Pathogenic C9ORF72 Antisense Repeat RNA Forms a Double Helix with Tandem C:C Mismatches

Transcription

1 Supporting Information Pathogenic C9ORF72 Antisense Repeat RNA Forms a Double Helix with Tandem C:C Mismatches David W. Dodd, Diana R. Tomchick, David R. Corey, and Keith T. Gagnon METHODS S1

2 RNA synthesis. RNA for crystallization was commercially synthesized by Oligo Factory and purified by ion exchange chromatography to 89% purity as determined by mass spectrometry. RNA for circular dichroism (CD) and differential scanning calorimetry (DSC) was synthesized and HPLCpurified by Integrated DNA Technologies. RNA sequences are listed in Table S2. RNA Crystallization. RNA was dissolved in 40 mm sodium cacodylate, ph 7.2, 50 mm KCl at a final concentration of 2 mm and refolded by heating to 95 C for 5 min then slow cooling at a rate of ~1 C/min. Crystal growth for [(CCCCGG) 3 CCCC] RNA was screened using a Nucleic Acid Mini Screen kit from Hampton Research. Refolded RNA at 2 mm was mixed 1:1, 1.5:1 or 2:1 with screening solutions in 2-3 µl drops and grown by a hanging-drop vapor diffusion method with equilibration over 0.5 ml of reservoir solution [35% v/v (+/-)-2-methyl-2,4-pentanediol]. Crystal A grew in condition #23 [10 % v/v (+/-)-2-methyl-2,4-pentanediol, 40 mm sodium cacodylate trihydrate, ph 7.0, 12 mm spermine tetrahydrochloride, 40 mm lithium chloride, 80 mm strontium chloride hexahydrate] and crystal B grew in condition #11 [10 % v/v (+/-)-2-methyl-2,4-pentanediol, 40 mm sodium cacodylate trihydrate, ph 6.0, 12 mm spermine tetrahydrochloride, 80 mm sodium chloride, 20 mm barium chloride]. Rectangular plate-like crystals appeared after 2 weeks at 4 C for crystal A and grew to their maximal extent by 4 weeks. Similar crystals grew after 4 weeks at 4 C for crystal B. Cryoprotection was performed by transferring the crystal to an aliquot of the reservoir solution, then flash freezing in liquid nitrogen. X-ray diffraction data collection Crystal A exhibited the symmetry of space group P with unit cell parameters of a=41.02 Å, b=47.62 Å, c=58.90 Å and contained one molecule of doublestranded RNA per asymmetric unit. Native crystals diffracted isotropically to a d min of 1.47 Å when exposed to synchrotron radiation. Crystal B exhibited the symmetry of space group P with unit cell parameters of a=41.13 Å, b=47.66 Å, c=59.50 Å and contained one molecule of double-stranded RNA per asymmetric unit. Native crystals diffracted isotropically to a d min of 1.75 Å when exposed to synchrotron radiation. Data were indexed, integrated and scaled using the HKL-3000 program package 1. Data collection statistics are provided in Table S3. S2

3 Phase determination, structure refinement, and structure analysis. Phases for the [(CCCCGG) 3 CCCC] RNA (Crystal A) were obtained via direct methods in the program ACORN 2 using non-anomalous data by artificially extending the data resolution to 1 Å. Phases were generated starting from a random atom, and after 188 cycles of refinement the mean phase error was 24.0 and the correlation coefficient for medium E-values was The final map had a correlation coefficient of An initial model containing about 84% of all nucleotides was automatically generated in the program Nautilus 3. Additional nucleotides and ions were manually modeled in the program Coot 4, and hydrogens were added in the calculated, riding positions in the program Phenix 5. Crystal B was isomorphous to crystal A. Refinement of both structures was performed using the program Phenix with a random 5% of all data set aside for an R free calculation. Model refinement statistics are provided in Table S1. To determine helical structure parameters, the web server version of 3DNA (W3DNA) was used 6. PDB files for crystal A, crystal B and an idealized A-form RNA were uploaded and parameters calculated using default settings. Circular Dichroism Spectroscopy. Circular dichroism (CD) spectroscopic analysis was carried out using a Jasco Model 860 spectropolarimeter with a 0.1 cm cuvette. RNA at 50 µm in 50 mm sodium cacodylate, ph 7.2, 5 mm KCl were heated to 95 C for 5 min followed by slow cooling to room temperature. Samples were scanned three times at 23 C from nm in 1 nm steps at a scan speed of 50 nm/min. The average of three scans was used for all analyses. Molar ellipticity increases proportionally with the size of an RNA helix, which is related to the chirality of the structure. Thus, to better compare CD spectra from repeat RNA of different lengths, raw molar ellipticity values were normalized by RNA length. Thermal Denaturation by Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) was performed on a MicroCal VP-DSC capillary cell microcalorimeter. RNA at 40 or 80 µm in degassed DSC buffer (20 mm Cacodylate, ph 7.2, 50 mm NaCl) were heated to 95 C for 5 min followed by slow cooling to room temperature to allow refolding. Samples were scanned from 25 C to 125 C at a scan rate of 90 C/h. Origin 7.0 with DSC Data Analysis (MicroCal) software was used to analyze and normalize data. Temperatures corresponding to the maximum peak in Cp of the RNA were taken as the melting temperature (T m ). Heat capacity (C p ) increases proportionally with the S3

4 size of an RNA helix, which reflects the number of stabilizing non-covalent bonds. Thus, to better compare C p curves from repeat RNA of different lengths C p values were normalized by RNA length. SUPPORTING FIGURES and TABLES Figure S1. Superpositioning of crystal A and crystal B. (A) Overall architecture and superpositioning of crystal A (orange) and crystal B (blue). (B) Crystal A with a coordinated Sr 2+ ion (blue), (C) crystal B with coordinated Ba 2+ ion (red), and (D) superposition of both crystal A and B. Hydrogen bonding of Sr 2+ and Ba 2+ ions are indicated by yellow dashes. Modeled waters are indicated by red non-bonded stars. The coordination of a single Sr 2+ ion in crystal A and a single Ba 2+ ion in crystal B at an isomorphous position occurs at a lattice contact between the O2 oxygen of cytosine 16 in chain B and the O2 oxygen of cytosine 10 in a symmetry-related chain B. The ions are coordinated to 5 additional water molecules, with bond lengths that range from 2.4 to 2.9 Angstroms. S4

5 Figure S2. CD and DSC for A-form and A-form mismatched RNA. (A) CD of a model c9ftd/als antisense RNA [(CCCCGG) 3 CCCC] annealed to a perfect RNA complement, [(GGGGCC) 3 GGGG], (orange) and CD of a (CUG) 6 repeat RNA (blue). (B) DSC of a model c9ftd/als antisense RNA [(CCCCGG) 3 CCCC] annealed to a perfect complement, [(GGGGCC) 3 GGGG]. S5

6 Table S1. Structure parameters for [(CCCCGG)3(CCCC)] RNA crystals. (values shown in red deviate from the normal range found in typical A-form helical RNA 6.) Table S2. RNA sequences for structural studies. S6

7 Table S3. Crystal structure data collection and refinement statistics. S7

8 SUPPORTING REFERENCES (1) Minor, W.; Cymborowski, M.; Otwinowski, Z.; Chruszcz, M. Acta Crystallogr D Biol Crystallogr 2006, 62, (2) Yao, J. X.; Dodson, E. J.; Wilson, K. S.; Woolfson, M. M. Acta Crystallogr D Biol Crystallogr 2006, 62, (3) Cowtan, K. Acta Crystallogr D Biol Crystallogr 2006, 62, (4) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr D Biol Crystallogr 2010, 66, (5) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr D Biol Crystallogr 2010, 66, (6) Zheng, G.; Lu, X. J.; Olson, W. K. Nucleic Acids Res 2009, 37, W S8