Random sequence DNA

The Watson-Crick structure is also referred to as B-form DNA, or B-DNA The B form is the most stable structure for a random-sequence DNA molecule under physiological conditions and is therefore the standard point of reference in any study of the properties of DNA Two structural variants that have been well characterized in crystal structures are the A and Z forms. These three DNA conformations are shown in Figure 8—19, with a summary of their properties. The A form is favored in many solutions that are relatively devoid of water. The DNA is still arranged in a right-handed double helix, but the helix is wider and the number of base pairs per helical turn is 11, rather than 10.5 as in B-DNA The... 

Deoxyribozymes (also called DNA enzymes or DNAzymes) are specific sequences of DNA that have catalytic activity. All currently known deoxyribozymes have been identified by in vitro selection from large random-sequence DNA pools (Joyce, 2004 Silverman, 2009). The catalytic range of DNA encompasses both oligonucleotide and nonoligonucleotide substrates (Baum and Silverman, 2008 Silverman, 2008). This report focuses on deoxyribozymes that are useful for reactions of RNA substrates, especially to assist studies of RNA structure, folding, and catalysis. 

Nucleic acids that can perform a wide variety of binding reactions have been selected from random sequence pools by affinity immobilization. Oliphant et al. [2] selected DNA molecules that could bind to the yeast transcriptional activator GCN4 from a random-sequence DNA pool that spanned nine positions. Since then, aptamers (nucleic acid ligands) have been selected against a variety of protein targets that naturally bind to nucleic acids, such as EF-Tu, ribosomal proteins, QP replicase, and reverse transcriptase (reviewed in Ref. 3). In addition, aptamers have been selected against intracellular and... 

The flexibility of the alkanediamine linkers in dinuclear platinating agents produces a broad array of adducts on random sequence DNA. The multiplicity of adducts formed complicates the detection and structural characterization of a specific lesion on double-stranded DNA. In order to control the sequence specificity of the DNA-targeting drugs more efficiently, alternative linkers exhibiting increased conformational rigidity are required. 

DNA libraries are obtained by a combination of the techniques of chemical solid-state synthesis and the combinatorial methods described later for peptides (see Sec. 9.3.2.2). DNA should, however, first be transformed to RNA libraries in order to yield useful recognition systems. Starting from a large pool of random-sequence DNA molecules, RNA sequences are obtained (see Fig. 8.4.3),... 

The principle of in vitro selection is governed by a number of the same principles that apply to the Darwinian theory of evolution, as shown in Figure 2. First, the random sequence DNA is prepared by automated solid-phase synthesis. A mixture of four types of nucleotide is added in a stepwise condensation reaction process. When necessary, this DNA library may be converted to an RNA library by in vitro transcription or to a peptide library by in vitro translation. Second, the prepared DNA, RNA, or peptide library is subjected to affinity selection, and the molecules that bind to a target molecule are selected. Because only a very small part of the library is selected in each selection, the selected fraction is then amplified by a polymerase chain reaction (PCR) or a reverse transcription PCR (RT-PCR) technique. Successive selection and amplification cycles bring about an exponential increase in the abundance of the targeting DNA, RNA, or peptide until it dominates the population. 

RNA polymerase holoenzyme has lower affinity for nonspecific DNA sequences than for promoter sequences. The nonspecific affinity, however, allows the enzyme to bind to random-sequence DNA and then slide along the molecule in a unidimensional random walk until it encounters a promoter sequence, for which its binding affinity is higher. Diffusion in one dimension is much faster than diffusion in three dimensions, thereby explaining the observed rapid rate constant for the binding of RNA polymerase holoenzyme to promoter sequences. If one measured the encounter of the polymerase with the nonspecific regions of the DNA rather than with promoter sequences, the value of the rate constant would be much lower and would fit our expectations for a three-dimensional, diffusion-limited reaction between macromolecules. 

UV absorption spectroscopy is further used to determine nucleic acid concentration. The average molecular weight of a nucleotide is 320 and 50[rgml concentration of a random sequence DNA provides the A260 absorbance of 1 in a 1 cm pathlength cell. In practice, the absorption coefficient of DNA is precisely determined by enzymatic hydrolysis of DNA to monomers and calculation using the absorption coefficients of the monomers, or by measurement of thermally denatured DNA. The absorption of the denatured DNA sample is calculated on the basis of absorption coefficients of the constituent mono- and dinucleotides. 

Fig. 13. Theoretical curves of transition width AtJ of a random sequence DNA versus %GC for the three models shown. Experimental points (A) are from bacterial DNA. transition curves in 0.1 SSC.

A comparison between theoretical transition curves and the experimental melting curve of E. coli DNA molecules is presented in Fig. 14. Theoretical curves were obtained by averaging the melting curves calculated from five sequences generated with the same initial instructions. Curves a, h, and c correspond respectively to a random sequence DNA using the MI model, a random sequence DNA using the LE model, and a block sequence DNA using the LE model. 

The initial instruction for a random sequence DNA is that each base pair has the probability of being an A-T, and (1 — pj of being a G-C. For a block sequence DNA, we divide an A-unit DNA into K blocks with base pairs in the ith block ... 

J. A. McClellan, E. Palecek, and D. M. Lilley, (A-T)n tracts embedded in random sequence DNA—formation of a structure which is chemically reactive and torsionally deformable, Nucleic Acids Research, vol. 14, no. 23, pp. 9291-9309, 1986. 

The homopolymer poly(dA) poly(dT) also gives a singlet P resonance (Table I Cohen et al., 1981) simil to those of random-sequence DNA, poly(dGdC) poly(dGdQ (Simpson and Shindo, 1980), and poly(dAdU) poly(dAdU) (Cohen et al., 1981) but at slightly higher field. 

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