Modified Polynucleotides

In recent years many other systematic modifications of the classical Crick-Watson structure have been effected. Work has been concentrated particularly on synthetic oligonucleotides which may prove to have valuable chemotherapeutic properties (e.g. antisense oligonucleotides) (Chapters 12.12 and 11.6). Various chemically modified forms of DNA are currently under investigation and some of these are described below. 

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T6. Tomasz, M., Barton, J. K., Magliozzo, C. C., Tucker, D., Lafer, E. M., and Stollar, B. D., Lack of Z-DNA conformation in mitomycin-modified polynucleotides having inverted circular dichroism. Proc. Natl. Acad. Sci. USA 80, 2874-2878 (1983). 

It is clear from these graphs that the modified polynucleotide, MPC, significantly inhibits the DNA polymerases present in both viral extracts furthermore, the inhibitory activity of MPC is very nearly the same in the two systems when poly (dA-dT) is used as the template (50% inhibition at 18 jxg/r.mix.), but in the presence of poly rA (dT)j4 as the template, MPC acts as a much more potent inhibitor of 3H-TMP incorporation in the MSV-M assay system (50% inhibition at 4 /rg/r.mix.) than in the FLV system (50% inhibition at 35 fig/r. mix.). In contrast, the unmodified polynucleotide, PC, stimulates DNA polymerase activity in both viral systems with poly (dA-dT) as the template, and it shows slight inhibitory activity (only 25 % inhibition, at 20-40 /ug/r.mix.) in the presence of the poly rA (dT)14 template. 

Many exotic duplexes, with modifications on the base and/or the sugar have been included. No attempt has been made, however, to deal with random copolymers and partially modified polynucleotides. Nor has there been an attempt to cover the numerous studies on nucleoside (nucleotide) - polynucleotide complexes [74tl] and the analogous oligonucleotide work [74t2]. The latter field is in full fling and new data appear every week. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Roth and Breaker1331 used in vitro selection to evolve DNA molecules of the type illustrated in Scheme 4, which self-cleave in the presence of histidine. (This is in principle mimicking a disabled mutant enzyme, of the sort mentioned in the introduction.1101) In this case a pool of 2 x 1013 modified DNAs was attached at the 5 -end, tightly but reversibly, to a solid support The polynucleotide was made up of a randomised sequence of 40 deoxynucleotides (N40 in Scheme 4) flanked by two regions of basepairing complementarity to the sequences on either side of a single upstream RNA linkage (rA). 

In this method the single-stranded DNA fragment to be sequenced is end-labelled by treatment with alkaline phosphatase to remove the 5 phosphate, followed by reaction with 32P-labelled ATP in the presence of polynucleotide kinase, which attaches 32P to the 5 terminal. The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base as follows. 

Figure 13 7 The two-metal-ion mechanism for polynucleotide polymerases. One metal ion (usually Mg2+) activates the 3 -OH group of the primer terminus and stabilizes one of the partly negatively charged equatorial oxygen atoms of the phosphoryl group, whereas the other binds the phosphoryl oxygen and the oxygen atoms of the pyrophosphate leaving group. The two metal ions are 3.9 A apart. This mechanism fits both RNA and DNA polymerases. [Modified from T. A. Steitz, Nature, Lond. 391,231 (1998).]...

Hildenbrand K, Schulte-Frohlinde D (1989) E.S.R. studies on the mechanism of hydroxyl radical-induced strand breakage of polyuridylic add. Int J Radiat Biol 55 725-738 Hildenbrand K, Schulte-Frohlinde D (1997) Time-resolved EPR studies on the reaction rates of per-oxyl radicals of poly(acrylic acid) and of calf thymus DNA with glutathione. Re-examination of a rate constant for DNA. Int J Radiat Biol 71 377-385 Hildenbrand K, Mirtsch S, Schulte-Frohlinde D (1993) H-NMR studies of y-irradiated polynucleotides and DNA in N20-saturated aqueous solutions. Release of undamaged and modified bases. Radiat Res 134 283-294... 

In order to obtain information about the origin of the peak at + 0.45 V, the GCE surface was modified with DNA-like sequences (po-lyguanylic and polyadenylic acids) that contain or not guanine residues [35]. These new types of biosensors were incubated in a quercetin solution and then conditioned (see Procedure 29 in CD accompanying this book). In this way, it was shown that the peak at + 0.45 V is directly related with the presence of guanine residues in the polynucleotidic chain and that it is due to the formation of 8-oxodGuo. 

Osmium tetroxide is an electroactive marker of the polynucleotide chain and is a good probe of the DNA structure since dsDNA is modified by osmium to a much lesser extent than single-stranded polynucleotides. The limit of detection of osmium-labelled DNA was below 5 ng cm-3 after 2 min accumulation time [66]. Adsorptive stripping linear sweep voltammetry of osmium tetroxide labelled DNA at a mercury electrode [66, 67] was shown to be a good sensor for hybridization of DNA. 

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