Crystallographic Analyses of Chemically Synthesized Modified Hammerhead RNA Sequences as a General A
Solid-support chemical synthesis of RNA (1 ), though costly, has the advantage of allowing the incorporation without restriction of any desired nucleotide sequence, including sequences which contain special modified nucleotides. For example, the crystal structure of an all-RNA hammerhead ribozyme containing a modified 2′-O -methylcytosine at the active site to prevent cleavage has recently been solved (2 ,3 ). Incorporation of the 2′-methoxyl moiety specifically at the cleavage site of the ribozyme can only be accomplished by using the chemical synthesis approach. Many “unnatural” hammerhead RNA sequence modifications, including modified purine and pyrimidine bases, 2′-fluoro- and 2′-amino-modified riboses, and phosphorothioates (among many other examples), have been synthesized by a variety of research groups for probing the hammerhead RNA reaction mechanism and structure. Modified sequences, including ones containing unnatural bases, can be synthesized and crystallized in the same conditions as those used to produce the original crystals for a variety of applications. For example, a hammerhead RNA substrate strand containing a photolabile moiety protecting the active site in a manner analogous to the 2′-O -methylcytosine at the active site, has recently been crystallized in the same conditions and space group as the originally solved crystal form. This will allow the structure of the modified hammerhead RNA to be solved with a single data set based on the (publicly distributed) coordinates of the original structure. The ultimate purpose of this particular modified hammerhead RNA is for time-resolved crystallographic experiments where removal of the photolabile protecting group with an Xe flashlamp will initiate the cleavage reaction in the crystal by exposing a free 2′-hydroxyl at the active site. Both dynamic and static crystallography experiments are thus made possible by combining the technique of RNA chemical synthesis with the previously elucidated structure and crystallization conditions. Such an approach therefore allows a completely general method for obtaining and analyzing crystals of modified hammerhead RNAs.