Synthetic Single-Stranded Polynucleotides

One well-established observation is that, under conditions where single-stranded polynucleotides give rise to a d.c. polarographic reduction wave, both native DNA and other double-helical natural and synthetic polynucleotides are inactive 22 23,46-47, 58,59,61) Tjjjs js rea(ji]y interpretable in that, in such helical structures, the adenine and cytosine residues are located in the interior of the helix, and hydrogen bonded in complementary base pairs (see below). Z-DNA, in which cytosine residues are at the surface of the helix, is of obvious interest in this regard, and the B - Z transition in the synthetic poly(dG dC) has been investigated with the aid of differential pulse polarography and UV spectroscopy 60). 

Polymer surfactant interaction has been examined by using sodium 2-(N-dodecyIamino)naphthalene-6-sulphonate as a probe. Solute-solvent interaction of free base phthalocyanine has been examined in both polyethylene and polystyrene by the effect of pressure on spectroscopic hole burning s Fluorescence has been used to indicate the onset of aggregation in water soluble polymers s interaction of pyrenylmethyltributylphosphonium bromide with single strand polynucleotides , and the interaction of indole compounds with synthetic polyelectrolytes. ... 

Using enzymatic methods with the substrate ADP, it has been possible to prepare a single-stranded polynucleotide containing only one kind of base, known as poly A . Similarly with UDP, single-stranded poly U has been synthesised. If dilute solutions of poly A and poly U are mixed, hydrogen bonding between the different chains takes place and a synthetic donble-stranded specimen is obtained (Figure 10.51). 

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... 

Studies of the specificities of experimentally induced antipolynucleotide antibodies have yielded some information on the antigenic determinants which could exist in a molecule of nucleic acid. Like the antibodies to proteins or synthetic polypeptides, antibodies to polynucleotides can be directed against different antigenic determinants derived from sequential regions as well as conformational factors in the molecule. Since antibodies in which the specificity is associated with the heterocyclic bases are obtained by use of single-stranded polynucleotides or certain components of nucleic acids it is thus evident that the bases themselves can be included among the antigenic determinants of polynucleotides. 

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. 

The ability of adenine and cytosine to undergo reduction in aqueous medium is at least partially retained at the level of oligo- and polynucleotides. In both acid and neutral media, such residues in single-stranded RNA, DNA and synthetic polynucleotides, when subjected to d.c. polarography, undergo irreversible reduction in a protonated, adsorbed, state with the transfer of four electrons to adenine... 

The density, p, of a double stranded DNA molecule, and hence its position in the gradient, depends primarily on its nucleotide composition (eq. 2.2 gives p= 1.660+0.098 (GC)). The relation does not hold for DNAs containing glucosylated, methylated or other modified residues, nor for DNAs of very simple sequence such as synthetic polynucleotides, crab poly dAT (Wells et al. 1970) and centromeric DNA. Single stranded DNA is denser than double stranded DNA and isopycnic centrifugation can be used to separate them. 

The two types of natural polynucleotides (nucleic acids) are classified according to the sugars they contain. Ribonucleic acid (RNA) contains exclusively P-o-ribose, while the sugar in deoxyribonucleic acid (DNA) is P-2-deoxy-D-ribose. Different nucleic acids can have from around 80 nucleotides (nt), as in transfer RNA (tRNA), to over 10 nucleotide-pairs in a single eukaryotic chromosome. The unit for size of nucleic acid is the base (for single-stranded species) or the base-pair (bp, for double-stranded species), with the unit Kb (thousand base-pairs) and Mb (million base-pairs). Examples of synthetic homopolynucleotides are poly(uridylate) or poly(deoxyadenylate), in poly(U) or poly(dA)... 

Homopolymar a polymer consisting of identical monomers, e. g. amylose and polyphenylalanine. In a narrower sense, H. are synthetic polynucleotides in which all the nucleotides contain the same base, e.g. polyadenylic acid, polyuridylic acid, polydeoxyade-nylic acid. Homopolynucleotides (usually single-stranded) are synthesized in vitro from nucleoside di-or triphosphates using the appropriate polymerases without a matrix. An oligonucleotide is ne ed as a... 

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. 

The realization that interferon inducers can modify antibody production developed early, with studies on synthetic polyneucleotides playing a pivotal role. jt was shown that natural nucleic acids were active in augmenting antibody formation to sheep red blood cells (SRBC) in mice, as were double-stranded synthetic polynucleotides. When single stranded synthetic polynucleotides were used, no augmentation was obtained. 

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. 

The hydrolysis step is more controversial, but one view (Roberts et al., 1969) is that it occurs by a reversal of the sequences followed during cyclization. Recently, Cozzone and Jardetzky (1977) studied the interaction of the single-stranded synthetic polynucleotide, poly(A), in the presence of RNase. They found that the enzyme can carry out the cyclization step on such polypurine nucleotides, but not the hydrolysis step. They interpreted their data to indicate that this nonoptimal substrate forms an enzyme-substrate complex which allows alignment with only one of the two active-site histidines. This emphasizes two important points for the mechanism of pancreatic RNase and other enzymes (1) binding must be correct to have the energetically optimal interaction with the enzyme that will lead to the formation of the transition state ... 

In contrast to RNA, DNA is polymorphic. Under low salt conditions or at high relative humidity, DNA adopts the B-form usually considered to be biologically active. With increasing addition of salt or of polar organic solvents (synonymous with reduced relative humidity or removal of available water of hydration), and with certain types of counterions, DNA and double-stranded synthetic polynucleotides transform from B-DNA to the A-, C-, D-, Z-forms (see Thble 24.1 and Fig. 24.1. Only the A-, B- and Z-DNA structures, which have thus far been determined in detail by single crystal diffraction methods, are of structural interest and they are considered in the following sections. 

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