Polynucleotides

Polynucleotides The polynucleotides were com-mercial products, obtained from Serva-Biochemica and Reanal. They have been used without further purification. (A)n (3.4/ 8S) (U)n ( 10S) (G)n (- 10 S)  

CT-DNA Calf-thymus DNA was a kind gift of Dr.G.Luck (Central Institute of Microbiology and Experimental Therapy, Academy of Sciences of the GDR) with a content of 0.28 % of protein, stabilized with chloroform. The stock solution was diluted to an OO26O 0.625 by means of a O.OOl M NaCl solution. 

Base-Pair Analogues 2.7-bis-[(dialkylamino- and oxa oligoalkylenimino)-acetylamino]-fluoren-9-ones ( f luoramides 1-5) were prepared via the corresponding 2.7-bis- [(chloroacetyl)-amino]-fluoren-9-one by a procedure described recently (56) see also (57). 

Vinylmonomers for Homo- and Copolymerization Reactions 1-Vinyluracil, 9-vinylhypoxanthine, and 9-vinyladenine were prepared by previously described methods (58-62), 1-vinylcytosine by the ammonolysis of l-vinyl-4-methylthiopyrimidine-2-one (62,63). 

Polynucleotide Strand-Analogues The following polymers (Tables I and II) were prepared by AIBN- or -ray induced (Laboratory Radiation Unit RCH-y-30 as a 60co -source) polymerization and copolymerization reactions, respectively, followed in case of (vA, IvOH]q 7)p by a polymeranalogous reaction. The starting amounts of N-vinylnucleobases and their comonomers were adjusted to yield a nearly l l-ratio in the copolymers. 

Polynucleotides are the group of natural polymers in which the monomeric units are formed by a type of biomolecule known as nucleotides. Nucleotides are basically nucleoside phosphates. Nucleoside is the part of a nucleotide that is formed by glycosidic linkage between a monosaccharide and a type of nitrogenous compound called a nucleotide base. The nucleotide base may be of two types a purine base or a pyrimidine base. Depending 

Purine and pyrimidine bases and their occurrence in polynucleotides 

CHEMICAL EVOLUTION TO HIGHER ORGANIZATION LEVELS 6.1. Polynucleotides 

Research on template-directed syntheses of polynucleotides has been reviewed by Orgel and Lohrman (1974). Briefly the technique involves using 

The second problem mentioned above indirectly raises some interesting questions. As already mentioned, the purines are formed very readily by spontaneous reactions of HCN in aqueous solution. Under the same conditions no biologically significant pyrimidines are formed except for small amounts of orotic acid. The available syntheses of uracil and thymine involve more steps than the purines, since the reactants (/3-alanine and urea) must first be formed, then undergo subsequent reaction on a clay surface. Cyto- 

Further potential polynucleotide-ligand interactions involve the negative surface charges (that can electrostatically interact with positively charged non-protein and protein ligands) and surface structural elements such as minor and major grooves and secondary structure loops. Thus, for example, polynucleotides can have palindromic complementary sequences, for example, 

Palindromic double strands of this kind can potentially form cruciform structures through the individual single strands forming looped out structures. 

A variety of plant substances with planar, polycyclic, aromatic structures can intercalate with DNA, examples being the quinoline alkaloid camptothecin and the furanocoumarin phenolic psoralen (Table 12.1). A variety of plant-derived anthraquinones and naphthoquinones bind to DNA and it is notable that the structurally related anthraquinones mitox-antrone and adriamycin are clinically employed as anticancer drugs (Table 12.1). DNA-binding compounds that interfere with DNA repair, DNA replication and gene expression are cytotoxic and have potential as anticancer agents (see Chapter 9). 

Ribonudease Tx Aspergillus oryzae RNA endonuclease splits d bonds where guanine nucleotides are in 3 position 

Deoxyribonuclease I Bovine pancreas DNA endonudease splits simple or double-stranded DNA spedfic for p bonds where pyrimidine nucleotides are in 3 position, produdng largely tetranudeotides 

Deoxyribonuclease II Porcine spleen Same as DNA as I, except that d bonds are broken and average product is a hexanudeotide 

Micrococcal nuclease Staphylococcus aureus RNA and DNA endonuclease splits d bonds in areas rich in adenosine, uracil, and thymine 

In the calculations on these systems (see Hoffmann and Ladik (1964) and Fraga and Valdemoro (1967)), the existence of the orthophosphoric acid and the sugar is not taken into account. That is, the treatments have been directed towards an investigation of the changes due to the possible Tt-bonding, if any, between the stacked bases. 

The large separation between any two consecutive bases seens to preclude any appreciable amount of overlapping between the orbitals of the independent bases. In fact, the results of Hoffmann and Ladik (1964) for dinucleotides confirm this point. The calculations of Fraga and Valdemoro (1967) show a similar situation for trinucleotides no 

10 The values obtained by Valdemoro and Fraga (1967b) for guanine-cytosine are not given in these tables, as they represent only preliminary results. 

11 Ldwdin (1964) gives a very sophisticated discussion of this problem, in terms of proton tunneling. 

The interest of the trinucleotides lies on the possibility of an interpretation of the genetic code. But before presenting the details for these systems, it is convenient to review the situation regarding dinucleotides and compare the results for some typical mono-, di-, and trinucleotides. Ladik and Appel (1966) have carried out Cl calculations for the dinucleotides GG, GC, CG, and CC. In these calculations the first sixteen singlet excited configurations have been used. (For comparison purposes, the calculations have been carried out for two sets of molecular orbitals those obtained after the first iteration and the true SCF orbitals). On the basis of these results, Ladik and Appel (1966) indicate the existence of a hypochromicity in the first absorption band for the four dinucleotides considered. 

Note the absence of a phosphate at the 5 end and the presence of deoxyribose. Another commonly used shorthand form for describing this structure is 

Note that this latter form does not define the nature of the sugar. EXAMPLE 7.14 

This structure contains six nucleotide units and is referred to as a hexanucleotide. The general term for structures containing a few nucleotides (10 or less) is oligonucleotide. 

Q The DNA in each of your cells (i.e. the human genome) contains about 3 X lO base pairs. How far would this stretch if laid out in a., straight line  

Many other polynucleotide conformations are possible, including the left-handed helical Z-DNA and more complicated structures thought to be involved in chain replication, together with supercoiling and more globular structures in single-stranded transfer RNA. 

In chloroform, carbon tetrachloride, and dimethyl sulfoxide, i.e., weak hydrogen-bonding solvents, nucleosides and nucleoside derivatives exist 

The coupling of nucleotides in vivo or in vitro to give polynucleotides does not occur via the monophosphates, but rather via the diphosphates and triphosphates, e.g., 

If all four 5 -triphosphates are present in vitro (d-ATP, d-CTP, d-GTP, and d-TTP), as well as a single-stranded DNA template (matrix), magnesium ions, and the enzyme DNA-polymerase, then high-molecular-weight poly(d-ribonucleotides) are produced. Since DNA synthesis will not occur without the DNA template, the template may be referred to as the primer. The composition and sequence of this newly formed DNA correspond to those of the native primer DNA, i.e., the DNA that was initially added. The DNA polymerase (Kornberg enzyme) may be obtained from Escherichia coli and added as a cell-free extract. 

The polymerization occurs very slowly if native (double-stranded) DNA is used as the primer. A DNA that has been treated with the enzyme pancrease-DNAse and then denatured at ll C is more effective. The fact that native DNA is a poor primer in this reaction supports the conclusion that DNA polymerase is what is known as a repairing enzyme. Indeed, in the presence of DNA polymerase, the necessary nucleoside triphosphos-phates, and a DNA template at 20°C, breaks in DNA molecules can be repaired. At higher temperatures, of course, branched polynucleotides occur. 

In DNA synthesis, each of the two strands duplicates itself in short segments of 1000 nucleotides. These segments (Okazi fragments) can then be joined together by the enzyme polynucleotideligase. Thus, DNA polymerase is not the only enzyme involved in DNA replication. 

Large-scale ab initio calculations on fragments of polynucleo- 

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The hydration shell is formed with the increasing of the water content of the sample and the NA transforms from the unordered to A- and then to B form, in the case of DNA and DNA-like polynucleotides and salt concentrations similar to in vivo conditions. The reverse process, dehydration of NA, results in the reverse conformational transitions but they take place at the values of relative humidity (r.h.) less than the forward direction [12]. Thus, there is a conformational hysteresis over the hydration-dehydration loop. The adsorption isotherms of the NAs, i.e. the plots of the number of the adsorbed water molecules versus the r.h. of the sample at constant temperature, also demonstrate the hysteresis phenomena [13]. The hysteresis is i( producible and its value does not decrease for at least a week. 

Besides the MDL Molfile formal, other file formats are often used in chemistry SMILES has already been mentioned in Section 2.3.3. Another one, the PDB file format, is primarily used for storing 3D structure information on biological macromolecules such as proteins and polynucleotides (Tutorial, Section 2.9.7) [52, 53). GIF (Crystallographic Information File) [54, 55] is also a 3D structure information file format with more than three incompatible file versions and is used in crystallography. GIF should not be confused with the Chiron Interchange Formal, which is also extended with. cif. In spectroscopy, JCAMP is apphed as a spectroscopic exchange file format [56]. Here, two modifications can be... 

PDB file. pdb Protein Data Bank file format for 3D stmcture information on proteins and polynucleotides nmm.rcsb.org 53... 

With the increase in hardware and software, larger systems can be handled with higher accuracy. Much work will continue to be devoted to the study of proteins and polynucleotides (DNA and RNA), and particularly their interactions with more sophisticated methods. Remember proteins and genes are chemical compounds and sophisticated theoretical and chemoinformatics methods should be applied to their study - in addition to the methods developed by bioinfor-maticians. 

Synthetic oligonucleotides may be used as "primers and be elongated stepwise with the aid of polynucleotide phosphorylase (PNPase) and nucleoside diphosphates. 

Adding nucleotides to the 3 oxygen of an existing structure is called elongation and leads ultimately to a polynucleotide The most important polynucleotides are ribonu cleic acid (RNA) and deoxyribonucleic acid (DNA) As we shall see m later sections the polynucleotide chains of DNA and some RNAs are quite long and contain hundreds of thousands of bases... 

In addition to hydrogen bonding between the two polynucleotide chains the double helical arrangement is stabilized by having its negatively charged phosphate groups on the outside where they are m contact with water and various cations Na" Mg and ammonium ions for example Attractive van der Waals forces between the... 

By analogy to the levels of structure of proteins the primary structure of DNA IS the sequence of bases along the polynucleotide chain and the A DNA B DNA and Z DNA helices are varieties of secondary structures... 

FIGURE 28 9 The new polynucleotide chain grows by reaction of its free 3 OH group with the 5 triphosphate of an appropriate 2 deoxyribonucleoside... 

Even if It could be shown that RNA preceded both DNA and proteins in the march toward living things that doesn t automatically make RNA the first self replicating molecule Another possibility is that a self replicating polynucleotide based on some carbo hydrate other than o ribose was a precursor to RNA Over many generations natural selection could have led to the replacement of the other carbohydrate by D ribose giving RNA Recent research on unnatural polynucleotides by Professor Albert Eschenmoser of the Swiss Federal Institute of Technology (Zurich) has shown for example that nucleic acids based on L threose possess many of the properties of RNA and DNA... 

The contents of each tube are then subjected to electrophoresis m separate lanes on the same sheet of polyacrylamide gel and the DNAs located by autoradiography A typical electrophoresis gel of a DNA fragment containing 50 nucleotides will exhibit a pattern of 50 bands distributed among the four lanes with no overlaps Each band cor responds to a polynucleotide that is one nucleotide longer than the one that precedes it (which may be m a different lane) One then simply reads the nucleotide sequence according to the lane m which each succeeding band appears... 

The mam use of PCR is to amplify or make hundreds of thousands—even mil lions—of copies of a portion of the polynucleotide sequence m a sample of DNA Sup pose for example we wish to copy a 500 base pair region of a DNA that contains a total of 1 million base pairs We would begin as described m Section 28 14 by cleaving the DNA into smaller fragments using restriction enzymes then use PCR to make copies of the desired fragment... 

The double stranded DNA shown m Figure 28 14(a) contains the polynucleotide sequence (the target region) we wish to amplify The DNA is denatured by heating to... 

Elongation of the primed polynucleotide fragments completes the second cycle and gives four DNAs... 

Oligonucleotides contain about 50 or fewer nucleotides held together by phosphodiester links polynucleotides can contain thousands of nucleotides... 

Section 28 7 Nucleic acids are polynucleotides present m cells The carbohydrate component is D nbose m ribonucleic acid (RNA) and 2 deoxy d ribose m deoxyribonucleic acid (DNA)... 

Section 28 11 Three RNAs are involved m gene expression In the transcription phase a strand of messenger RNA (mRNA) is synthesized from a DNA tern plate The four bases A G C and U taken three at a time generate 64 possible combinations called codons These 64 codons comprise the genetic code and code for the 20 ammo acids found m proteins plus start and stop signals The mRNA sequence is translated into a prescribed protein sequence at the ribosomes There small polynucleotides called... 

Section 28 14 The nucleotide sequence of DNA can be determined by a technique m which a short section of single stranded DNA is allowed to produce its complement m the presence of dideoxy analogs of ATP TTP GTP and CTP DNA formation terminates when a dideoxy analog is incorporated into the growing polynucleotide chain A mixture of polynucleotides dif fermg from one another by an incremental nucleoside is produced and analyzed by electrophoresis From the observed sequence of the comple mentary chain the sequence of the original DNA is deduced... 

DNA (deoxyribonucleic acid) (Section 28 7) A polynucleotide of 2 deoxynbose present in the nuclei of cells that serves to store and replicate genetic information Genes are DNA... 

Messenger RNA (mRNA) (Section 28 11) A polynucleotide of nbosethat reads the sequence of bases in DNA and inter acts with tRNAs in the nbosomes to promote protein biosynthesis... 

Nucleic acid (Section 28 7) A polynucleotide present m the nu clei of cells... 

Oligomer (Section 14 15) A molecule composed of too few monomer units for it to be classified as a polymer but more than in a dimer trimer tetramer etc Oligonucleotide (Section 28 6) A polynucleotide containing a relatively small number of bases Oligosaccharide (Section 25 1) A carbohydrate that gives three to ten monosacchandes on hydrolysis Optical activity (Section 7 4) Ability of a substance to rotate the plane of polanzed light To be optically active a sub stance must be chiral and one enantiomer must be present in excess of the other... 

Reverse transcriptase (Section 28 13) Enzyme that catalyzes the transcription of RNA to DNA Ribozyme (Section 28 11) A polynucleotide that has catalytic activity... 

Transcription (Section 28 11) Construction of a strand of mRNA complementary to a DNA template Transfer RNA (tRNA) (Section 28 11) A polynucleotide of n hose that is bound at one end to a unique amino acid This ammo acid is incorporated into a growing peptide chain Transition state (Section 3 1) The point of maximum energy in an elementary step of a reaction mechanism Translation (Section 28 12) The reading of mRNA by van ous tRNAs each one of which is unique for a particular ammo acid... 

The lUBMB Commission on Nomenclature has issued a number of recommendations dealing with areas of a more biochemical nature (72), such as peptide hormones (86), conformation of polypeptide chains (87), abbreviations for nucleic acids and polynucleotides (88), iron—sulfur proteins (89), enzyme units (90), etc. The Commission has also produced rules and recommendations for naming enzymes (91,92). 

The nucleotides are linked together via the phosphate groups, which connect the 5 -hydroxyl group of one nucleotide and the 3 -hydroxyl group of the next to form a polynucleotide chain (Fig. la). DNA is not a rigid or static molecule rather, it can adopt a variety of hehcal motifs. 

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