Computational protocol: Click Modification of RNA at Adenosine: Structureand Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosinein RNA

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Protocol publication

[…] X-ray diffraction data from all three crystals were collected on beamline 7-1 at the Stanford Synchrotron Radiation Lightsource (SSRL) at 100 K. Diffraction intensities were processed and scaled with the software packages XDS and XSCALE, respectively. Originally the data were indexed, processed, and scaled in space group R32, with unit cell parameters a = b = 43.2 Å and c = 125.8 Å (hexagonal setting) and with one RNA 16 mer in the crystallographic asymmetric unit (the crystal 2-fold generated the palindromic duplex). Although the data scaled well as R32 (Rmerge < 5%), subsequent refinement after structure solution resulted in the Rfree value incapable of decreasing less than ∼30%, possibly indicating the wrong space group. Therefore, data was reanalyzed and reprocessed in the lower symmetry space group C2, with unit cell parameters a = 75.0 Å, b = 43.1 Å, c = 48.8 Å, and β = 120.98°, with three RNA 16 mers (1.5 duplexes) in the crystallographic unit (VM = 2.0 Å3/Da, solvent content ∼60%). Strands B and C generated one duplex, and strand A was situated at the crystal 2-fold to generate an A–A duplex. Subsequent structure refinement resulted in Rfree values ∼24%, suggesting that the correct space group is C2 with pseudo R32 symmetry. Data collection and processing statistics are listed in Table 1 in the . The original native structure was solved by molecular replacement using the program PHASER. The search model consisted of a single-stranded 8 mer RNA structure (PDB ID: 1YZD) that had the same palindromic sequence as the unknown but was modified by 2′-amino ribose on position 6. The native structure was used as a phasing model to solve the two base-modified structures: 7-EAA modified and 7-EAA-triazole modified 16 nt RNAs. The atomic models were built with the molecular graphics program COOT and refined using REFMAC. Geometric restraint libraries, used in refinement for modified bases, were generated using CCP4 programs Library Sketcher and libchek to create dictionary files. Refinement was carried out with local noncrystallographic symmetry (NCS) restraints. Removing the NCS restraints resulted in increasing the Rfree value and did not improve the electron density. Final refinement statistics are listed in Table 1 in the . The 7-EAA-modified base showed clear electron density to incorporate the two extra carbon atoms in the adenine-modified base at position 5. The 7-EAA-triazole-modified structure showed clear electron density for the triazole group off carbon-7. However, weak broken electron density in the major groove near the triazole group prevented us from confidently building the piperidine moiety and suggests that the piperidine group is flexible or adopts multiple conformations. This weak piperidine electron density is observed in all three RNA strands in the asymmetric unit for the C2 crystal and was completely absent in the R32 space group refinement. […]

Pipeline specifications

Software tools XDS, Coot
Applications Small-angle scattering, Protein structure analysis
Diseases Bird Diseases
Chemicals Adenosine, Alkynes, Copper, Ribonucleosides, Uridine