Polynucleotide complexes poly

Nucleic Acids - The use of nucleic acids as adjuvants to the immune response was first indicated in the demonstration of a restorative capacity of nucleic acid-rich tissues in animals rendered immunodeficient following x-irradiation . Their ability to act as adjuvants to the normal immune response to unrelated antigens has also been documented and shown to be attributable to low molecular weight oligonucleotides. Extended study over the past 5 years has shown the more well-defined polynucleotide complexes (poly A U and poly I C) to be powerful adjuvants to many antigens... 

We shall conclude this section by a comparison of Equations (30) and (34) with published data. Using a dye indicator for Mg , Krakauer [17] measured a quantity 6 which is identical to the l.h.s. of these equations. In Figure 1 is portrayed his data for the synthetic polynucleotide complex Poly (A+2U), characterized [18] by the... 

Deeble DJ, Schulz D, von Sonntag C (1986) Reactions of OH radicals with poly(U) in deoxygenated solutions sites of OH radical attack and the kinetics of base release. Int J Radiat Biol 49 915-926 Denk O, Washino K, Schnabel W (1983) OH radical-induced chemical reactions of polynucleotide complexes. Makromol Chem 184 165-173... 

Sakurai K, Mizu M, Shinkai S (2001) Polysaccharide-polynucleotide complexes. 2. Complementary polynucleotide mimic behavior of a natural polysaccharide schizophyllan in the macromolecular comple with a single strand RNA poly(C). Biomacromolecules 2 641-650... 

Langer [3] coupled 1,4-butanediol diacrylate with poly(A, A -dimethylethyl-enediamine), (III), piperazine, and 4,4 -trimethylenedipiperidine to prepare poly(p-amino esters) that were particularly suited for the delivery of polynucleotides. Nanoparticles containing polymer/polynucleotide complexes were also prepared. Hubbell [4] and Zhao [5] prepared polymeric biomaterials by the nucleophilic addition of cysteine, (IV), and polyethylenimine, (V), respectively, to cx,p-unsaturated macromolecular diacrylates. 

The inhibitions described above occurred only when the analog and polynucleotide contained complementary bases. These combinations are not the only ones in which the interaction can occur, e.g., affinity methods detect some interaction between the non-complementary poly-9-vinyladenine and polyadenylate Apparently, such complexes are too unstable to affect the enzymatic reactions nevertheless, extensive modification of the analog can increase the stability of the polymer-polynucleotide complex to the point where such a polymer can effectively inhibit the reaction. Thus, omisssion of the amino group from poly-9-vinyladenine leads to poly-9-vinylpurine and the latter polymer inhibits the reverse transcription of polyadenylate and polyuridylate The introduction of a dimethylamino group in place of the amino group of poly-9-vinyladenine abolMies all of its inhibitory effects All these effects can be correlated with the ability of polymers to form complexes with templates. 

Synthetic polynucleotide complexes have been shown to be effective immune response modulators in animals and man (Braun et al. 1971, Johnson 1979). The polynucleotides are formed foUowing the action of an enzyme, polynucleotide phosphor-ylase on the synthetic mononucleotide diphosphates. Complexing takes place following the mixing of polymers composed of opposite base pairs. Two have been utilised, polyinosinic acid complexed with polycytidylic acid (poly I poly C) and polyad-enylic acid complexed with polyuridylic acid (poly A poly U). The single strands mononucleotides are ineffective. 

Several additional results have arisen from these studies. Polynucleotides can not only form Watson-Crick helical double-stranded complexes but may also form helical structures between themselves which can have more than two strands, as well as non-Watson-Crick base pairs, like the complex poly(l) poly(A)-poly(l). Furthermore, numerous polymers of base and sugar analogues have been prepared and studied. 

The four-stranded poly(l) structure surprisingly shows a 2 -endo puckering in contrast with all other ribopolynucleo-tldes studied so far (Table 6.1) and also in contrast with the 3 -endo puckering of the three-stranded polynucleotide complexes. 

Arnott s group is currently studying all these polynucleotide complexes by X-ray fiber diffraction, and the results will clarify some ambiguities. Thus, poly(dG) poly(dC) in fibers was found to be preferentially in the A form. The alternating polymers poly(dA-dT) and poly(dG-dC) show a rather unusual D form, with eight base pairs per turn (Table 6.1). Although the D- A transition is observed at low relative humidities, the A form is metastable, and the transition is never complete, returning rapidly to the D form (27). 

Some other polynucleotide complexes. Polyxanthylic acid [poly(X)l forms complexes of various stoichiometries (1 1, 1 2, 2 1) with poly(U) and/or poly(l) (30). 

In order to define the specificity of the antibodies and to attempt to identify the antigenic determinants (or groups of antigenic determinants) Nahon-Merlin et al. (1973 a) studied the cross-reactions of anti-poly I poly C antibodies with different polynucleotides and polynucleotide complexes by direct precipitation and by specific absorption of antibodies. The rabbits respond to immunization with poly I poly C — MBSA, with a production of antibodies which varies according to the rabbit the sera contain from 700 to 1000 [i.g/ml of antibodies. 

Analysis of the specific absorption of anti-poly I poly C antibodies by double-stranded polynucleotide complexes reinforces both the idea of specificity of these antibodies for double-stranded structures and also the immunochemical differences between the three double-helical complexes. Thus poly rA poly rU, poly dG poly dC and poly rG poly rC absorb, respectively, 81.5, 73.3 Sind 57.7% of the antibodies in the homologous system. In order to inhibit 50 % of the reaction with the homologous antigen, 5-7 [xg of poly A poly U, 12.5 xg poly dG poly dC, and 85 [xg of poly rG poly rC... 

It is essentially the cross-reactions with another double-helical complex, poly A poly U, which have been studied with immune sera of mice and hamsters. These cross-reactions have been observed very frequently with immune sera of RAP mice and B/W mice and to a lesser degree with the sera of hamsters poly A poly U is the best inhibitor of the reaction between anti-poly I poly C of mice and the homologous antigen (Lacour et al., 1971 Steinberg et al., 1971). The role of the bases in this immunoreaction does not appear to be essential. It is probable that, as in rabbit, these antibodies recognize double-helical structures. While there is similarity in the reactions of the sera of the three species with synthetic polynucleotide double-helical complexes, the cross-reactions of the anti-poly I poly C antibodies with nucleic acids are very different in the rabbit, the mouse, and the hamster (Table 4). 

Antibodies induced by the equimolar complex poly A poly U precipitate one of the components of the homologous antigen, poly A (Nahon et al., 1967 a, b). This reaction was not observed by Schwartz and Stollar (1969) A quantitative analysis of the reactions of these antibodies with the homologous antigen and with its component polynucleotides, and of their reaction with related antigens as well as the analysis of results of inhibition studies have allowed a better characterization of these antibodies (Nahon-Merlin et al., 1973 b). The level of antibodies reacting with poly A-poly U varies... 

Anti-poly G poly C antibodies have been demonstrated by immunodiffusion and by complement fixation in the sera of rabbits immunized with poly G-poly C — MBSA (Michelson et al., 1971 Nahon-Merlin et al., 1971). The anti-poly G poly C antibodies react not only with poly G poly C but also with a large number of double-helical complexes such as poly A poly U, poly I poly C and poly dG poly dC. It is nevertheless to be noted that the complex poly A poly I in which two purine polynucleotides are involved is not precipitated by these antibodies, which is readily explained by the special stereochemical structure of this complex. The anti-poly G poly C antisera nevertheless precipitate poly iso A poly I, but in this case it can be considered that displacement of the glycosyl-hnkage from N to in polyisoadenylic acid converts this polynucleotide into an analogue of poly C. The complex is thus effectively between a poly purine ribonucleotide and a poly pyrimidine ribonucleotide as in the case of other complexes such as poly A poly U and poly I poly C. Immune sera against poly G poly C also react with the triple-stranded complex 2 poly G poly C. In addition they can precipitate one or the other of the component polynucleotides of the homologous complex. 

Immune sera against poly A poly U, anti-poly I poly C and anti-poly G were used as controls. Anti-poly G does not precipitate any of the RNAs tested, whereas antibodies to the two complementary complexes precipitate all the RNAs, regardless of origin or fraction (Table 7 to 9). Thus antipoly G poly C antibodies show a specificity for ribosomal RNA from animal cells, and for certain viral RNAs furthermore they distinguish between rRNA and tRNA. In contrast, antibodies obtained by immunization with other polynucleotide complexes such as poly A poly U and poly I poly C show no specificity for any RNA whatever its type or origin, with the exception however of double-stranded viral RNA (Stollar, 1970). 

Excited state resonance Raman spectra of CuTMPyP bound to DNA or poly[d(A-T)] have been recorded [167,168], These are assigned to an exciplex formed between the porphyrin and the A-T sites of the polynucleotide. The excited state lifetime is estimated to be ca. 20 ps. Weak emission from CuTMPyP" bound to DNA has been reported and has been assigned to originate in a tripdoublet or tripquartet level [169]. It is believed that the emissive complexes are intercalated, whereas groove-bound CuTMPyP does not emit because of solvent quenching of the excited state. 

Just as zinc reacts with the phosphates, silver(I) reacts with the bases. Figure E shows some titration curves for 1 1 mixtures of silver ion and polynucleotides. Below alkaline pH, silver ions do not compete with any protons on Poly A and Poly C (A and B) on the other hand, Poly I and Poly U (C and D) compete rather effectively. This conclusion is borne out in a comparison of the ultraviolet spectra of the silver complexes of Poly I and Poly A with the spectra of the uncomplexed polynucleotides (Figures F and G). 

Figure F. Ultraviolet spectra of silver complexes of Poly I and Poly A compared with the spectra of the uncomplexed polynucleotides...

Complexes between chiral polymers having ionizable groups, and achiral small molecules become, under certain conditions, optically active for the absorption regions of the achiral small molecules. Dyes such as acridine orange and methyl orange have been used as achiral species, since they are in rapport with biopolymers through ionic coupling. This phenomenon has been applied to the detection of the helix chirality in poly-a-amino acids, polynucleotides, or polysaccharides when instrumental limitations prevent direct detection of the helices. 

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