W0227: RNA Crystallization: An Irrational Approach

RNA Crystallization: An Irrational Approach. Joseph D. Ng ¹ and Richard Giegé ², ¹Department of Biological Sciences, Structural Biology, University of Alabama at Huntsville, Huntsville, Alabama 35899 USA, ²UPR 9002, IBMC du CNRS, 15 rue René Descartes, 67084 Strasbourg Cedex, France

Combinatorial selection-amplification has been used to study RNA to determine if any nucleic acid (having a minimal size) can exist to serve a particular biochemical function(1). In vitro selection techniques have isolated high affinity molecules to a target from RNA oligonucleotide libraries(2). Selection-amplification methods, on the other hand, have never been used strictly for in vitro selection of crystallizable RNA sequences. Sequence variations have proven to be more effective in changing the quality of the crystals than variations in crystallization conditions, often making the difference among obtaining true single crystals, multiple crystalline material or not obtaining any crystals at all. Because the packing of each RNA sequence is unique and not known before hand, it is difficult to predict which sequence modifications will improve or give rise to crystals of a particular RNA or RNA-protein complex. For example, it has also been demonstrated that adding over hanging bases and varying their number and type is non-disruptive and leads to drastic changes in crystal packing (3).

We present preliminary data of a pool of randomized ribo-oligonuleotides screened for crystallization under a narrow set of crystal growth conditions. Those molecules that do crystallize are examined with the following questions in mind. 1) Do we see favored space groups during crystallization events as observed in proteins? 2) What particular biochemical function, if any, would be or could be associated with the crystallized RNA molecules? 3) Would there be any new structural motifs that may correspond to unanticipated shapes in their three-dimensional structures? 4) What RNAs of different size, sequence, complexity and intrinsic flexibility can exist that would be able to crystallize?

The screening of randomized ribo-oligonucleotide pools also included the usage of positively charged additives (4). RNA crystals that have been obtained in the presence of additives were optically evaluated and X-ray diffraction analysis were performed on selective crystals to compare their space group, cell parameters, and diffraction limit with those of controls. Whereas no changes in space group nor cell parameters were observed for most RNA analyses, changes in diffraction limit were found when certain RNA’s were crystallized with certain synthetic polyamines.