Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

Transcription

1 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

2 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.

3 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, , 2014).

4 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, , 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.

5 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.

6 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.

7 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),

8 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, , 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, , 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).

9 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}

10 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

11 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

12 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

13 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

14 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}

15 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 C C G G A A T U T U T U T U T U T U G G G G C C G G C C C C A A A A A A A A A A A A T U C C G G A 6 -DNA m1g A 6 -RNA m1g C C G G A A T U T U T U T U T U T U G G10 n.d. G G C C G G C C C C A A A A A A A A A A

16 A A T U C C G G gc-dna m1a gc-rna m1a G G C C A A G G T U G G G G C C G G C C C C A A C C T U G G C C A 2 -DNA m1a A 2 -RNA m1a G G C C A A T U C C G G A A T U T U G G G G C C G G C C C C A A A A T U C C G G A A T U G G C C n.d. not detected due to significant line-broadening

17 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 ± ± ± ±0.1 A 2 -DNA m1a duplex ± ± ± ±0.4 A 2 -RNA duplex ± ± ± ±0.8 A 2 -RNA m1a duplex ± ± ± ±0.1 A 6 -DNA duplex ± ± ± ±0.1 A 6 -DNA duplex ± ± ± ±0.3 A 6 -DNA(*) duplex ± ± ± ±0.2 A 6 -DNA m1a duplex ± ± ± ±0.2 A 6 -DNA m1a duplex ± ± ± ±0.2 A 6 -DNA m1a (*) duplex ± ± ± ±(<0.1) A 6 -DNA m1g duplex ± ± ± ±0.1 A 6 -RNA duplex ± ± ± ±0.3 A 6 -RNA duplex ± ± ± ±0.1 A 6 -RNA(*) duplex ± ± ± ±0.1 A 6 -RNA m1a duplex ± ± ± ±0.3 A 6 -RNA m1a (*) duplex ± ± ± ±(<0.1) A 6 -RNA m1g duplex ± ± ± ±0.1 gc-dna duplex ± ± ± ±0.1 gc-dna m1a duplex ± ± ± ±0.3 gc-rna duplex ± ± ± ±0.3 gc-rna m1a duplex ± ± ± ±0.3 hp-a 6 -DNA hairpin ± ± ± ±0.5 hp-a 6 -DNA m1a hairpin ± ± ± ±0.4 hp-a 6 - m1g DNA hairpin ± ± ± ±0.6 hp-a 6 -RNA hairpin ± ± ± ±0.2 hp-a 6 -RNA m1a hairpin ± ± ± ±0.3 hp-a 6 - m1g RNA hairpin ± ± ± ±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± ± ±0.3 A 6 -DNA m1a (*) 5.5± ± ±0.3 A 6 -DNA m1g 6.8± ± ±0.2 hp-a 6 -DNA m1a 17.3± ± ±0.6 hp-a 6 -DNA m1g 17.9± ± ±0.8

18 A 6 -RNA m1a 24.4± ± ±0.3 A 6 -RNA m1a (*) 20.3± ± ±0.1 A 6 -RNA m1g 44.9± ± ±0.3 hp-a 6 -RNA m1a 19.2± ± ±0.4 hp-a 6 -RNA m1g 25.2± ± ±0.3 A 2 -DNA m1a 1.0± ± ±0.4 A 2 -RNA m1a 42.6± ± ±0.8 gc-dna m1a 19.9± ± ±0.4 gc-rna m1a 35.2± ± ±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, , 2007).

19 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= C; ph= C; ph= C; ph=5.4 Magnetization Alignment GS Average GS Average Fitted parameters ω (ppm) 2.73± ± ± ±0.14 p ES (%) 2.466± ± ± ±0.009 k ex (s -1 ) 39±8 595± ± ±210 k forward (s -1 ) 0.96± ± ± ±0.44 k backward (s -1 ) 38.0± ± ± ±209.6 R 1 (s -1 ) 1.62± ± ± ±0.04 R 2 (s -1 ) 33.85± ± ± ±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= C; ph= C; ph= C; ph=5.4 Magnetization Alignment GS Average GS Average Fitted parameters ω (ppm) 2.08± ± ± ±0.13 p ES (%) 1.151± ± ± ±0.052 k ex (s -1 ) 481±75 442± ± ±191 k forward (s -1 ) 5.54± ± ± ±1.30 k backward (s -1 ) 475.5± ± ± ±189.9 R 1 (s -1 ) 2.2± ± ± ±0.09 R 2 (s -1 ) 29.1± ± ± ± State Fitting ES1 ω ES1 (ppm) 1.27± ±0.14 p ES1 (%) 11.05± ±0.229 k ex1 (s -1 ) 32±5 634±320 ES2 ω ES2 (ppm) 7.3± ±0.97 p ES2 (%) 0.102± ±0.022 k ex2 (s -1 ) 8698± ±1146 R 1 (s -1 ) 2.74± ±0.09 R 2 (s -1 ) 26.82± ±0.39

20 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, , 2011).

21 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± ±1.9 hp-a 6 -RNA-HG (run1) hp-a 6 -RNA-HG (run2) A 6 -DNA m1a -HG hp-a 6 -RNA m1a -HG A 6 -DNA ra -HG 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.

22 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

23 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 ( 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: ) 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.)

24 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, , (2002). 2 Zhou, H. et al. New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey. Nucleic Acids Res. 43, , (2015). 3 Nikolova, E. N. et al. Transient Hoogsteen base pairs in canonical duplex DNA. Nature 470, , (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, , (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, , (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, , (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, , (2013). 8 Ferner, J. et al. Structures of HIV TAR RNA Ligand Complexes Reveal Higher Binding Stoichiometries. ChemBioChem 10, , (2009).

25 9 Peterson, R. D. & Feigon, J. Structural Change in Rev Responsive Element RNA of HIV-1 on Binding Rev Peptide. J. Mol. Biol. 264, , (1996). 10 Rich, A. The Era of RNA Awakening: Structural biology of RNA in the early years. Q. Rev. Biophys. 42, , (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, , (2008).