Polynucleotide complexes, synthetic double-stranded

Like double-stranded DNA, synthetic polynucleotide complexes containing complementary bases show cooperative melting curves, i.e., the property studied (UV absorption, optical activity, viscosity, etc.) shows a sharp transition at a specific temperature. This Tm depends on ionic strength and, to a certain degree, on pH. If the temperature is increased above the Tm, however, a further continuous variation in the property studied will be observed. It is only in recent years that an understanding of such noncooperative phenomena is emerging. Here the studies of oligomers by CD and NMR were of critical importance. 

In order to give DNA immunizing properties Plescia et al. (1964, 1965) complexed the material to MBS A. A certain number of polynucleotides such as denatured DNA, poly dAT, and tRNA associated with MBS A have been used as immunogens by these authors. This method of preparation has been used for other polynucleotides, particularly for single-stranded synthetic polynucleotides and the various double- and triple-hehcal complexes formed from such polyribonucleotides. It appears that secondary structure of these complexes is generally maintained on adsorption onto MBS A, although some of their characteristics could well be modified in this electrostatic interaction. 

A second structural requirement for the interferon-inducing capacity (antiviral activity) of synthetic polynucleotides is a stable, highly ordered secondary, hence double-stranded structure based on complementary base-pairing. The stability of the complex is reflected in its Tm (thermal stability) value. From a comparative study of different double-stranded RNA duplexes, De Clercq and Merigan61-1 and De Clercq et al. 53-) concluded that a Tm value higher than 60 °C (calculated for 0.15M Na+) was needed for full expression of antiviral activity. Since thermal stability represents a valuable measure of the overall stability of double-stranded RNAs116 double-stranded RNAs with Tm values higher than 60 °C may also be considered to be the most stable ones at 37 °C. 

The anti-poly I poly C antibodies react not only with RNA of reovirus but also, though to a lesser degree, with RNA extracted from mammalian cells (Fig. 4). Comparison of the efficiency of inhibition of the cross-reaction with mammahan RNA by RNA of reovirus, by double-helical complexes of synthetic polynucleotides and by single-stranded polynucleotides, has shown that the antibodies anti-poly I poly C react especially with double-stranded conformational determinants of the RNA. None of the single-stranded polynucleotides is capable of totally inhibiting the reaction with RNA whatever... 

Differences in the capacity of inhibition by polynucleotides not involved in complementary hydrogen bonds and by double-helical complexes of synthetic polyribonucleotides, or double-stranded viral RNA allow the conclusion that it is above all the regions of associated base pairs which are recognized in the RNA by anti-poly I poly C antibodies. Such complementary double-stranded helical regions have been described especially in tRNA but they have also been shown to exist in ribosomal RNA. These two kinds of RNA were therefore isolated and studied separately. Although both fractions precipitate anti-poly I poly C antibodies, their reactivity is nevertheless very different and rRNA precipitates eight times as much antibody as tRNA. Since tRNA possesses an important tertiary structure, this low reactivity could be explained by the non-accessibility of antigenic sites. 

The existence of conformational determinants has been clearly demonstrated through the use of synthetic polynucleotides in double-hehcal structures and in triple-stranded complexes. It has also been shown that the specificity of antibodies is determined by the macromolecular conformation of the immunogen. Thus single-stranded polynucleotides induce formation of antibodies specific for the single-strand, while double-stranded hehcal complexes of polynucleotides induce antibodies specific for these structures, and antibodies specific for triple-strand structures are induced by the triple-stranded polynucleotide complexes. 

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