SYNTHESIS OF A BIOACTIVE FLUORESCENT DYE AND ENZYMATIC LABELING AT THE 3-TERMINI OF RNAS - AN APPLICATION FOR THE CHARACTERIZATION OF THE THERMAL-STABILITY OF TRANSFER-RNAS AND OLIGONUCLEOTIDES USING FLUORESCENCE ANISOTROPY MEASUREMENTS

A novel fluorescent nucleotide analogue, 2'-O-anthraniloylcytidine 3', 5'-diphosphate (Ant-pCp) was synthesized from cytidine 3',5'-diphospha te and isatoic anhydride. Using T4 RNA ligase, Ant-pCp was easily join ed to the 3' terminus of the various oligonucleotides and tRNAs, for e xample, (pA)(4), (pA)(6), (pA)(8), yeast tRNA(Phe), B. subtilis tRNA(T hr), and B. subtilis unfractionated tRNA. It seems that anthraniloyl g roup does not influence the ligation reaction. Steady-state fluorescen ce anisotropy (r(s)) of these labeled oligonucleotides or tRNAs were m easured in the presence or absence of Mg2+ in the temperature range 5 to 60 degrees C. The complex formation between fluorescent labeled oli gonucleotides and poly(U) and the thermal denaturation profiles of the complexes were successfully detected by fluorescence anisotropy at lo w nucleotide concentration (10(-6 )M) in the presence of MgCl2. Fluore scent labeled tRNA showed characteristic thermal denaturation profiles in the absence of Mg2+, indicating that tRNA has a secondary and/or t ertiary structure. The anisotropy values of the fluorescent probe decr eased in the order: oligo(A):poly(U) complex > tRNA (+Mg2+) > oligo(A) . These results indicate that the mobility of the 3' terminal of tRNA is more restricted than that of the single strand of oligo(A) in solut ion. (C) 1998 Elsevier Science Ltd. All rights reserved.