Interferon induction by poly(inosinic acid).cntdot.poly(cytidylic acid) segmented by spin-labels

Abstract

Poly(inosinic acid).poly(cytidylic acid) [(I)n.(C)n] duplexes of which the (C)n strand was modified to various degrees chemically or enzymatically with nitroxide radicals (spin-labels) were evaluated for interferon-inducing activity. Upon annealing of the chemically modified (C)n, (1C,Cx)n (X = 1000 or 16), with (I)n, the interferon-inducing activity was similar to that of (I)n.(C)n in PRK cell cultures. However, to overcome hydrolysis of the spin-label linkage in (1C,Cx)n, and enzymatic approach was taken to synthesize (1S(4)U,Cx)n copolymers with x = 100, 38, 16, and 8. The (1s(4)U,Cx)n copolymers were chemically stable, and upon annealing with (I)n the correlation time of the nitroxide moiety in (I)n. (1s(4)U,Cx)n was determined. A comparison of this correlation time with that measured for (RUGT,U100)n.(A)n, which contains the nitroxide moiety in position 5 of the U moiety, suggests that the 1s(4)U residue is in a nonintrahelical conformation and partitions the duplex into double-helical segments of varying size. The interferon-inducing activity of (I)n. (1s(4)U,Cx)n was evaluated in primary rabbit kidney, human skin fibroblast (strain VGS), and mouse L-929 cell cultures as well as in rabbits. The 1s(4)U residue did not cause a significant change in the interferon induction as compared to (I)n.(C)n in most systems tested unless x less than 16. These findings indicate that double-helical segments of approximately 16 base pairs partitioned by nonintrahelical 1s(4)U residues suffice to trigger the interferon response in all systems studied.

Internal motions in ribonucleic acid duplexes as determined by electron spin resonance with site-specifically spin-labeled uridines

“…Therefore, the substantial decrease in mobility for the 5-substituted (U)" as compared to the 4substituted (ls4U,U)" upon complexation strongly supports the hypothesis that 5-substituted bases assume intrahelical base pairing upon complexation with (A)". A similar conclusion was reached when comparing the correlation times of (I)"-(ls4U,C)" with those of (A)"(RUGT,U100)" (Bobst et al, 1981b). Furthermore, the observed decrease in mobility in the case of (RUTT,U)"(A)" supports the hypothesis that rotation around bond 2 or any bond parallel to it in the spin-label does not correspond to the dominant motion for xx, or else the difference between a nonintrahelical and an intrahelical conformation could not have been observed.…”

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—Gene 5 Protein Complexes

“…The experimental ESR spectra of (DUTT,dT)n and (DUMBT,dT)n in the presence and absence of gene 5 protein were also determined, and the parameters needed for their simulations are given in Table I Table I. The experimental ESR spectra of the spin labeled single strands (DUAP,dT)n containing various percentages of DUAP reveal lineshapes similar to those of (DUMPT,dT) 0 • As noted on previous occasions (6,11,12) some broadening of the lineshape is noticeable when the spin labeled nucleotide to unlabeled nucleotide ratio is larger than a few percents. Complexation of the various (DUAP,dT)n containing variable amounts of DUAP results in some ESR intensity loss and the remaining signal is similar to that of (DUMPT,dT) 0 -gene 5 protein shown in Fig.…”

Redox-active daunomycin-spin-labeled nucleic acid complexes

“…The enzymatically spin-labeled nucleic acids were prepared by approaches developed in this laboratory. The singlestranded RNAs (ls4U,C)" with different ls4U/C ratios were obtained according to Bobst et al (1981). (DUAT,dT-dA)" was prepared according to Toppin et al (1986).…”