Nair T M, Myszka D G, Davis D R
Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
Nucleic Acids Res. 2000 May 1;28(9):1935-40. doi: 10.1093/nar/28.9.1935.
Surface plasmon resonance (BIACORE) was used to determine the kinetic values for formation of the HIV TAR-TAR* ('kissing hairpin') RNA complex. The TAR component was also synthesized with the modified nucleoside 2-thiouridine at position 7 in the loop and the kinetics and equilibrium dissociation constants 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 hairpin (12.5 nM). This is a result of a 2-fold faster k(a) (4.14x10(5) M(-1) s(-1) versus 2.1x10(5) M(-1) s(-1)) and a 4-fold slower k(d) (6.55x10(-4) s(-1) versus 2.63x10(-3) s(-1)). (1)H NMR imino spectra show that the secondary structure interactions involved in complex formation are retained in the s(2)U-modified complex. Magnesium has been reported to significantly stabilize the TAR-TAR* complex and we found that Mn(2+) and Ca(2+) are also strongly stabilizing, while Mg(2+) exhibited the greatest effect on the complex kinetics. The stabilizing effects of 2-thiouridine indicate that this base modification may be generally useful as an antisense RNA modification for oligonucleotide therapeutics which target RNA loops.
表面等离子体共振（BIACORE）用于确定HIV TAR - TAR*（“亲吻发夹”）RNA复合物形成的动力学值。TAR组分还在环的第7位合成了修饰核苷2 - 硫尿苷，并将其动力学和平衡解离常数与未修饰的TAR发夹进行比较。BIACORE数据显示，含有s(2)U修饰TAR发夹的复合物的平衡解离常数为1.58 nM，比亲本发夹（12.5 nM）低8倍。这是由于结合速率常数k(a)快2倍（4.14×10⁵ M⁻¹ s⁻¹ 对 2.1×10⁵ M⁻¹ s⁻¹）和解离速率常数k(d)慢4倍（6.55×10⁻⁴ s⁻¹ 对 2.63×10⁻³ s⁻¹）的结果。¹H NMR亚氨基谱表明，复合物形成过程中涉及的二级结构相互作用在s(2)U修饰的复合物中得以保留。据报道，镁能显著稳定TAR - TAR*复合物，我们发现Mn(2+)和Ca(2+)也有很强的稳定作用，而Mg(2+)对复合物动力学的影响最大。2 - 硫尿苷的稳定作用表明，这种碱基修饰作为针对RNA环的寡核苷酸治疗药物的反义RNA修饰可能具有普遍用途。
