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

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Poly(inosinic acid).poly(cytidylic acid) [Poly(I).Poly(C)] is a synthetic RNA duplex known to induce interferon.
• Chemical or enzymatic modification of Poly(I).Poly(C) with nitroxide radicals (spin-labels) can alter its properties and biological activity.
• Understanding the structure-activity relationship of modified Poly(I).Poly(C) is crucial for developing novel immunomodulatory agents.

Purpose of the Study:

• To evaluate the interferon-inducing activity of Poly(I).Poly(C) duplexes modified with spin-labels.
• To investigate the structural implications of spin-label incorporation on the duplex conformation.
• To determine the minimum structural requirements for interferon induction by modified Poly(I).Poly(C).

Main Methods:

• Chemical and enzymatic synthesis of modified Poly(I).Poly(C) duplexes with varying degrees of spin-label incorporation.
• Annealing of modified (C)n strands with Poly(I)n to form duplexes.
• Electron paramagnetic resonance (EPR) spectroscopy to determine the correlation time of the nitroxide moiety.
• Interferon induction assays in various cell cultures (PRK, VGS, L-929) and in rabbits.

Main Results:

• Chemically modified Poly(I)n.(1C,Cx)n duplexes showed similar interferon-inducing activity to unmodified Poly(I)n.(C)n.
• Enzymatically synthesized Poly(I)n.(1s(4)U,Cx)n copolymers were chemically stable.
• EPR analysis indicated that 1s(4)U residues are in a nonintrahelical conformation, partitioning the duplex into segments of varying sizes.
• Interferon induction by Poly(I)n.(1s(4)U,Cx)n was comparable to Poly(I)n.(C)n unless the spin-label density (x) was less than 16.
• Double-helical segments of approximately 16 base pairs, separated by nonintrahelical 1s(4)U residues, were sufficient for interferon response.

Conclusions:

• Spin-label modification of Poly(I).Poly(C) duplexes does not abolish interferon-inducing activity.
• The nonintrahelical conformation of 1s(4)U residues and the resulting segmented duplex structure are key features for interferon induction.
• Approximately 16 base pairs of double-helical structure are sufficient to trigger the interferon response in multiple biological systems.