Polyribonucleotides, double-stranded

Polynucleotides. Polynucleotides are potent interferon inducers. A mismatched, double-stranded synthetic polyribonucleotide ampligen and the double-stranded acids, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acids have been widely studied for cancer therapy(ii). Although these materials elicit excellent activity with murine rodents, therapeutic effects are dramatically decreased within primates. 

The enzyme is very sensitive to the secondary structure of the substrate. Native calf thymus DNA is quite resistant to enzymic attack by spleen exonuclease, being split at less than 4% the rate at which alkali-denatured DNA is split (11). Long deoxyribonucleotides (average chain length 20-50), which still have complementary double-stranded structure, are rather resistant to the enzyme (26). Some limited results obtained with synthetic polyribonucleotides (11) are rather puzzling since poly C was found to be completely resistant, whereas poly A, poly I, and poly U were degraded at comparable rates. In the solvent used (0.15 M acetate buffer-0.01 M EDTA, pH 5.0), poly A and poly C are believed to have... 

Bustamante C, Gurrieri S, Pasternack RF et al (1994) Interaction of water-soluble porphyrins with single- and double-stranded polyribonucleotides. Biopolymers 34 1099-1104... 

That resistance to nucleolytic degradation may play an important part in the interferon-inducing capacity of poly(I) poly(C) and other double-stranded polyribonucleotides is suggested by the aforementioned findings pointing to a parallel increase of interferon production and resistance to nucleases upon... 

Among the synthetic interferon inducers described (polycarboxylates, polynucleotides, low molecular weight compounds), polynucleotides and more specifically double-stranded polyribonucleotides such as poly(I) poly(C) are efficient interferon inducers in vitro (cell cultures). Therefore, studies on the mechanism of interferon production have almost exclusively been performed with ds—RNAs (or viruses). 

In contrast to studies on DNA as a thera-I peutic agent, substantially more work has been reported on RNA and polyribonucleotides. Much of the effort focused on the ability of polynucleotides to induce interferon (5) and the most thoroughly studied polynucleotide in this regard is double-stranded polyribo-inosine polyribonocytidine (poly rI poly rC)... 

The same authors noted that almost 20% of the polyribonucleotides obtained in vitro formed ribonuclease-resistant double-stranded RNA after self-annealing. They concluded that this ribonuclease-resistant RNA arose from partly symmetric transcription and that at most 12-15 of the RNA formed in vitro was complementary to the L strand. The asymmetry of transcription was thought to result from damage of the organelles. These experiments were extended (Aloni and Attardi, 1971). Using HeLa... 

Fig. 7. Comparison of calculated and observed CD for different sequences of polyribonucleotides. The geometry of double stranded RNA was used for all sequences. The alternating sequences poly AU poly AU and poly GC poly GC probably do not have this sequence. (Figure from C.L. Cech, Ph.D. thesis, University of California, Berkeley, 1975).

In order to elucidate the role of conformation of the immunogen, animals were immunized with complexes of polyribonucleotides (double- or triple-stranded) associated with MBS A. The antibodies obtained reacted specifically with double- or triple-stranded complexes and it was concluded that the specificity was determined by the macromolecular conformation of the immunogen (Nahon et al., 1967 Lacour et al., 1968 Michelson et al., 1971). Immunization of rabbits with DNA-MBSA or with double-stranded polynucleotide complexes adsorbed to MBS A ehcits antibodies belonging to the macroglobulin class (Stollar and Sandberg, 1966 Nahon-Merlin et al., 1973). 

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. 

Thus poly A poly U and poly I poly C induce antibodies specific for the double-stranded structure, and reciprocal cross-reactions between these two polyribonucleotide complexes are constant and quantitatively comparable. A third complex, poly G poly C reacts only feebly with the two preceding immune sera. The h3q)othesis that this immunochemical difference correlates with a stereochemical difference of structure implies that this complex, if it has immunogenic capacity, may induce antibodies with a particular specificity. The validity of this concept has been demonstrated and wiU be discussed next. 

Narrower specificities have been obtained with antibodies to some unusual helical structures. Poly(dG)-poly(dC) induces antibodies specific for the immunogen and unreactive with other deoxyribonucleotide polymers, such as poly(dAT) or native DNA. Double-helical polyribonucleotides with modified furanoses, such as poly(A)-poly(2 -0-methylU), induce antibodies that react with a number of polymers bearing 2 -furanose substitutions (such as methyl or ethyl groups on either the purine or pyrimidine-containing strand). Poly(G)-poly(C) induced antibodies of narrow specificity in our studies, but Lacour and co-workers obtained anti-poly(G)-poly(C) that cross-reacted with several forms of viral RNA. ... 

Table 3 (section 3.2.2.2) contains melting temperatures of polyribonucleotide hetero-complexes (including base-modified complexes), both double and triple stranded. 

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. 

Free DDA concentration (x10 ) M Figure 10.2 Binding isotherms of DDA with native (1) and denatured (2) DNA, with single-stranded polyribonucleotides poly (U) (3), poly(A) (5), as well as with double helix poly(U) poly(A) (4). (From Ref. [10].)... 

---