Surface plasmon resonance kinetic studies of the HIV TAR RNA kissing hairpin complex and its stabilization by 2-thiouridine modification |
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| Authors: | Nair T M Myszka D G Davis D R |
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| Affiliation: | Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA. |
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| Abstract: | 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 s2U 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 ka (4.14 × 105 M–1 s–1 versus 2.1 × 105 M–1 s–1) and a 4-fold slower kd (6.55 × 10–4 s–1 versus 2.63 × 10–3 s–1). 1H NMR imino spectra show that the secondary structure interactions involved in complex formation are retained in the s2U-modified complex. Magnesium has been reported to significantly stabilize the TAR–TAR* complex and we found that Mn2+ and Ca2+ are also strongly stabilizing, while Mg2+ 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. |
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