Small Oligonucleotides

The number of studies on ssODNs is rather low, but important information as to the formation of tandem lesions has been obtained with these systems. 

In the reaction of OH with d(TpApCpG) in oxygenated aqueous solution the Fo lesion, the Cyt-derived l-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine modification and 8-oxo-G were observed (Paul et al. 1988). In the presence of GSH and in the absence of O2, the major detected modification is 50HH2Thy (Paul et al. 1990). 

With d(ApCpGpT) X-irradiated in N2-saturated solution the H2Thy, 60HH2Thy, Tg, 8-oxo-G and cA modifications were detected (Schroder et al. 1995). In the presence of 02, the observed modifications were 8-oxo-G, 8-oxo-A, 

In an extension of this study, some further tandem lesions were detected in d(CpGpTpA) and d(CpApTpG) (Box et al. 1998). The cross-linked tandem base lesions shown below are only formed in the absence of 02. 

It is reasonable to assume that the H-- and -OH-adducts at C(5) of C bind to C(8) of the neighboring G, and the resulting adduct radical is oxidized in subsequent free-radical termination reactions. While the H--adduct product (on the right) 

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In Section II we provide an overview of the current status of nucleic acid simulations, including studies on small oligonucleotides, DNA, RNA, and their complexes with proteins. This is followed a presentation of computational methods that are currently being applied for the study of nucleic acids. The final section of the chapter includes a number of practical considerations that may be useful in preparing, performing, and analyzing MD simulation based studies of nucleic acids. 

The enzyme-resistant residue left after ribonuclease action is not dialyz-able and was believed to have a high molecular weight. This has now been shown to be due not to molecular size but to electrostatic effects, and in the presence of salts the enzyme-resistant core readily dialyzes.212 Moreover, it consists of a mixture of relatively small oligonucleotides containing some di- and tri-nucleotides.96 Some of these oligonucleotides have been charac-... 

Any enzyme that catalyzes breakage of a phosphodiester linkage at one or the other end of a polynucleotide chain, resulting in the release of single nucleotides or small oligonucleotides. See specific exonuclease... 

RNA and the synthetic polynucleotides can be cleaved in the absence of divalent metal ions by basic hydrolysis as shown in Figure C, in which the formation of small oligonucleotides from highly polymeric nucleotide chains is followed. In the absence of divalent metal, the reaction rates are approximately the same for RNA, Poly I, and Poly A. 

The Maxam-Gilbert method doesn t require synthesis of a primer and it sometimes works well for sequences that are difficult to obtain with the Sanger -Coulson procedure. The two methods may both be used to provide additional certainty about a sequence. The Maxam-Gilbert method is very convenient for sequencing small oligonucleotides which often react poorly with the polymerase used for the chain termination method. The method usually requires that a restriction map be prepared. 

Sequencing RNA. The first known RNA sequence, that of an alanine tRNA, was determined by Holley and associates in 1965. The RNA was subjected to partial hydrolysis with pancreatic ribonuclease and ribonuclease Tj (Fig. 5-43). Tire small oligonucleotide fragments were separated by ion exchange chromatography under denaturing conditions (7 M urea) and were then characterized individually.645 Tire availability... 

Excision of a group I intron to form a circular RNA and a small oligonucleotide. (B) Excision of a group II intron from yeast mitochondrial pre-mRNA as a circular RNA. See Cech and Bass.581... 

No small oligonucleotides such as di- or trinucleotides with 3 -terminal Ap are produced throughout the process of digestion, but those with 3 -terminal Gp or Up are produced in the course of digestion. 

One way to detect whether these treatments had any effect on the nucleic acid is to subject it to electrophoresis on an agarose gel. Free nucleotides, or even small oligonucleotides, are not visible on agarose gels, whereas the original viral nucleic acid should yield one (or more) discrete high molecular weight bands. 

Apart from NMR, other techniques - such as CD68,75 and IR142 - may be used, often complementary, to study the structure and conformation of small oligonucleotides. 

The VCD features for a number of larger peptide models have been calculated in the course of our efforts to define their solution conformation. These calculations proceeded exactly in the same manner as the ones described for small oligonucleotides. Cartesian coordinates from X-ray experiments, or from the program MacroModel [21), were used, along with a vibrational frequency for an unperturbed, single amide I or amide I vibration. The dipole transition moment for the amide I vibration was taken somewhat lower than that of the nucleotide base carbonyl stretching vibration, in agreement with observed data and literature values. Details of these calculations will also be provided in Section 4. 

For many years, mass spectrometry has had an important role in the analysis of nucleic acids. However, older work was limited to the analysis of nucleotides because the El and Cl methods did not allow oligonucleotide analysis. With the advent of FAB and PD, small oligonucleotides comprising up to about 10 bases have been analysed. 

The error on the measured molecular mass obtained by MALDI varies between 0.01 to 0.05 % for small oligonucleotides. This error increases to 0.5 % or even more for large nucleotides. Indeed, at higher masses the signals from adducts or resulting from the loss of a base are more abundant and cannot be resolved, any more, causing a broadening of the peak. The error on the centroid determination increases. One could consider that the maximum size that can be analysed reasonably by MALDI is around 50 bases for DNA. For RNA more than 100 bases have been analysed with success. [164]... 

THE TETRAMERS GCAT AND CGTA AND OTHER SMALL OLIGONUCLEOTIDES... 

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