Surface plasmon resonance kinetic studies of the HIV TAR RNA kissing hairpin complex and its stabilization by 2-thiouridine modification

Surface plasmon resonance (BIACORE) was used to determine the kinetic value s for formation of the HIV TAR-TAR* ('kissing hairpin') RNA complex. The TA R component was also synthesized with the modified nucleoside 5-thiouridine at position 7 in the loop and the kinetics and equilibrium dissociation co nstants compared with the unmodified TAR hairpin. The BIACORE data show an equilibrium dissociation constant of 1.58 nM for the complex containing the s(2)U modified TAR hairpin, which is 8-fold lower than for the parent hair pin (12.5 nM). This is a result of a 2-fold faster k(a) (4.14 x 10(5) M-1 s (-1) versus 2.1 x 10(5) M-1 s(-1)) and a 4-fold slower k(d) (6.55 x 10(-4) s(-1) versus 2.63 x 10(-3) s(-1)), H-1 NMR imino spectra show that the seco ndary structure interactions involved in complex formation are retained in the s(2)U-modified complex. Magnesium has been reported to significantly st abilize the TAR-TAR* complex and we found that Mn2+ and Ca2+ are also stron gly stabilizing, while Mg2+ exhibited the greatest effect on the complex ki netics. The stabilizing effects of 2-thiouridine indicate that this base mo dification may be generally useful as an antisense RNA modification for oli gonucleotide therapeutics which target RNA loops.