Supplementary Figure 1 Resonance assignment and NMR spectra for hairpin and duplex A 6 constructs. (a) 2D HSQC spectra of hairpin construct (hp-a 6 -RNA) with labeled assignments. (b) 2D HSQC or SOFAST-HMQC spectra of m 1 A or m 1 G modified RNA (in violet) duplexes with labeled assignments overlaid on their unmodified counterparts (in grey). Arrows indicate significant chemical shift perturbations induced by m 1 A or m 1 G. Resonances that are exchanged broadened out of detection due to m 1 A
or m 1 G are highlighted on the corresponding resonance of the unmodified duplexes with a dashed circle. In A 6 -RNA m1a, G13C-C1 has a 6.0 p.p.m. upfield shift suggesting the ribose adopts C2 -endo conformation rather than the C3 -endo conformation typical in A-RNA. (c) Overlay of 1D 1 H spectra showing the imino G-H1 and T/U-H3 resonances at high temperature (35 C). Modified and unmodified duplexes are shown in color and grey, respectively.
Supplementary Figure 2 Lack of detectable exchange across diverse RNA sequence and structural contexts. (a) Secondary structures with bps showing no detectable RD highlighted in red. (b) Off-resonance RD profiles for the highlighted bps with error bars representing experimental uncertainty (one s.d.) estimated from mono-exponential fitting of n = 6 independently measured peak intensities using a Monte-Carlo based method (Methods). RD profiles not shown for wttar have been reported previously (Lee, J. et al., Proc Natl Acad Sci USA. 111, 9485 9490, 2014).
Supplementary Figure 3 1 H assignment of duplex A 6 constructs with m 1 A and m 1 G. Chemical structures of m 1 da dt and m 1 dg dc + HG bps and color-coded duplexes as in Fig. 3a and Fig. 3b, respectively. Representative 2D NOESY spectra showing NOE connectivity (labeled in orange) indicating HG hydrogen-bonding. Residues in grey are broadened out of detection. Non-exchangeable and exchangeable 1 H 1 H sequential connectivity used to generate assignments and intra-nucleotide H1 H8 NOEs used to assess syn conformation are labeled for each residue on the spectra. Resonance assignments for corresponding DNA duplexes A 6 -DNA m1a and A 6 -DNA m1g were reported in other studies (Nikolova, E.N. et al., Nature. 470, 498 502, 2011). The NOE walk is indicated using grey arrows on the duplex structure. Interruption of the sequential walk due to exchange broadening of resonances or syn conformation is shown using dashed lines on the spectra as well as the duplex structure.
Supplementary Figure 4 Resonance assignment and NMR spectra for duplex gc and A 2 constructs. 2D HSQC or SOFAST-HMQC spectra of m 1 A or m 1 G modified DNA (in blue) and RNA (in violet) duplexes with labeled assignments overlaid on their unmodified counterparts (in grey). Folded resonances are indicted using an asterisk. Arrows indicate significant chemical shift perturbations induced by m 1 A or m 1 G. Resonances that are exchanged broadened out of detection due to m 1 A or m 1 G are highlighted on the corresponding resonance of the unmodified duplexes with a dashed circle. Note that in gc-rna m1a, there is an additional resonance showing characteristic chemical shifts of a flipped out uridine based on C6-H6 (in red) and C5-H5 (data not shown) chemical shifts. This resonance is likely U5 that is complementary to m 1 A.
Supplementary Figure 5 1 H assignment of duplex gc and A 2 constructs with m 1 A. Representative 2D NOESY spectra showing NOE connectivity (labeled in orange) indicating HG hydrogen-bonding or syn purine conformation. Residues in grey are broadened out of detection. Non-exchangeable and exchangeable 1 H 1 H sequential connectivity used to generate assignments and intra-nucleotide H1 H8 NOEs used to assess syn conformation are labeled for each residue on the spectra. Resonance assignments for A 2 -DNA m1a will be reported in other study (B.S., H.Z., Y.X., H.M.A., unpublished). Color-coded duplexes are shown as in Fig. 3b. The NOE walk is indicated using grey arrows on the duplex structure. Interruption of the sequential walk due to exchange broadening of resonances or syn conformation is shown using dashed lines on the spectra as well as the duplex structure.
Supplementary Figure 6 NMR analysis of ribonucleotide-substituted A 6 -DNA. (a) Shown are overlays of 2D HSQC spectra of A 6 -DNA ra (in red), A 6 -DNA rg (in green), and unmodified A 6 -DNA (in black) with arrows indicating significant chemical shift perturbations induced by the ra16 or rg10. (b) Shown are 2D HSQC spectra of A 6 -DNA m1ra (in red),
A 6 -DNA m1rg (in green), overlaid with unmodified (in black) A 6 -DNA ra and A 6 -DNA rg respectively, with arrows indicating significant chemical shift perturbations induced by the m 1 ra16 or m 1 rg10. A 6 -DNA ra and A 6 -DNA rg duplexes with 13 C/ 15 N labeled residues (Methods) colored in red. ra16 and rg10 are shown in bold. Sites used as NMR RD probes are highlighted with a circle. (c) Comparison of 2-state and 3-state BM fitting of RD profiles measured on dc15-c6 in A 6 -DNA rg. Error bars refer to one s.d. estimated from mono-exponential fitting of n = 6 independently measured peak intensities using the Monte-Carlo method (Methods). The data is statistically better satisfied using a 3-state rather than 2-state fit though this minimally impacts the exchange parameters obtained for the HG state (Supplementary Table 4). (d) van t Hoff analysis (Nikolova, E.N. et al., Nature. 470, 498 502, 2011), using combined data from the current study (da16-c8 RD at 10ºC) with previously published varying temperature A16-C8 RD data (Nikolova, E.N. et al., Nature. 470, 498 502, 2011), shows that RD of da16-c8 at 10ºC is in better agreement with the 2-state exchange model with AVG than GS initial alignment of magnetization during the BM fitting. (e) Shown are chemical shift perturbations ( = Hoogsteen Watson- Crick) for syn purine-c8 and purine-c1 in A-RNA, apical loop or mispairs from experimental data ( EXPT ) and DFT calculations ( DFT ) (Supplementary Note). Error bars shown for fitted parameters from RD measurements represent experimental uncertainty (one s.d.) estimated from mono-exponential fitting using the Monte-Carlo method (Methods).
Supplementary Table 1 Spin lock powers and offsets used in the RD experiments. Nuclei/pH/T/Mg 2+ /Field [spin lock power]{offset frequencies} //( C)/ (mm)/ (MHz) [ω SL 2π -1 (s -1 )]{Ω 2π -1 (s -1 )} ra16-c8/5.4/25/0/600 ra16-c8/6.8/25/0/700 ra16-c8/6.8/35/0/700 ra16-c8/6.8/25/4/600 rg11-c8/6.8/25/4/600 hp-a 6 -RNA [200]{±10,78,156,234,312,390,468,546,624,702} [400]{±10,156,312,468,624,780,936,1092,1248,1404} [1000]{±10,389,778,1167,1556,1945,2334,2723,3112,3501} [150,200,300,400,500,600,700,800,1000,1500,2000,3000]{0} [150]{±50,100,150,200,250,300,350,400} [400]{±100,200,300,400,500,600,700,800} [1000]{±100,200,400,600,800,1100,1400,2000,3000} ra3-c8/6.8/5/4/600 [100,150,200,250,300,400,600,800,1000,150 ra3-c1 /6.8/5/4/600 ra3-c8/5.4/25/0/600 ra16-c1 /5.4/25/0/600 ra3-c1 /5.4/25/0/600 [100,150,200,250,300,400,600,800,1000,150 [200]{±10,78,156,234,312,390,468,546,624,702} [400]{±10,156,312,468,624,780,936,1092,1248,1404} [1000]{±10,156,312,468,624,780,936,1092,1248,1404} [200]{±10,78,156,234,312,390,468,546,624,702} [400]{±10,156,312,468,624,780,936,1092,1248,1404} [1000]{±10,156,312,468,624,780,936,1092,1248,1404} [200]{±10,78,156,234,312,390,468,546,624,702} [400]{±10,156,312,468,624,780,936,1092,1248,1404} [1000]{±10,156,312,468,624,780,936,1092,1248,1404}
ra3-c1 /6.8/25/0/700 [150,200,250,300,350,400,500,600,700,800,900,1000,1200,1400,1600,180 rg10-c8/5.4/25/0/600 [200]{±10,78,156,234,312,390,468,546,624,702} [400]{±10,156,312,468,624,780,936,1092,1248,1404} [1000]{±10,156,312,468,624,780,936,1092,1248,1404} rg10-c8/6.8/25/0/700 rg10-c8/6.8/35/0/700 [150,200,300,400,500,600,700,800,1000,1500,2000,3000]{0} [150]{±50,100,150,200,250,300,350,400} [400]{±100,200,300,400,500,600,700,800} [1000]{±100,200,400,600,800,1100,1400,2000,3000} rg11-c8/6.8/25/4/600 rg10-n1/5.4/13/0/600 [100,150,200,250,300,350,400,450,500,550,600,700,800,900,1000,1200,14 00,1600,1800,2000]{0} E-gc ra7-c8/6.8/5/0/600 ra7-c8/6.8/10/0/600 ra7-c8/6.8/25/0/700 ra7-c8/5.4/10/0/700 ra7-c8/5.4/25/0/700
ra7-c8/5.4/35/0/700 ru9-n3/6.8/5/0/600 rg1-c8/5.4/10/0/700 rg1-c8/5.4/35/0/700 rg19-c8/8.4/25/0/700 rg7-c8/8.4/25/0/700 rg19-c1 /8.4/25/0/700 rg7-c1 /8.4/25/0/700 rc29-c6/5.4/25/0/600 [150]{±50,100,150,200,250,300,350,400} [400]{±100,200,300,400,500,600,800,1000} [1000]{±100,200,400,600,800,1100,1400,1800,2200} [100,150,200,250,300,350,400,450,500,550,600,700,800,900,1000,1200,14 00,1600,1800,2000]{0} [150]{±25,50,100,150,200,250,300,400} [400]{±50,100,150,200,250,350,500,800,1000} [1000]{±50,150,250,350,450,550,800,1100,1300,1600} [150]{±50,100,150,200,250,300,350,400} [400]{±100,200,300,400,500,600,800,1000} [1000]{±100,200,400,600,800,1100,1400,1800,2200} [150,200,300,400,500,600,700,800,1000,1500,2000,3000]{0} [150]{±50,100,150,200,250,300,350,400} [400]{±100,200,300,400,500,600,800,1000} [1000]{±100,200,400,600,800,1100,1400,1800,2200} hp-gc GU [150]{±60,90,120,150,180,240,300,360,390,420} [250]{±50,100,200,300,350,400,450,500,600,720} [350]{±100,200,300,400,450,500,600,700,800,1000} [800]{±100,200,400,500,600,800,1100,1400,1800,2400} [150,200,250,300,350,400,500,600,700,800,1000,1200,1400,1600,1800,20 00,2500,3000]{0} [150]{±60,90,120,150,180,240,300,360,390,420} [250]{±50,100,200,300,350,400,450,500,600,720} [350]{±100,200,300,400,450,500,600,700,800,1000} [800]{±100,200,400,500,600,800,1100,1400,1800,2400} [150,200,250,300,350,400,500,600,700,800,1000,1200,1400,1600,1800,20 00,2500,3000]{0} [150]{±60,90,120,150,180,240,300,360,390,420} [250]{±50,100,200,300,350,400,450,500,600,720} [350]{±100,200,300,400,450,500,600,700,800,1000} [800]{±100,200,400,500,600,800,1100,1400,1800,2400} wttar
rc41-c6/5.4/25/0/600 ra27-c8/6.8/10/0/700 ra27-c8/6.8/25/0/700 ra27-c1 /6.8/10/0/700 ra27-c1 /6.8/25/0/700 rg28-c8/6.8/10/0/700 rg28-c8/6.8/25/0/700 TAR-UUCG GU [150,200,250,300,350,400,500,600,700,800,900,1000,1200,1400,1600,180 [150,200,250,300,350,400,500,600,700,800,900,1000,1200,1400,1600,180 [200]{±50,100,150,200,250,300,400,500,600,700} [300]{±100,200,300,400,500,600,700,800,900,1000} A 6 -DNA
da16-c8/5.4/10/0/600 [100]{-420,-360,-330,-300,-270,-240,-210,-180,-150,-120,-90,-60,- 30,30,60,90,120,150,180,210,240,270,300,360,420} [150]{-540,-510,-480,-450,-420,-390,-360,-330,-300,-270,-240,-200,-160,- 120,-80,-40,40,80,120,160,200,240,300,360,420,480} [200]{-660,-630,-600,-570,-540,-510,-480,-450,-420,-390,-360,-300,-270,- 240,-210,-180,-150,-120,-90,-60,- 30,30,60,90,120,150,180,210,240,270,300,360,420,480,540,600} [250]{-720,-660,-600,-540,-480,-450,-420,-390,-360,-300,-270,-240,-180,- 120,-60,60,120,180,240,300,360,420,480,540,600,660,720} [300]{-1000,-800,-650,-600,-550,-500,-450,-400,-350,-300,-250,-200,-150,- 100,-50,50,100,150,200,250,300,350,400,450,500,600,800,1000} [500]{±75,150,225,300,375,450,550,650,800,1000,1200,1400,1600} A 6 -DNA ra da16-c8/5.4/10/0/600 [100]{-360,-330,-300,-270,-240,-210,-180,-150,-120,-90,-60,- 30,360,300,240,180,120,90,60,30,-500,-450,-400,-350,-250,-200,-100,- 50,500,450,400,350,250,200,150,100,50,-540,-510,-480,-420,-390,480,420} [150]{-540,-510,-500,-480,-450,-420,-400,-390,-360,-350,-300,-250,-240,- 200,-180,-150,-120,-100,-60,- 50,50,60,100,120,150,180,200,240,250,300,350,360,400,420,450,480,500} [200]{-660,-630,-600,-570,-540,-510,-480,-450,-420,-390,-360,-300,-270,- 240,-180,-120,-60,60,120,180,240,270,300,360,420,480,540,600} [250]{-720,-660,-630,-600,-570,-540,-510,-480,-450,-420,-400,-360,-350,- 300,-240,-200,-180,-150,-120,-100,-60,- 50,50,60,100,120,150,180,200,240,300,350,360,400,420,450,480,540,600, 660,720} [500]{-1600,-1400,-1200,-1000,-800,-640,-600,-560,-520,-480,-440,-400,- 300,-200,-100,100,200,300,400,500,600,800,1000,1200,1400,1600} A 6 -DNA rg
dc15-c6/5.4/25/0/600 [150,200,250,300,350,400,450,500,550,600,650,700,800,900,1000,1500,20 00,2500,3000,3500]{0} [150]{±10,48,96,144,192,240,289,337,385,433,481,530} [200]{±10,63,127,190,254,318,381,445,509,572,636,700} [250]{±10,80,160,240,320,400,480,560,640,720,800,880} [300]{±10,95,190,286,381,477,572,668,763,859,954,1050} [400]{±10,95,190,286,381,477,572,668,763,859,954,1050}
Supplementary Table 2 Chemical shift perturbations induced by m 1 A and m 1 G. Per nucleotide chemical shift perturbations ( ω residue ) are computed as described in methods. Also shown is the average chemical shift perturbation ( ω avg ) computed by averaging the ω residues measured for the two bps above and below the site of the m1a or m1g modification. Sequence ω residue (p.p.m.) ω avg (p.p.m.) Sequence ω residue (p.p.m.) ω avg (p.p.m.) A 6 -DNA m1a A 6 -RNA m1a C1 0.022 0.085 C1 0.004 0.122 G2 0.020 G2 0.003 A3 0.022 A3 0.004 T4 0.016 U4 0.005 T5 0.017 U5 0.027 T6 0.025 U6 0.027 T7 0.068 U7 0.027 T8 0.050 U8 0.138 T9 0.822 U9 0.147 G10 0.046 G10 0.148 G11 0.037 G11 0.123 C12 0.053 C12 0.056 G13 0.035 G13 0.764 C14 0.036 C14 0.078 C15 0.247 C15 0.122 A16 0.997 A16 0.642 A17 0.165 A17 0.253 A18 0.028 A18 0.087 A19 0.029 A19 0.041 A20 0.015 A20 0.004 A21 0.015 A21 0.011 T22 0.016 U22 0.004 C23 0.019 C23 0.004 G24 0.022 G24 0.005 A 6 -DNA m1g A 6 -RNA m1g C1 0.026 0.084 C1 0.105 0.114 G2 0.019 G2 0.065 A3 0.014 A3 0.038 T4 0.020 U4 0.028 T5 0.018 U5 0.026 T6 0.025 U6 0.009 T7 0.022 U7 0.010 T8 0.049 U8 0.038 T9 0.129 U9 0.057 G10 0.700 G10 n.d. G11 0.148 G11 0.086 C12 0.069 C12 0.161 G13 0.040 G13 0.100 C14 0.087 C14 0.139 C15 0.320 C15 0.352 A16 0.099 A16 0.258 A17 0.051 A17 0.077 A18 0.034 A18 0.020 A19 0.019 A19 0.018 A20 0.016 A20 0.006
A21 0.016 A21 0.010 T22 0.059 U22 0.015 C23 0.022 C23 0.013 G24 0.032 G24 0.030 gc-dna m1a gc-rna m1a G1 0.007 0.083 G1 0.051 0.088 C2 0.031 C2 0.020 A3 0.064 A3 0.037 G4 0.056 G4 0.092 T5 0.160 U5 0.160 G6 0.084 G6 0.163 G7 0.013 G7 0.095 C8 0.017 C8 0.020 G9 0.009 G9 0.024 C10 0.047 C10 0.035 C11 0.184 C11 0.087 A12 0.766 A12 0.683 C13 0.184 C13 0.157 T14 0.030 U14 0.036 G15 0.035 G15 0.014 C16 0.014 C16 0.015 A 2 -DNA m1a A 2 -RNA m1a G1 0.015 0.060 G1 0.006 0.103 C2 0.006 C2 0.004 A3 0.005 A3 0.004 T4 0.033 U4 0.010 C5 0.012 C5 0.005 G6 0.033 G6 0.014 A7 0.014 A7 0.043 T8 0.019 U8 0.136 T9 0.062 U9 0.095 G10 0.071 G10 0.120 G11 0.025 G11 0.100 C12 0.016 C12 0.035 G13 0.009 G13 0.021 C14 0.037 C14 0.023 C15 0.233 C15 0.104 A16 0.988 A16 0.658 A17 0.004 A17 0.214 T18 0.076 U18 0.082 C19 0.016 C19 0.014 G20 0.029 G20 0.003 A21 0.006 A21 0.012 T22 0.033 U22 0.003 G23 0.039 G23 0.000 C24 0.007 C24 0.022 n.d. not detected due to significant line-broadening
Supplementary Table 3 Thermodynamic parameters from UV melting. Construct structure C T (µm) ph Tm ( C) -ΔH (kcal mol -1 ) -ΔS (e.u.) -ΔG 25 C (kcal mol -1 ) A 2 -DNA duplex 2.0 6.8 47.6±0.3 93.0±1.5 262.5±4.6 14.7±0.1 A 2 -DNA m1a duplex 2.0 6.8 41.2±0.6 92.0±6.6 265.3±21.0 12.9±0.4 A 2 -RNA duplex 3.0 6.8 56.8±0.1 106.0±8.1 294.5±24.6 18.2±0.8 A 2 -RNA m1a duplex 3.0 6.8 43.6±0.3 63.4±2.0 173.7±6.2 11.7±0.1 A 6 -DNA duplex 3.0 5.4 38.8±0.2 84.5±0.9 244.1±2.5 11.7±0.1 A 6 -DNA duplex 3.0 6.8 42.1±0.3 87.3±4.2 250.4±13.2 12.6±0.3 A 6 -DNA(*) duplex 2.0 6.8 50.0±0.2 79.2±2.4 217.6±7.4 14.3±0.2 A 6 -DNA m1a duplex 3.0 5.4 30.6±0.6 75.8±3.3 223.4±10.4 9.2±0.2 A 6 -DNA m1a duplex 3.4 6.8 32.1±0.3 69.6±3.9 201.5±12.4 9.5±0.2 A 6 -DNA m1a (*) duplex 3.0 6.8 41.1±0.1 73.7±0.8 207.8±2.5 11.7±(<0.1) A 6 -DNA m1g duplex 6.4 5.4 31.8±0.3 77.7±3.5 229.7±11.6 9.2±0.1 A 6 -RNA duplex 3.0 5.4 43.0±0.2 83.7±4.6 237.9±14.4 12.7±0.3 A 6 -RNA duplex 2.3 6.8 42.2±0.1 100.8±2.6 293.9±8.4 13.2±0.1 A 6 -RNA(*) duplex 2.0 6.8 54.0±0.1 102.1±1.3 284.5±4.0 17.2±0.1 A 6 -RNA m1a duplex 3.0 6.8 30.4±0.3 76.4±11.8 226.6±38.6 8.9±0.3 A 6 -RNA m1a (*) duplex 2.0 6.8 37.4±0.2 81.8±1.0 235.9±3.2 11.5±(<0.1) A 6 -RNA m1g duplex 3.4 5.4 17.6±2.6 38.8±14.0 106.8±47.2 7.0±0.1 gc-dna duplex 4.0 6.8 34.9±0.3 57.1±2.9 159.2±9.3 9.6±0.1 gc-dna m1a duplex 3.0 6.8 14.6±3.5 37.2±7.0 103.3±22.8 6.4±0.3 gc-rna duplex 3.5 5.4 48.7±0.3 75.4±1.6 207.9±4.6 13.4±0.3 gc-rna m1a duplex 3.5 5.4 28.1±1.9 40.2±4.6 107.0±14.7 8.3±0.3 hp-a 6 -DNA hairpin 3.0 6.8 71.8±0.3 94.1±3.6 272.7±10.3 12.8±0.5 hp-a 6 -DNA m1a hairpin 2.0 6.8 67.1±0.3 76.8±3.3 225.9±9.9 9.5±0.4 hp-a 6 - m1g DNA hairpin 2.0 5.4 63.0±0.2 76.2±5.5 226.7±16.4 8.6±0.6 hp-a 6 -RNA hairpin 2.5 6.8 69.9±0.3 94.9±1.0 276.5±2.7 12.4±0.2 hp-a 6 -RNA m1a hairpin 2.0 6.8 61.4±0.3 75.7±2.3 226.4±6.7 8.2±0.3 hp-a 6 - m1g RNA hairpin 2.0 5.4 55.8±0.2 69.7±2.9 211.9±8.8 6.5±0.2 T=25 C Construct ΔΔH (kcal mol -1 ) TΔΔS (kcal mol -1 ) ΔΔG 25 C (kcal mol -1 ) A 6 -DNA m1a 17.7±5.7 14.5±5.4 3.1±0.3 A 6 -DNA m1a (*) 5.5±2.5 2.9±2.3 2.6±0.3 A 6 -DNA m1g 6.8±3.6 4.3±3.5 2.5±0.2 hp-a 6 -DNA m1a 17.3±4.9 14.0±4.2 3.3±0.6 hp-a 6 -DNA m1g 17.9±6.6 13.8±5.7 4.2±0.8
A 6 -RNA m1a 24.4±12.1 20.1±11.8 4.3±0.3 A 6 -RNA m1a (*) 20.3±1.6 14.5±1.5 5.8±0.1 A 6 -RNA m1g 44.9±14.7 39.1±14.7 5.7±0.3 hp-a 6 -RNA m1a 19.2±2.5 15.0±2.1 4.2±0.4 hp-a 6 -RNA m1g 25.2±3.1 19.3±2.8 5.9±0.3 A 2 -DNA m1a 1.0±6.8-0.8±6.4 1.8±0.4 A 2 -RNA m1a 42.6±8.3 36.0±7.5 6.5±0.8 gc-dna m1a 19.9±7.6 16.5±7.3 3.4±0.4 gc-rna m1a 35.2±4.9 30.1±4.6 5.1±0.4 Errors shown represent one s.d. (n=3 independent measurements); hp-a 6 -DNA represents a hairpin construct having A 6 -DNA sequence with C1 G24 capped by a GAA apical loop (5 -G24GAAC1); (*) indicates data collected in the buffer containing 15mM sodium phosphate, 100mM NaCl, 4mM Mg 2+ and ph=6.8 with errors shown representing one s.d. from curve-fitting (Methods) of a single measurement (n=1) and the uncertainty in the calculated thermodynamic parameters were determined by error propagation as reported previously (Siegfried, N.A. et al., Biochemistry. 46, 172 181, 2007).
Supplementary Table 4 Exchange parameters from fitting RD data in A 6 -DNA, A 6 - DNA ra and A 6 -DNA rg with 2- or 3-state Bloch-McConnell equations with various initial magnetization alignment. 2-State Fitting Construct A 6 -DNA A 6 -DNA A 6 -DNA ra A 6 -DNA ra Nuclei da16-c8 da16-c8 ra16-c8 ra16-c8 Condition 10 C; ph=5.4 10 C; ph=5.4 10 C; ph=5.4 10 C; ph=5.4 Magnetization Alignment GS Average GS Average Fitted parameters ω (ppm) 2.73±0.07 2.8±0.07 3.79±0.14 3.73±0.14 p ES (%) 2.466±0.343 0.298±0.04 0.175±0.008 0.182±0.009 k ex (s -1 ) 39±8 595±106 2504±212 2325±210 k forward (s -1 ) 0.96±0.24 1.77±0.40 4.38±0.42 4.23±0.44 k backward (s -1 ) 38.0±7.8 593.2±105.7 2499.6±211.6 2320.8±209.6 R 1 (s -1 ) 1.62±0.03 1.59±0.04 1.64±0.03 1.63±0.04 R 2 (s -1 ) 33.85±0.08 33.42±0.08 42.27±0.13 42.28±0.14 Construct A 6 -DNA A 6 -DNA A 6 -DNA rg A 6 -DNA rg Nuclei dc15-c6* dc15-c6* dc15-c6 dc15-c6 Condition 26 C; ph=5.2 25 C; ph=5.2 25 C; ph=5.4 25 C; ph=5.4 Magnetization Alignment GS Average GS Average Fitted parameters ω (ppm) 2.08±0.06 2.09±0.05 2.24±0.12 2.31±0.13 p ES (%) 1.151±0.122 1.328±0.149 0.59±0.051 0.582±0.052 k ex (s -1 ) 481±75 442±65 1289±181 1294±191 k forward (s -1 ) 5.54±1.04 5.87±1.08 7.60±1.25 7.53±1.30 k backward (s -1 ) 475.5±74.1 436.1±64.1 1281.4±180.0 1286.5±189.9 R 1 (s -1 ) 2.2±0.11 2.08±0.11 2.78±0.08 2.76±0.09 R 2 (s -1 ) 29.1±0.15 29.02±0.14 27.71±0.26 27.62±0.28 3-State Fitting ES1 ω ES1 (ppm) 1.27±0.15 1.84±0.14 p ES1 (%) 11.05±2.008 0.784±0.229 k ex1 (s -1 ) 32±5 634±320 ES2 ω ES2 (ppm) 7.3±3.72 6.21±0.97 p ES2 (%) 0.102±0.053 0.09±0.022 k ex2 (s -1 ) 8698±3526 2394±1146 R 1 (s -1 ) 2.74±0.06 2.6±0.09 R 2 (s -1 ) 26.82±0.92 27.26±0.39
Errors represent experimental uncertainty (one s.d.) estimated from mono-exponential fitting using a Monte-Carlo based method (Methods). * data published previously (Nikolova, E.N. et al., Nature. 470, 498 502, 2011).
Supplementary Table 5 Averaged percentage of trajectory time for HG hydrogen bonding formation in unbiased MD simulations of B-DNA and A-RNA. N7---H-N3 ( %) O4---H-N6 (%) A 6 -DNA-HG 89.0±7.0 97.5±1.9 hp-a 6 -RNA-HG (run1) 2.4 99.3 hp-a 6 -RNA-HG (run2) 82.7 37.5 A 6 -DNA m1a -HG 61.6 99.7 hp-a 6 -RNA m1a -HG 6.6 37.5 A 6 -DNA ra -HG 97.5 98.5 Errors represent one s.d. n=5 independent MD trajectories for A 6 -DNA-HG; others shown are data taken from a single trajectory for the corresponding construct.
Supplementary Note syn purines in A-form helices The PDB survey identified one helical ra ru HG bp in RNA which is surrounded by non-canonical motifs in the X-ray structure of the P4-P6 domain of the Group I intron RNA (PDBID: 1L8V 1 ). Interestingly, the χ-angle ( 70º) for the syn ra is similar to χ-angles found in DNA HG bps ( 70º), which in turn differs by 170º from typical anti χ-angles in B-DNA but by 130º from typical anti χ-angles in A- RNA. Accommodation of this HG bp is accompanied by significant changes in the torsion angles α and γ which resemble those in Z-form DNA duplexes. In addition, the residue 3 to the syn ra adopts a DNA-like C2 -endo sugar pucker. The survey also identified nine HG-like bps with syn purines in A-RNA duplexes that do not form HGtype H-bonds. Similar HG-like conformations were previously reported in B-DNA 2. In these HG-like bps, the C1 C1 distance ( 10.5 Å) is not constricted as in HG bps ( 8.5 Å) but remains WC-like ( 10.7 Å). The syn purine is accommodated through changes in backbone angles α, γ, δ and ε. In addition, rg syn ra anti and rg syn rg anti HG mismatches were identified in A-RNA with even larger C1 C1 distance (11.3±0.1 Å). Here, the syn purine is accommodated through changes in the torsion angles α and γ. Interestingly, the χ-angle for these syn purine bases ( 15º) differs by 180º relative to the anti χ-angle in A-RNA ( -160º). These results indicate that while syn purines can be accommodated within A-RNA, constriction of the C1 C1 distance may require substantial changes in the A-RNA structure. We did not identify any additional steric clashes when adding a methyl group at the N 1 -position of the syn purine base in A-RNA or B-DNA, indicating that the lower stability of HG bps in m 1 ra and m 1 rg containing A-RNA is not due to
unique steric contacts involving the methyl group as verified in molecular dynamics (MD) simulations (Supplementary Movies 5 and 6). HG chemical shifts in RNA In B-DNA, HG bps result in downfield shifts ( 3 p.p.m.) in the sugar-c1, purine-c8, and dc-c6 carbon chemical shifts and smaller ( 1.8 p.p.m.) upfield shifts in the dg-n1 and dt-n3 imino nitrogen chemical shifts 3,4. The downfield shift in sugar-c1 5,6 and purine-c8 are both attributed to the change in the χ-angle from anti to syn conformation whereas the upfield shift in dt-n3 and dg-n1 is attributed to weaker H-bonds 4. The downfield shift in dc-c6 is due to protonation of dc-n3 7. The absence of detectable RD in WC bps within A-RNA is unlikely to be due to smaller differences in chemical shifts between WC and HG for the various spins targeted for RD measurements. First, significant (1.4 5.8 p.p.m.) downfield shifted purine-c8 and/or purine-c1 chemical shifts have been reported for syn rg in the UUCG tetraloop (e.g. BMRID: 16431 8 ) which forms a reverse wobble bp ( UUCG ), rg anti rg syn mispairs near a bulge in the HIV-1 Rev responsive element ( RRE ) 9, transient HG bps in ra16 and rg10 substituted A 6 -DNA ( RD ), and trapped HG bp in m 1 rg10 substituted A 6 -DNA (Supplementary Fig. 5e). Second, DFT calculations on a variety of HG bp configurations suggest significant differences in chemical shifts for WC and HG bps (Supplementary Fig. 5e). These HG configurations include the ra ru HG bp in P4-P6 (PDBID: 1L8V 1 ); tertiary rg rc + HG bp in the structure of 23S ribosomal RNA-protein complex (PDBID: 3U56 10 ); snapshots of ra ru HG bp from the biased MD simulations (Methods); and rg syn rg anti HG mismatches (PDBID: 3CZW 11 ) (Supplementary Fig. 5e). In all cases, we observe sizeable downfield shifts in purine-c8 (4.3±2.8 p.p.m.) and C1 (2.8±1.9 p.p.m.)
consistent with the values observed in BMRB. Finally, sizeable chemical shift changes are expected for base N1/N3 and protonated dc + -C6 even for these specific HG configurations. We also carried out DFT calculations on a number of different HG configurations in RNA that feature C3 -endo or C2 -endo sugar with and without N 1 -methyl. These data show that for most configurations, large changes in chemical shifts are expected in either or both the base and sugar chemical shifts due to WC-HG transitions (data not shown). In general, the calculations indicate that the N 1 -methyl induces a downfield shift for ra-c8 in both anti and syn purine conformations, analogous to what is observed in DNA (data not shown), but that it minimally affects the chemical shifts of anti or syn rg- C8 and the sugar-c1. Supplementary References 1 Battle, D. J. & Doudna, J. A. Specificity of RNA RNA helix recognition. Proceedings of the National Academy of Sciences 99, 11676-11681, (2002). 2 Zhou, H. et al. New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey. Nucleic Acids Res. 43, 3420-3433, (2015). 3 Nikolova, E. N. et al. Transient Hoogsteen base pairs in canonical duplex DNA. Nature 470, 498-502, (2011). 4 Nikolova, E. N., Gottardo, F. L. & Al-Hashimi, H. M. Probing transient Hoogsteen hydrogen bonds in canonical duplex DNA using NMR relaxation dispersion and single-atom substitution. J. Am. Chem. Soc. 134, 3667-3670, (2012). 5 Xu, X.-P. & Au Yeung, S. C. F. Investigation of chemical shift and structure relationships in nucleic acids using NMR and density functional theory methods. The Journal of Physical Chemistry B 104, 5641-5650, (2000). 6 Fonville, J. M. et al. Chemical shifts in nucleic acids studied by density functional theory calculations and comparison with experiment. Chemistry A European Journal 18, 12372-12387, (2012). 7 Nikolova, E. N., Goh, G. B., Brooks, C. L., III & Al-Hashimi, H. M. Characterizing the protonation state of cytosine in transient G C Hoogsteen base pairs in duplex DNA. J. Am. Chem. Soc. 135, 6766-6769, (2013). 8 Ferner, J. et al. Structures of HIV TAR RNA Ligand Complexes Reveal Higher Binding Stoichiometries. ChemBioChem 10, 1490-1494, (2009).
9 Peterson, R. D. & Feigon, J. Structural Change in Rev Responsive Element RNA of HIV-1 on Binding Rev Peptide. J. Mol. Biol. 264, 863-877, (1996). 10 Rich, A. The Era of RNA Awakening: Structural biology of RNA in the early years. Q. Rev. Biophys. 42, 117-137, (2009). 11 Rypniewski, W., Adamiak, D. A., Milecki, J. & Adamiak, R. W. Noncanonical G(syn) G(anti) base pairs stabilized by sulphate anions in two X-ray structures of the (GUGGUCUGAUGAGGCC) RNA duplex. RNA 14, 1845-1851, (2008).