HELIX: a new modular nucleic acid crystallization screen

Abstract

Crystallization of nucleic acids remains a bottleneck to their structural characterization by X‐ray crystallography. A new 96‐well‐format initial screen for nucleic acids, called HELIX, has been developed at UCL School of Pharmacy, London, on the basis of a detailed analysis of the crystallization conditions from 1450 nucleic acid structures deposited in the Protein Data Bank (PDB), combined with observations and experience acquired in the authors' nucleic acids crystallography laboratory during the crystallization of DNA/RNA quadruplexes and ligand complexes. Despite using traditional buffers, precipitants and salts, the resulting modular screen is designed to offer a variety of approaches to enhance successful crystallization of oligonucleotides with a diverse range of topologies, sequences and molecular weights. HELIX includes a set of 24 conditions divided into four sets that can be mixed (inter‐ and intra‐set) to provide a customizable orthogonal screening tool for experienced users, termed VariX. Additionally, mindful of synchrotron anomalous data collection, cacodylate buffers are avoided in the formulations and an optimized cryocrystallization module is included. This article reviews the crystallization trends and data derived from the PDB and discusses the HELIX screen layout, formulation and results from in‐house crystallization trials.

Native de novo structural determinations of non-canonical nucleic acid motifs by X-ray crystallography at long wavelengths

“…The starting oligonucleotide solution was annealed in the buffer (20 mM potassium cacodylate at pH 6.5, 50 mM potassium chloride) by heating to 95°C for 5 min, followed by gradual cooling to room temperature. The i-motif DNA sequence 5′-d(TAACCCTAA)-3′ (tel-i-motif) and 5′-d(TAACCC Br TAA)-3′ were purchased from Eurofins, HPLC purified and prepared for crystallization as previously described ( 25 ). Meso-Tetra ( N -methyl-4-pyridyl) porphine tetrachloride (TMPyP4, CAS# 92739-63-4) was purchased from Frontier Scientific (Logan, UT, USA).…”

Structure and ligand binding of the SAM-V riboswitch

“…SAM (Sigma-Aldrich) was added to a final concentration of 5 mM. Crystals were grown by sitting drop vapor diffusion at 7°C using drops prepared by mixing 0.3 μl of the RNA–ligand complex with 0.3 μl of a reservoir solution comprising 50 mM Bis-Tris (pH 7.0), 200 mM KCl, 10 mM CaCl 2 dihydrate and 40% v/vPEG 400 (HELIX26) ( 16 ) using a Mosquito robot (TTP LabTech). Crystals (of approximate dimensions 50 × 50 × 50 μm 3 ) with space group P4 1 22 appeared after 7 days.…”

Interactions Between Metal Ions and DNA

“…On a somewhat more day-to-day level, the crystallisation of a typical small synthetic oligonucleotide requires the use of a crystallisation screen, several of which are now commercially available. The classic sparse matric screen 81 has been expanded more recently to a 96 well screen 82 . This screen combines sparse matrix, incomplete factorial approaches and modified orthogonal arrays.…”