures of 1, obtained at 2.0-Å and 1.9-Å resolution at room temperature (RT) and cryo temperature (LT), respectively, were refined jointly with the 1.56-Å (RT) and 1.65-Å (LT) X-ray crystallographic data to produce joint XN structures 1_RT and 1_LT (Tables S1 and S2). All exchangeable H atoms were observed as D, and we were able to model 29 and 39 heavy water (D2O) molecules in 1_RT and 1_LT, respectively. 1 adopts the A-DNA form in the crystal, having C3′-endo ribose sugar ring puckering, a narrow deep major groove, and a wide shallow minor groove (Figure 1). Conversely, in B-DNA the sugar rings prefer C2′-endo puckering, whereas the geometric characteristics of the grooves are opposite to those in the A-DNA. The B-DNA's major groove is wide and the minor groove is narrow. As usually observed in A-DNA structures (Wahl and Sundaralingam, 1997Wahl M.C. Sundaralingam M. Crystal structures of A-DNA duplexes.Biopolymers. 1997; 44: 45-63Crossref PubMed Scopus (0) Google Scholar), the deep major groove is hydrated to a higher extent than the shallow minor groove, as illustrated in Figure S1, with several water molecules H-bonded to the backbone phosphates. There are only eight ordered D2O molecules observed in the minor groove close to the oligonucleotide's 3′ and 5′ termini in each 1_RT and 1_LT structure. These water molecules are conserved, but move slightly, when the crystal is flash-frozen in liquid nitrogen. By comparison, the major groove contains at least 30 water molecules in each structure. In addition, the major groove harbors two Mg(D2O)2+ complexes positioned close to the middle of the oligonucleotide between the nucleobases of Gua4 and Cyt6. Each Mg ion interacts through the outer sphere contacts with the nucleobases of Gua3 and Gua4, with H-bond distances of 2.6–2.9 Å. Metal ions stabilize the multiple negative charges on the DNA backbone phosphates but are rarely seen in crystal structures (Wahl and Sundaralingam, 1997Wahl M.C. Sundaralingam M. Crystal structures of A-DNA duplexes.Biopolymers. 1997; 44: 45-63Crossref PubMed Scopus (0) Google Scholar). The 2′-SeCH3 modification of Cyt5 is clearly beneficial for crystal growth, as the SeCH3 group forms intermolecular van der Waals contacts with the 5′-Gua1 nucleotide, which help create tight packing of the DNA molecules in the crystal (Figure S2). We did not observe H/D exchange of C8-H proton on any of the three guanine nucleobases in 1_RT or 1_LT, although significant exchange of this proton with D in guanine was previously documented in the neutron structure of hexameric Z-DNA (Chatake et al., 2005Chatake T. Tanaka I. Umino H. Arai S. Niimura N. The hydration structure of a Z-DNA hexameric duplex determined by a neutron diffraction technique.Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 1088-1098Crossref PubMed Scopus (42) Google Scholar). In addition, unlike in the previous neutron structure of the decameric A-DNA (Leal et al., 2010Leal R.M.F. Callow S. Callow P. Blakeley M.P. Cardin C.J. Denny W.A. Teixeira S.C.M. Mitchell E.P. Forsyth V.T. Combined neutron and X-ray diffraction studies of DNA in crystals and solutions.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 1244-1248Crossref PubMed Scopus (9) Google Scholar), we did not observe protonation of N7 in guanine or N3 in adenine nucleobases in our current XN structures. Unexpectedly, we detected protonation of the RP oxygen of Ade7 phosphate in the RT XN structure 1_RT (Figure 2). A strong peak is observed in the difference FO-FC neutron-scattering density length map located at a distance about 1 Å from the RP oxygen. The density peak was interpreted as D, because no extra electron density was seen near this oxygen. The D-atom occupancy was refined to 67%; thus, the RP-oxygen atom is two-thirds protonated. The RP oxygen is weakly H-bonded with a water molecule that bridges the phosphates of Ade7 and Cyt6, with O-D⋅⋅⋅O distances of 2.3–2.4 Å. Such phosphate-bridging water molecules are common in A-DNA structures. When the crystal of oligonucleotide 1 was flash-frozen in liquid nitrogen and the XN structure 1_LT determined, to our surprise we find that the Ade7 backbone phosphate is no longer protonated. Instead, Mg2+ ion hydrated with five D2O molecules binds to the RP oxygen, completing its octahedral coordination sphere (Figure 3). The metal and water molecules are clearly visible in the neutron scattering length density and electron density maps, while there is no indication of a D-atom presence near the backbone phosphate. Thus, the D atom bound to RP oxygen of Ade7 in 1_RT is replaced with metal coordination at cryo temperature in 1_LT. The water molecule that acted as a bridge between Ade7 and Cyt6 phosphates in 1_RT is pushed away from Ade7 by the incoming metal complex (W6 in Figure 3), losing its H bond with the former but keeping the H bond with Cyt6 in 1_LT. Neutron structures of nucleic acids have been studied mainly by fiber diffraction (Fuller et al., 2004Fuller W. Forsyth T. Mahendrasingam A. Water-DNA interactions as studied by X-ray and neutron fibre diffraction.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 1237-1248Crossref PubMed Scopus (65) Google Scholar). There are only four neutron crystallographic structures of DNA reported in the literature (Chatake et al., 2005Chatake T. Tanaka I. Umino H. Arai S. Niimura N. The hydration structure of a Z-DNA hexameric duplex determined by a neutron diffraction technique.Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 1088-1098Crossref PubMed Scopus (42) Google Scholar, Leal et al., 2010Leal R.M.F. Callow S. Callow P. Blakeley M.P. Cardin C.J. Denny W.A. Teixeira S.C.M. Mitchell E.P. Forsyth V.T. Combined neutron and X-ray diffraction studies of DNA in crystals and solutions.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 1244-1248Crossref PubMed Scopus (9) Google Scholar, Arai et al., 2005Arai S. Chatake T. Ohhara T. Kurihara K. Tanaka I. Suzuki N. Fujimoto Z. Mizuno H. Niimura N. Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements.Nucleic Acids Res. 2005; 33: 3017-3024Crossref PubMed Scopus (97) Google Scholar, Fenn et al., 2011Fenn T.D. Schnieders M.J. Mustyakimov M. Wu C. Langan P. Pande V.S. Brunger A.T. Reintroducing electrostatics into macromolecular crystallographic refinement: application to neutron crystallography and DNA hydration.Structure. 2011; 19: 523-533Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), while there are still no neutron structures of RNA. Well-diffracting crystals of Z-DNA oligonucleotides can usually be grown, and the 1.4- and 1.6-Å resolution neutron structures have been published (Arai et al., 2005Arai S. Chatake T. Ohhara T. Kurihara K. Tanaka I. Suzuki N. Fujimoto Z. Mizuno H. Niimura N. Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements.Nucleic Acids Res. 2005; 33: 3017-3024Crossref PubMed Scopus (97) Google Scholar, Fenn et al., 2011Fenn T.D. Schnieders M.J. Mustyakimov M. Wu C. Langan P. Pande V.S. Brunger A.T. Reintroducing electrostatics into macromolecular crystallographic refinement: application to neutron crystallography and DNA hydration.Structure. 2011; 19: 523-533Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). On the contrary, much lower 2.4- and 3-Å resolution neutron structures of A- and B-form DNA oligonucleotides have been obtained from crystals of several cubic millimeters in volume. The 2′-SeCH3 modification introduced on the ribose sugar ring of Cyt5 in A-DNA oligonucleotide 1 resulted in high-diffraction-quality crystals and made it possible to obtain high-resolution neutron diffraction data—2.0 Å at RT and 1.9 Å at 100 K—from crystals that are about an order of magnitude smaller in volume. In addition, we were able to investigate the effect of temperature on the crystal structure and protonation states in DNA, via neutrons. Neutrons are the perfect probe to visualize D atoms in biological macromolecules. With the neutron scattering power of D being as good as that of C, N, and O, positions of virtually all D atoms can be determined in a neutron structure, whereas X-rays often cannot provide this information even at ultra-high resolutions, especially for water molecules (Brzezinski et al., 2011Brzezinski K. Brzuszkiewicz A. Dauter M. Kubicki M. Jaskolski M. Dauter Z. Hihg regularity of Z-DNA revealed by ultra high-resolution crystal structure at 0.55 Å.Nucleic Acids Res. 2011; 39: 6238-6248Crossref PubMed Scopus (47) Google Scholar). In the RT XN structure 1_RT obtained using a crystal grown at pH 5.6, we unequivocally observed protonation of the RP oxygen atom of the Ade7 backbone phosphate (Figure 2). The D atom on the phosphate is not involved in any H-bonding interactions and the O-D group is rotated inward, facing the adenine nucleobase. It is interesting to note that unusual protonation of guanine at N7 nitrogen and protonation of adenine nucleobase were demonstrated in the neutron structure of another A-DNA oligonucleotide (Leal et al., 2010Leal R.M.F. Callow S. Callow P. Blakeley M.P. Cardin C.J. Denny W.A. Teixeira S.C.M. Mitchell E.P. Forsyth V.T. Combined neutron and X-ray diffraction studies of DNA in crystals and solutions.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 1244-1248Crossref PubMed Scopus (9) Google Scholar). Protonated and positively charged cytosine and adenine are well known and have been implicated in affecting polymerase fidelity and ribozyme general acid-base catalysis (Wilcox et al., 2011Wilcox J.L. Ahluwalia A.K. Bevilacqua P.C. Charged nucleobases and their potential for RNA catalysis.Acc. Chem. Res. 2011; 44: 1270-1279Crossref PubMed Scopus (68) Google Scholar, Wilcox and Bevilacqua, 2013aWilcox J.L. Bevilacqua P.C. A simple fluorescence method for pKa determination in RNA and DNA reveals highly shifted pKa’s.J. Am. Chem. Soc. 2013; 135: 7390-7393Crossref PubMed Scopus (46) Google Scholar, Wilcox and Bevilacqua, 2013bWilcox J.L. Bevilacqua P.C. pKa shifting in double-stranded RNA is highly dependent upon nearest neighbours and bulge positioning.Biochemistry. 2013; 52: 7470-7476Crossref PubMed Scopus (19) Google Scholar). However, protonation of either the backbone phosphate or the guanine nucleobase is unexpected, because the pKa values of these protonated functional groups must be much lower than 5. Perhaps the A-conformation of DNA might induce the increase in the intrinsic basicity of some nucleobases and the phosphate backbone, shifting their intrinsic pKa upward. In addition, H-ion concentration in the vicinity of DNA may be significantly higher than in the bulk solution, as suggested by theoretical calculations (Lamm and Pack, 1990Lamm G. Pack G.R. Acidic domains around nucleic acids.Proc. Natl. Acad. Sci. USA. 1990; 87: 9033-9036Crossref PubMed Scopus (106) Google Scholar, Jayaram et al., 1989Jayaram B. Sharp K.A. Honig B. The electrostatic potential of B-DNA.Biopolymers. 1989; 28: 975-993Crossref PubMed Scopus (258) Google Scholar). Moreover, protonation of the phosphate moiety has been previously shown for uracil and thymine residues (Wu et al., 2017Wu R.R. Hamlow L.A. He C.C. Nei Y.-W. Berden G. Oomens J. Rodgers M.T. The intrinsic basicity of the phosphate backbone exceeds that of uracil and thymine residues: protonation of the phosphate moiety is preferred over the nucleobase for pdThd and pUrd.Phys. Chem. Chem. Phys. 2017; 19: 30351-30361Crossref PubMed Google Scholar), and the DNA alkylating antitumor agents are believed to be activated by the DNA backbone phosphate protonating the drug's carbonyl to generate a reactive carbocation (Hurley and Needham-VanDevanter, 1986Hurley L.H. Needham-VanDevanter D.R. Covalent binding of antitumor antibiotics in the minor groove of DNA. Mechanism of action of CC-1065 and the pyrrolo(1,4)benzodiazepines.Acc. Chem. Res. 1986; 19: 230-237Crossref Scopus (187) Google Scholar, Warpehoski and Harper, 1994Warpehoski M.A. Harper D.E. Acid-dependent electrophilicity of cyclopropyl-pyrroloindoles. Nature’s masking strategy for a potent DNA alkylator.J. Am. Chem. 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Metal ions are believed to have essential biological roles in nucleic acid folding and enzymatic reactions (Aoki and Murayama, 2012Aoki K. Murayama K. Nucleic acid-metal ion interactions in the solid state.in: Interplay between Metal Ions and Nucleic Acids Metal Ions in Life Sciences. vol. 10. Springer Science, 2012: 43-102Google Scholar, Sigel and Sigel, 2013Sigel R.K.O. Sigel H. Metal-ion interactions with nucleic acids and their constituents.in: Comprehensive Inorganic Chemistry II. vol. 3. Elsevier, 2013: 623-660Google Scholar). Metal ion interactions with nucleic acids are important for counterbalancing the high concentration of charged phosphate groups in DNA and RNA, but usually only a handful of metal ions are sufficiently ordered to be observed in crystal structures (Wahl and Sundaralingam, 1997Wahl M.C. Sundaralingam M. Crystal structures of A-DNA duplexes.Biopolymers. 1997; 44: 45-63Crossref PubMed Scopus (0) Google Scholar, Leonarski et al., 2017Leonarski F. D’Ascenzo L. Auffinger P. Mg2+ ions: do they bind to nucleobase nitrogens?.Nucleic Acids Res. 2017; 45: 987-1004Crossref PubMed Scopus (56) Google Scholar). In 1_RT, two Mg2+ ions are observed as hexahydrated Mg(D2O)62+ complexes bound in the major groove (Figure 1). Mg ions interact through the outer-sphere contacts with the nucleobases of Gua3 and Gua4, which is typical for A-DNA oligonucleotide structures (Aoki and Murayama, 2012Aoki K. Murayama K. Nucleic acid-metal ion interactions in the solid state.in: Interplay between Metal Ions and Nucleic Acids Metal Ions in Life Sciences. vol. 10. Springer Science, 2012: 43-102Google Scholar, Robinson et al., 2000Robinson H. Gao Y.-G. Sanishvili R. Joachimiak A. Wang A.H.-J. Hexahydrated magnesium ions bind in the deep major groove and at the outer mouth of A-form nucleic acid duplexes.Nucleic Acids Res. 2000; 28: 1760-1766Crossref PubMed Scopus (68) Google Scholar). Thus, Mg2+ ions and the protonated Ade7 backbone phosphates provide six positive charges to balance the fourteen negative charges present in the double helix of oligonucleotide 1, with the rest of charge balancing presumably coming from the metal ions that are disordered in the structure. Surprisingly, in the low-temperature XN structure 1_LT, the Ade7 backbone phosphate protonation is replaced with metal coordination. We detected no D atom near the Rp oxygen in the nuclear density map in 1_LT. Instead, there is clear evidence in the electron and nuclear density maps for a pentahydrated Mg ion, Mg(D2O)52+, bound to this oxygen using its sixth available coordination site (Figure 3). Such inner sphere binding of Mg2+ ions to the backbone phosphate is also commonly observed in low-temperature A-DNA oligonucleotide crystal structures (Aoki and Murayama, 2012Aoki K. Murayama K. Nucleic acid-metal ion interactions in the solid state.in: Interplay between Metal Ions and Nucleic Acids Metal Ions in Life Sciences. vol. 10. Springer Science, 2012: 43-102Google Scholar). This observation suggests that, while the DNA duplex retains the original two hydrated Mg2+ ions, one Mg(D2O)62+ complex (for each" @default.
• W2890696136 created "2018-09-27" @default.
• W2890696136 creator A5011183452 @default.
• W2890696136 creator A5035426386 @default.
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• W2890696136 date "2018-12-01" @default.
• W2890696136 modified "2023-10-09" @default.
• W2890696136 title "Temperature-Induced Replacement of Phosphate Proton with Metal Ion Captured in Neutron Structures of A-DNA" @default.
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• W2890696136 doi "https://doi.org/10.1016/j.str.2018.08.001" @default.
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