Double-stranded synthetic polynucleotide

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. 

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. 

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. 

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. 

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). 

Just as main-chain NH 0=C hydrogen bonds are important for the stabilization of the a-helix and / -pleated sheet secondary structures of the proteins, the Watson-Crick hydrogen bonds between the bases, which are the side-chains of the nucleic acids, are fundamental to the stabilization of the double helix secondary structure. In the tertiary structure of tRNA and of the much larger ribosomal RNA s, both Watson-Crick and non-Watson-Crick base pairs and base triplets play a role. These are also found in the two-, three-, and four-stranded helices of synthetic polynucleotides (Sect. 20.5, see Part II, Chap. 16). 

Most native DNAs and certain synthetic polynucleotides are able to undergo a B A conformational transition. In films and fibers, this transition may be induced by decreasing the relative humidity to which the sample is exposed. This is demonstrated by the Raman spectra of fibers (Erfurth et al., 1975 Martin and Wartell, 1982 Prescott et al., 1984) and by the IR spectra of films (Pilet and Brahms, 1973 Taillandier et al., 1985) made of native DNAs. In the case of polynucleotides, the decrease of relative humidity affords different results, depending on the sequence of the polymer. Thus, poly d(A-T) and poly d(A-C). poly d(G-T) - double stranded DNA with one strand adenine and cytosine and in the other guanine and thymine - show a classical B A transition (IR Brahms et al 1976 Taillandier et al., 1984a Adam et al., 1987. Raman Thomas and Benevides, 1985 Jenkins et al., 1986). 

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. 

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. 

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). 

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)... 

Hilleman and his co-workers have demonstrated interferon production following the administration of reovirus RNA or the replicative form of RNA isolated from E. coli infected with MS2 coliphage. These BNAs are effective in inducing resistance to virus infection m vitro and vivo. The induction of interferon and the broad-spectrum protection against viral infection conferred by double-stranded RNA suggested the use of synthetic polynucleotides, including polyriboinosinic-polyribocytidilic acid, which possess a double-stranded conformation. Chemically modified RNA may also induce the production of interferon. ... 

A synthetic short double-stranded RNA such as Polyinosinic-polycytidylic acid (poly(I)-poly(C)) is widely used as a inducer in interferon production. The presence of DEAE-Dextran in the reaction medium increases the uptake of poly(I)-poly(C) by the cells and also makes the polynucleotide less susceptible to degradation from RNase. In addition, the... 

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. 

We have been interested in the conformations of simple sequence synthetic polynucleotides. Figure 7 shows a comparison between calculated and observed CD of four RNA double strands [19]. The agreement is not very good but poly rA poly rlJ and poly rG poly rC give reasonable agreement. We conclude that these two polynucleotides have RNA geometry this conformation is similar to A-form DNA. However, the alternating sequences... 

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. 

Until recently, only right-handed double helices were known. A left-handed helix structure was observed for the first time in the synthetic hexameric DNA containing alternating cytosine and guanine bases [d(CGCGCG)]. Both polynucleotide strands are wound around themselves in a left-handed sense. The phosphate groups show a zick-zack-type pattern along the screw this structure therefore was named Z-DNA (Fig. 2). 

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