IRIDIUM HEXAMMINE VERSUS COBALT HEXAMMINE: IMPROVING CRYSTAL DIFFRACTION IN NUCLEIC ACID / M(NH₃)\O(₆;³⁺) COMPLEXES (M=Co, Rh OR Ir)

Thierry Prangé^1,2,William Cruse¹, William Shepard² and Roger Fourme²

1Chimie Structurale Biomoléculaire (URA-1430 CNRS), UFR Biomédicale, 74, rue M. Cachin, 93012-BobignyCedex, France
2LURE, Bât 209d Université Paris-Sud, 91405 ORSAY Cedex, France.

Oligonucleotides folded sequences which include repeat pyrimidine- or purine-rich motifs like C(UUCG)G or CGAAAGC have been described as extraordinarily stable in solution.

They usually adopt loop structures when they are flanked by a number of Watson-Crick steps. However, in the crystal state, they form duplex structures with C-U and G-A or A-A mismatches, as demonstrated in the two RNA sequences r-(GGACUUCGGUCG) and r-(GGCCGAAAGGCC) [1].

Attempts to crystallise shorter sequences like r-(GCUUCGGC)-d(^XU) (A) or d-(GCGAAAGCT) (B) were only possible in the presence of hexammine trichloride salts of cobalt, rhodium or iridium. The mechanical stability as well as the limit of diffraction of these crystals increase in the order Co < Rh < Ir. For instance, a resolution of 2.2 Ä is observed for Co(NH3)\o(₆;³⁺) complexes while Ir(NH₃)\o(₆;³⁺) complexes may diffract up to a 1.6 Ä.

Several variations of A were crystallised as duplexes including different halide atoms at the last uracil (X= F, Cl or Br) and complexed with either cobalt, rhodium or iridium hexammines. The structures are all isomorphous except when fluoro-Uracil is used at position 9 [2]. Furthermore, two different forms may be isolated depending upon the conditions of crystallisation : A1 is monoclinic, C2 space group with a=53.8, b=19.4, c=50.3Ä and b=109°9; and A2 is hexagonal, P6₁22 with a=b=22.1, c=139 Ä. The first form was solved by molecular replacement [2], the second form is under study using the iridium anomalous signal of its Ir(NH₃)\o(₆;³⁺) complex.

In the case of B, a new zipper-like structure of DNA was discovered instead of a standard duplex or a loop structure. The structure of B was solved by MAD methods at the bromine K-edge using a bromo-5 cytosine-2 derivative [3].

The role of the M(NH₃)\o(₆;³⁺) cation in the stability of the crystal packing will be discussed as a function of M=Co, Rh, Ir.

1. Holbrook et al Nature (London) 353 (1991) 579-581; Bayens et al Proc Natl. Acad. Sci. USA 93 (1996) 12851-12855
2. Cruse et al Proc. Natl. Acad. Sci. USA 91 (1994) 4160-4164
3. Shepard et al Structure (1998) submitted