SUPPLEMENTARY INFORMATION. Pistol Ribozyme Adopts a Pseudoknot Fold. Facilitating Site-specific In-line Cleavage
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1 UPPLEMENTAY INFMATIN Pistol ibozyme Adopts a Pseudoknot Fold Facilitating ite-specific In-line Cleavage Aiming en 1,2,4, Nikola Vušurović 3,4, Jennifer Gebetsberger 3, Pu Gao 2, Michael Juen 3, Christoph Kreutz 3, onald Micura 3 and Dinshaw J. Patel 2 1 Life ciences Institute, Zhejiang University, Hangzhou, , China 2 tructural Biology Program Memorial loan-kettering Cancer New York, NY, Institute of rganic Chemistry Leopold-Franzens University and Center of Molecular Biosciences CMBI Innsbruck A-6020, Austria 4 These authors made equal contributions *Corresponding authors: pateld@mskcc.org (DJP) and ronald.micura@uibk.ac.at (M)
2 UPPLEMENTAY EULT upplementary Table 1. Crystallographic statistics for env25 pistol ribozyme Crystal Native [Ir(NH 3 ) 6 ] 3+ soak Mn 2+ soak Data collection 24-ID-C 24-ID-C 24-ID-C pace group P322 1 P322 1 Cell dimensions a, b, c (Å) 56.2, 56.2, , 56.5, , 56.0, 89.5 α, β, γ (º) 90, 90, , 90, , 90, 120 Peak Wavelength (Å) esolution (Å) ( )* ( ) ( ) pim 0.037(0.727) 0.038(0.618) (0.589) I/σI 12.8 (0.9) 14.9 (1.2) 9.4 (1.1) Completeness (%) 99.8 (100) 99.8 (100) 95.7 (98.5) edundancy 6.2 (6.7) 6.2 (6.4) 3.1 (3.2) efinement esolution (Å) No. reflections work/ free 0.20/ / /0.29 No. atoms NA Cations Water B-factors NA Cations Water m.s deviations Bond lengths (Å) Bond angles (º) *Values for the highest-resolution shell are in parentheses. 2
3 UPPLEMENTAY FIGUE CAPTIN upplementary Figure 1. Long-range pairing alignments in the structure of the env25 pistol ribozyme. a, epresentation of the continuous stacking alignment involving stem segments (starting from the bottom) P3, non-canonical paired segment, PK and P1. b, Hydrogen-bond network involving the U17-A18-A19-A20-A21-(U20)-A23 segment and the minor groove of stem P1. c, The minor groove triple involving the minor groove edge of G24 and the Watson-Crick G5-C12 pair. G5 is a highly conserved residue in the pistol ribozyme. d, Mg 2+ binding site close to, but not directly coordinated to, the catalytic cleavage G53-U54 site in the pistol ribozyme. e, An expended view of the bound Mn 2+ cation-binding site at the G53-U54 cleavage site in the pistol ribozyme, with the Mn 2+ anomalous electron density map contoured at σ level. Mn 2+ cation is shown as a purple sphere. upplementary Figure 2. elf-cleavage of the env25 pistol ribozyme. a, Cartoon presentation of the cleavage assay. b, HPLC trace of wild type (WT), and mutants c, A32U, and d, A32G. Cleavage analyzed at 55 µm NA each strand; 2 mm MgCl 2, 100 mm KCl, 30 mm HEPE, ph 7.5, 23 C. 47-nt ribozyme, 11-nt substrate; C1 and C2, 6-nt and 5-nt cleavage products. HPLC conditions: Dionex DNAPac colum (4 x 250 mm), 80 C, 1 ml min -1, 0 60% buffer B in 45 min. Buffer A: Tris HCl (25 mm), urea (6 M), ph 8.0. Buffer B: Tris HCl (25 mm), urea (6 M), NaCl 4 (0.5 M), ph 8.0. upplementary Figure 3. NM spectroscopic demonstration of stem P2 formation of the env25 pistol ribozyme in solution. a, Chemical structures of 3
4 15 N1/3-labeled U26 used. b, 1 J( 1 H, 15 N) HQC spectra of the 15 N-U26 pistol complex showing the appearance of a correlation corresponding to substrate binding. upporting Figure 4. NM spectroscopic analysis of the env25 pistol ribozyme in solution reference experiments. a, Chemical structure of the 13 C2-labeled adenosine used. b, 1 J( 1 H, 13 C) HQC spectra of a short NA duplex with labeled A in a Watson-Crick base pair at varying ph values; the protonated form is in slow exchange; estimated pk a value as indicated. c, Exemplary 1 J( 1 H, 13 C) HQC spectra of phdependent NM experiments of a 13 C2-A labeled single-stranded NA (left), chemical shift changes of 13 C2-A, with changes in the ph value derived from 1 J( 1 H, 13 C) HQC spectra (right); the line represents the least-square fit (also see reference 44 ) estimated pk a values as indicated. Conditions: c(na) = 80 to 120 µm; 25 mm NaCl, 2 mm MgCl 2, 15 mm sodium phosphate, H 2 /D 2 9:1, 298 K. upplementary Figure 5. Temperature dependence of the cleavage kinetics of the env25 pistol ribozyme. a, Chemical structure of Ap nucleoside used. b, Quantitative assay for rate determination at 20 C. Exemplary fluorescence time course recorded at two different temperatures can be compared as shown in panel b (20 C) and Fig. 6c (15 C). 4
5 a C1 G16 U3 C13 C12 G11 G4 G5 C34 PK G33 G42 C36 U7 U44 G10 A8 A23 U37 A38 U3 A21 G5 c G40 U43 P3 U17 A39 C41 A14 G2 G9 C35 G16 C15 C13 G2 U17 C15 A14 P1 b A20 C12 G4 U18 A19 G5 G52 G45 A51 A46 C50 U47 U49 G24 C34 e d G33 G40 G40 U54 G33 U54 G53 G53 up. Fig. 1
6 a b c d G U 5' 5' Mg 2+ mau x WT start A32U 10 min A32G U-H-5' mau x min 5' 2',3'-cp-G 3' C2 C1 5' mau x Time (min) Time (min) Time (min) up. Fig. 2
7 a NA U26 P 5' 15 N 15 NH b N / ppm H NA H / ppm up. Fig. 3
8 a b 5'-GCUUAAGCCCU UCCCGAAUUCG-5' c NA P A32 ph 2.43 ph 3.39 NH 2 N N N N 3' H 13 CH NA 5'-GAAUU ph 3.89 ph 4.43 ph C / ppm ph 6.73 ph 3.72 ph 4.97 ph 3.58 pka A ~3.6 ph 3.84 ph H / ppm 1H / ppm 1H / ppm 5'-GAAUU pka A ~ C / ppm 13C / ppm 13C / ppm H / ppm ph value 6.5 up. Fig. 4
9 a NA P N N N N NH 2 A57Ap H NA b Fluorescence (a.u.) mm Mg 2+ k obs = 2.72 ± 0.38 min C t (s) up. Fig. 5
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