Polydeoxynucleotide

An efficient template-guided polycondensation of a decadeoxynucleotide into long-chain double-stranded polydeoxynucleotides was achieved with the thymidine 3 -phos-phoric imidazolides [161]... 

The dissociation of 7 (Scheme 5) from poly[(G-C)] showed three relaxation times and the amplitude corresponded to the total signal, while the dissociation of 7 from poly[(A-T)] was faster and only two relaxation times, corresponding to 70% of the total signal were observed in the stopped-flow experiment. The biological activity of this class of molecules was correlated to the presence of four relaxation times when the dissociation process is measured with DNA, in particular the presence of the long-lived component of hundreds of milliseconds.86,104 105,132 143 The difference in the dissociation kinetics observed with the two polydeoxynucleotides indicates that intercalation into G-C sites is responsible for the biological activity. The dissociation of 7 from ct-DNA led to four relaxation times, a result that is in line with the relaxation times observed with poly[(G-C)] and poly[(A-T)]. 

Table 5 Association and dissociation rate constants for anthraquinone derivatives with ct-DNA and polydeoxynucleotides from ref. 101...

The first evidence of the special structure of DNA was the observation that the amounts of adenine and thymine are almost equal in every type of DNA. The same applies to guanine and cytosine. The model of DNA structure formulated in 1953 explains these constant base ratios intact DNA consists of two polydeoxynucleotide molecules ( strands ). Each base in one strand is linked to a complementary base in the other strand by H-bonds. Adenine is complementary to thymine, and guanine is complementary to cytosine. One purine base and one pyrimidine base are thus involved in each base pair. 

By far the most common form is B-DNA (2). As discussed on p. 84, this consists of two antiparallel polydeoxynucleotide strands intertwined with one another to form a right-handed double helix. The backbone of these strands is formed by deoxyribose and phosphate residues linked by phosphoric acid diester bonds. 

Hurwitz, J. The enzymatic incorporation of ribonucleotides into polydeoxynucleotide material. J. Biol. Chem., 234, 2351-2358 (1959)... 

All of the bacterial deoxyribonucleases that have been examined in detail possess a specificity directed in varying degrees toward the secondary structure of the polydeoxynucleotide. With one recent exception, none of the deoxyribonucleases shows a simple base specificity whereby they attack phosphodiester bonds adjacent to a single base. However, it is now clear that several of the endonucleases may possess an extremely high order of specificity and have the capacity to recognize and attack one or a few phosphodiester bonds in polydeoxynucleotide chains composed of many thousands of internucleotide linkages. 

As noted above, the enzymes to be discussed are those which attack polydeoxynucleotides exclusively. Thus, nucleases from, for example, Bacillus subtilis (3-4), Serratia marcescens (5), and Staphylococcus aureus (0), which attack both RNA and DNA will not be considered. Such an enzyme from S. aureus is, however, the subject of Chapter 7 by Cotton and Hazen and Chapter 8 by Anfinsen et al. in this volume. 

The bacterial DNA exonucleases which have been purified and examined in detail are with two exceptions derived from E. coli and E. coli infected with bacteriophages all show a strong specificity for the secondary structure of their polydeoxynucleotide substrate. 

Barcellona M L, Cardiel G, Gratton E. Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides. Biochem Biophys Res Commun. 170 270-80, 1990. 

DNA is a polydeoxynucleotide and among the largest of the biological macromolecules some DNA molecules comprise more than 108 nucleotides. They contain adenine, thymine, guanine, and cytosine as the bases, and the genetic information is encoded within the nucleotide sequence, which is precisely defined over the entire length of the molecule. One of the simplest methods for determining the nucleotide sequence of DNA makes use of an enzyme, DNA polymerase, which catalyzes the synthesis of DNA. The properties of this enzyme are discussed in Chap. 16. 

DNA in the chromosomes of most organisms is double-helical-, i.e., it consists of two polydeoxynucleotide chains (or strands) twisted around one another in the form of a helix. The genetic information is contained in the sequence of nucleotides along one of the chains, with the sequence in one being complementary to that in the other (Chap. 7). A replica of DNA is one that is an exact copy of itself. 

A characteristic feature of the DNA polymerases is their inability to initiate synthesis of a polydeoxynucleotide chain they can only extend an existing chain. How is initiation of the nascent fragments within the discontinuously synthesized DNA strand achieved ... 

Figure 2.1. A polydeoxynucleotide chain and the structure of DNA. (A) A polydeoxynucleo-tide chain composed of four different deoxynucleotides covalently linked to one another by a phosphodiester bonds (note orientation of the 5 and 3 end of the molecule) (B) Watson-Crick double helix (top) and simple representation of the antiparallel strands (bottom). [Adapted from Volgelstein, B., and Kinzler, K. W. (Eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, 1998.]...

The possibility that this compound may react directly with DNA was indicated by the cytogenetic studies of Green and West57). Tilorone was found to inhibit mitosis significantly at 3.0 pg/ml, and produced chromosomal abnormalities at 1.5 pg/ml. Soon it was discovered by Chandra et a/.26 28)that tilorone does form molecular complexes with DNA and polydeoxynucleotides. Some of these studies will be described here. 

Table 1. Binding constants for the interaction of tilorone hydrochloride with DNAs and synthetic polydeoxynucleotides...

The interaction of tilorone with DNA and synthetic polydeoxynucleotides can be influenced by modifying tilorone structure. Such studies are indeed, important to elucidate the role of structural entities in their complex formation with DNA. Structure-activity relationship of tilorone derivatives (Fig. 6) has recently been studied.7,60,62 ... 

In DNA, two polydeoxynucleotide strands are coiled about one another in a double-helical structure as originally proposed by the Watson-Crick (W-C) model (Figure 23-6). The important features of the W-C model are as follows ... 

Figure 2. Section of a sieving gel on which polydeoxynucleotides have been separated. The polyacrylamide concentration is 67o. The numbers to the right of the arrows indicate the number of bases in the polymer at this point. The letters G, A, T, C indicate which base is present at that particular position in the sequence. Reproduced with permission from Ref. 6. Copyright 1984 Biochem. Biophys. Method.

Figure 1.59 Full structural illustration of a oligodeoxynucleotide (left) and various shorthand structures (right) commonly used to define oligo-polydeoxynucleotide structures simply. Similar short hand structures exist for oligo-polynucleotide structures except that "d" for "deoxy" is dispensed with.

Figure 1.71 Main conformational preferences of 2 -deoxy-p-D-ribofuranose rings in polydeoxynucleotides (right). Cartoon illustration of these main envelope conformations (left) (illustrations from Sinden, 1994, Fig. 1.4).

The entire oligo- or polydeoxynucleotide is then detached from the solid support and de-protected to complete the synthesis. After synthesis of the oligo- or polydeoxynucleotide... 

Figure 2.5 Solid Phase DNA Synthesis Cycle (Contd.). Most frequently used base protecting groups are shown Bz Af-6 benzoyl (adenine), W-4 benzoyl (cytosine) N-2 isobutyroyl (guanine). All are base sensitive. DNA chain is built up from 3 to 5 on controlled-pore glass (CPG) bead solid support. Post global deprotection and resin removal, the desired product oligo-/polydeoxynucleotide is then separated initially by precipitation by means of an agent such as ethanol and purified finally by reversed phase liquid chromatography, or ion exchange chromatography as appropriate (see later in Chapter 2).

---