Crystallization of the 10-23 DNA enzyme using a combinatorial screen of paired oligonucleotides

Abstract

One of the most dif®cult steps in the X-ray crystallography of nucleic acids is obtaining crystals that diffract to high resolution. The choice of the nucleotide sequence has proven to be more important in producing high-quality crystals than the composition of the crystallization solution. This manuscript describes a systematic procedure for identifying the optimal sizes of a multi-stranded nucleic acid complex which provide high-quality crystals. This approach was used to crystallize the in vitro evolved 10-23 DNA enzyme complexed with its RNA substrate. In less than two months, 81 different enzyme±substrate complexes were generated by combinatorial mixing and annealing of complementary oligonucleotides which differed in length, resulting in duplexes of varying length, with or without nucleotide overhangs. Each of these complexes was screened against a standard set of 48 crystallization conditions and evaluated for crystal formation. The screen resulted in over 40 crystal forms, the best of which diffracted to 2.8 A Ê resolution when exposed to a synchrotron X-ray source.

Alternative conformations of a nucleic acid four-way junction

“…All oligonucleotides were puri®ed as described (Nowakowski et al, 1999a). The particular sequence of the RNA-DNA complex used for crystal structure determination was identi®ed through a combinatorial sequence screen (Nowakowski et al, 1999b). Crystals were grown at 24.5 C from 50 mM sodium cacodylate (pH 6.0), 20 mM MgCl 2 , 1 mM spermine, and 25 % (v/v) methyl-2,4-pentanediol (MPD) using the vapor diffusion method.…”

Mismatch discrimination and efficient photomodulation with split 10–23 DNAzymes

“…However, the structure of the loop and its catalytic mechanism require further characterization. The existing crystal structures are not believed to represent the active conformation of the loop, which is likely very flexible [18, 19]. Our lab’s recent discovery that the 10-23 DNAzyme can work as two separate a and b strands (Fig.…”

RNA Crystallization

“…A survey of the RNA structures deposited in the PDB reveals general trends that can be used to narrow down the search: a majority of RNAs crystallize at pH 6-7 (slightly acidic to prevent RNA hydrolysis), in the presence of sodium cacodylate buffer, magnesium ions, and crystallants such as 2-methyl-2,4-pentandiol (MPD), PEG 400 or 4000, or ammonium sulfate ( Figure 23.2). This observation triggered the design of first sparse matrices dedicated to the crystallization of nucleic acids [29][30][31][32][33][34][35]. Nowadays, many commercial screens are available in a 96-well format that facilitates the initial search for appropriate conditions.…”