Rapid determination of the affinity of 28- and 14-mer phosphorothioate oligonucleotides for HIV-1 reverse transcriptase by fluorescence spectroscopy.
Intrinsic fluorescence of human immunodeficiency virus type 1 reverse transcriptase (E.C. 2.7.7.49) and displacement experiments of a fluorescent template.primer probe were used to study the interaction of the enzyme with several types of 28- and 14-mer normal or phosphorothioate oligodeoxycytidinylates and their duplexes with poly(rI). The two methods gave convergent results and allowed in each case fast determinations of ligand affinities for the enzyme. The dissociation constants (Kd) obtained from intrinsic fluorescence changes were slightly lower than those determined from the less direct competitive displacement experiments. In all cases, the enzyme displayed better recognition of the hybrid than of the unannealed oligonucleotide. The Kd values of phosphorothioate oligomers and their hybrids were lower than those of the corresponding normal oligomers and hybrids, but the difference was not as significant as in the case of the Ki constants for (dC)28 and S(dC)28 (Majumdar et al. (1989) Biochemistry 28, 1340). The affinities of the annealed phosphorothioate oligodeoxycytidinylates for the enzyme were found to be larger than for any other compounds in this series (Kd of poly(rI).S(dC)28: 0.28 nM at 25 degrees C). Changing the beta stereochemistry of the oligomer bases to alpha did not alter the affinity of the oligodeoxycytidinylate and its hybrids for the enzyme.
Maury
G
G
Université de Montpellier II, URA 488 du C.N.R.S., Montpellier, France.
Divita
G
G
Morvan
F
F
Imbach
J L
JL
Goody
R S
RS
D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D054303:HIV Reverse Transcriptase; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology*; D011069:Poly I / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D018894:Reverse Transcriptase Inhibitors; D013050:Spectrometry, Fluorescence; D013873:Thionucleotides / Q000737:chemistry / Q000494:pharmacology*
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D013873:Thionucleotides; C059536:Sd(C)28; D011069:Poly I; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase