Concerted dynamics of metallo-base pairs in an A/B-form helical transition

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-HgII-T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-HgII-(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-HgII-(N4)C base pairs and greater A-form characteristics. Ten-fold different 1J coupling constants (15N,199Hg) are observed for (N3)C-HgII (114 Hz) versus (N4)C-HgII (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-HgII-T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding HgII, demonstrating that C-HgII-T has unique switching potential in DNA-based materials and devices.


Supplementary Figures
Supplementary Figure 1 | MALDI-MS analysis of unlabeled ODN 1 "C-T" and 15 N-labeled ODN 1 * "C*-T*". 15  sequence and imino region of 1 H NMR spectrum. Blue and green bases indicate C-T mismatches and red bases indicate residues flanking C-T mismatches. b) Assignment of thymidine residues flanking (red) the C-T mismatch (blue and green) according to NOE cross peaks between NH imino protons of thymidine. c) Assignment of thymidine imino protons according to NOE cross peaks between NH imino signals of thymidine residues and H2 of adenine residues. d) Assignment of guanosine residues of "C-T" according to NOE cross peaks between NH imino protons. The DNA sample contained 0.5 mM duplex DNA in aqueous buffer (200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 7.0). Spectra were recorded at 4 °C. Figure 5 | Assignment of NH imino protons of ODN 1 "C-T" in the presence of 1.5 equiv Hg II . a) "C-T" sequence and imino region of 1 H NMR spectrum. Blue and green bases indicate C-T mismatches and red bases indicate residues flanking C-T mismatches. b) Assignment of thymidine imino protons according to NOE cross peaks between NH imino signals of thymidine residues and H2 of adenine residues. c) Assignment of thymidine and guanosine imino proton signals in the presence of Hg II according to NOE cross peaks between NH imino protons. The DNA sample contained 0.5 mM duplex DNA and 1.5 mM Hg II (1.5 equiv relative to mismatch) in aqueous buffer (200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 7.0). Spectra were recorded at 4 °C. Equiv of Hg II given relative to mismatch. b) 2 J 1 H, 15 N coupling between thymidine N1 and H6 (green) and 3 J-and 2 J 1 H, 15 N coupling between cytosine N1 and H5 and H6  DNA samples contained 0.5 mM duplex DNA (ODN 1 * "C*-T*") and 1.5 mM Hg II (1.5 equiv relative to mismatch) in aqueous buffer (200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 7.8) and were measured at the indicated temperature.  Figures 9 and 10). b) NH2,H5 and NH,H5 NOE cross peaks and NH2,NH exchange cross peaks observed in the 15 N-coupled (red) and 15 N-decoupled (blue) [ 1 H, 1 H]-NOESY spectra at 25 °C. The DNA sample contained 1 mM duplex DNA (ODN 1 * "C*-T*") and 3 mM of Hg(ClO4)2 (1.5 equiv Hg II relative to mismatch) in aqueous buffer (200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 7.8). HSQC spectra of ODN 1 * "C*-T*" upon addition of 199 Hg-isotopically enriched Hg(ClO4)2 provided direct evidence for Hg IInucleobase coordination sites. a) 1 J 15 N, 199 Hg coupling according to 15 N NMR spectrum of ODN 1 * "C*-T*" with 1.5 equiv of 199 Hgenriched Hg(ClO4)2 (79 % enriched, Supplementary Figure 13). b) 1 J 15  (1.5 equiv Hg II relative to mismatch) in aqueous buffer (200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 7.8), and the spectra were measured at 4 °C. and ODN 1 "C-Hg II -T" sequence. Blue and green bases indicate C-T mismatches. Sequential walking along H1' and aromatic protons in the [ 1 H, 1 H]-NOESY spectra through the entire sequence from residues C1 to G14 for apo-(b), major-(c) and minor (d) duplex DNA. The DNA sample of the apo duplex (ODN 1 "C-T") contained 0.3 mM duplex DNA in an aqueous solution of NaClO4 (50 mM, D2O, pD = 7.75) and was measured at 25 °C. The DNA sample of the "C-Hg II -T" contained 0.4 mM duplex DNA (ODN 1 "C-T") and 1.2 mM Hg II (1.5 equiv Hg II relative to mismatch) in an aqueous solution of NaClO4 (50 mM, D2O, pD = 7.75) and was measured at 25 °C. COSY spectrum used to determine coupling constants for Hg II -bound residues C4 and T11 and coupling constants of T3 as a selected example for a Hg II unbound residue. b) H1',H2' and H1',H2'' cross peaks in the magnitude processed [ 1 H, 1 H]-COSY spectrum for Hg II -coordinated residues. c) H1',H2' and H1',H2'' cross peaks of the major duplex in the magnitude processed [ 1 H, 1 H]-COSY spectrum illustrating similar shape of T11 cross peaks of the minor duplex species and C4 cross peaks of the major duplex species. The DNA sample contained 0.4 mM duplex DNA (ODN 1 "C-T") and 1.2 mM Hg II (1.5 equiv Hg II relative to mismatch) in an aqueous solution of NaClO4 (50 mM, D2O, pD = 7.75) and was measured at 25 °C.  Table 5). According to the Karplus relationship a pseudorotation phase angle (P) 2 of 85° and 202° was calculated for the Hg II -coordinated cytosine residue of the major duplex species. A pseudorotation phase angle of 85° corresponds to an O4'-endo sugar pucker. 2,5 Φ1'2' determined from the solution structures represents the mean value and standard deviation of the 20 lowest energy conformations. Figure was  the middle of a base pair step (Zp). c) P-P distance across the helix. d) P-P distance along the helix. C1'-C1' distance, phosphate displacement, and P-P distance along the helix were calculated using 3DNA (a-b) 11,12 and P-P distance across the helix were determined using PyMOL. 6 Standard A-form (red) and B-form (blue) values for C1'-C1' distance and phosphate displacement were taken from (ref 13) and (ref 14), respectively. Reference A-form and B-form values for P-P distance across the helix were determined by generating an ideal A-form and B-form using scfbio-iitd software 15 and for P-P distance along the helix from (ref 11,12).

Supplementary
Reported values are mean and standard deviation of the 20 lowest energy conformations of major-(black), minor-(grey) and apo (green) duplex species. Source data are provided as a Source Data file.

Supplementary Figure 22 | Base pair parameters X-and Y-displacement, shift and slide and curvature analysis. a)
Translation of base pair towards grooves (a, x-displacement) and perpendicular to the grooves (b, y-displacement). Local dimer step parameters shift (c, Dx) and slide (d, Dy) describing relative positioning of successive base pairs with respect to the x-and y-axis of the relative base pair reference frame. e) axial bend and f) curvature analysis of major-, minor-, and apo duplexes. Curvature is a dimensionless quantity normalizing the curvature to DNA on the nucleosome (curvature ~ 1). 16 All parameters were analyzed using (1.5 equiv Hg II relative to mismatch) in aqueous buffer (200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 6 -9).  name duplex sequence ε260
Sugar pucker conformations were determined using Curves+. 7 A single C3'-endo sugar pucker was observed in one of the 20 models of the major duplex. Otherwise no C3'-endo sugar puckers were observed.  Figure 5a and Supplementary Figures 39-42. Equiv of Hg II are given relative to mismatch.

Synthesis and Folding of Duplex DNA
Unmodified oligonucleotides were purchased from Sigma-Aldrich as HPLC-purified sequences. 15 Figure 1). Oligonucleotide stock solutions were prepared in deionized water and their concentrations were determined by absorbance at 260 nm using a molar extinction coefficient (ε260) calculated using a nearest-neighbor model. 18,19 For calculated extinction coefficients see Supplementary

Melting Temperature Analysis (Tm) and CD Spectroscopy
Thermal denaturation temperatures of duplex DNA were determined by measuring the absorbance at 260 nm as a function of temperature in a 1 mm path length thermo-controlled strain-free quartz cuvette on a JASCO J-715 spectrometer equipped with a temperature control system. Solutions of pre-folded duplex H2O / D2O) and the pH was adjusted to 7.75 by addition of an aqueous solution of NaOH. The samples were annealed as described above and if necessary the pH was readjusted to pH = 7.75. Samples measured in D2O were prepared in an aqueous solution of NaClO4 (50 mM) and the pH was adjusted to 7.75 by addition of an aqueous solution of NaOH and annealed as described above. The samples were lyophilized, dissolved in 99.9% D2O and the pD was adjusted to 7.35 by addition of a solution of NaOD in 99.9 % D2O. DNA samples used for structure determination measurements and containing Hg II were treated with Chelex-100 (BIO-RAD) for 10 min after addition of Hg II (1.5 equiv with respect to mismatch) to remove unspecific Hg II coordination. 20 Samples measured at 4 °C were equilibrated at 4 °C for 15 min prior to measuring.

NMR Spectra Measurements
1 H NMR spectra were recorded on a Bruker Avance II 500 MHz spectrometer equipped with a TXI zaxis gradient probe head using excitation sculpting for water suppression. Proton chemical shifts were referenced to the water line at 4.70 ppm at the given temperature. The spectra were processed with a line broadening factor of 10 Hz. [ 1 H, 1 H]-NOESY spectra for unlabeled oligonucleotides in 200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 7.0 and for 15 N-labeled oligonucleotides (200 mM NaClO4, 50 mM cacodylic acid in H2O / D2O (9:1) at pH = 7.8) were recorded on a Bruker Avance 600 MHz spectrometer equipped with a TCI z-axis gradient CryoProbe at 4 °C with mixing times of 100 ms and 120 ms, respectively. Proton chemical shifts were referenced to the water line at 4.70 ppm at the given temperature. 1D 15 N NMR spectra were recorded on a Bruker Avance II 500 MHz spectrometer equipped with a BBO z-axis gradient CryoProbe at 4 °C or 25 °C using either inverse gated or no proton decoupling.
An inter-scan delay of 1 s was used with a nominal 30° pulse for excitation and the pulse sequence employed a gradient spin echo of 2.5 ms duration prior to acquisition in order to get rid of the strong background signal. The total experiment time was typically on the order of 72 h. 15 N chemical shifts were indirectly referenced against 1 H using Ξ = 0.101329118. 21 The spectra were processed with a line-broadening factor were recorded on a Bruker Avance 600 MHz spectrometer equipped with a TCI z-axis gradient CryoProbe.

Calculation of Pseudorotation Phase Angle
The pseudorotational phase angle (P) was determined from a plot of 3 J H1',H2' as a function of P. 2 The dihedral angle Φ1' 2' was calculated according to the Karplus relation. 3   with td = delay time, R1 = autorelaxation rate, k1 and k-1 = rate constants of interconversion and a0 , c, and c0, q = constants.

Variable Ionic Strength
To

Rate Constants of Global Helix Interconversion
To determine rate constants of global interconversion of the duplexes, we measured