PubMed · 4687708
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J C Catlin, F Cramer. 1973-01-26. Deoxy oligonucleotide synthesis via the triester method.. https://doi.org/10.1021/jo00942a011
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To evaluate the in vivo mutagenicities of damaged DNA precursors (deoxyribonucleoside 5'-triphosphates) produced by exposure to nitric oxide (NO) and ionizing radiation, five damaged deoxyribonucleotides (deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, dUTP, and 8-hydroxy-dATP) were introduced into competent Escherichia coli cells. Their mutagenic potentials were assayed using the chromosomal rpoB gene as a mutagenesis target. In contrast to 8-hydroxy-dGTP and 2-hydroxy-dATP, which were examined in an earlier study, none of these damaged deoxyribonucleotides significantly increased the rpoB mutant frequency. These results suggest that these five damaged deoxyribonucleotides are weakly mutagenic in vivo if at all. Thus their contributions to mutations induced by NO and ionizing radiation may be small.
Electrospray ionization (ESI) is a dynamic process that, when coupled with mass spectrometry (MS), serves as an invaluable tool for analysis of biomolecules. Our group, as well as others, has observed that there is a bias in signal intensity for one strand of a PCR amplicon over the complementary strand in an ESI mass spectrum. In this report, we have investigated the contributions of hydrophobicity and free energy of solvation to relative signal intensities in ESI-MS spectra of nucleic acids. We developed approaches for predicting which strand of the PCR amplicon will be the most intense: one based on a rate equation for calculating ion flux using values from the literature for hydrophobicity and free energy of solvation and the other based on the percentage of the relatively hydrophilic guanines present in the strand. A trend in signal intensity for deoxyribonucleotide triphosphates, oligonucleotides, and PCR amplicons was observed that was consistent with our model. On the basis of the observation that increased hydrophobicity correlates with greater signal intensity, we selectively enhanced the signal intensity of a 20-mer with the addition of an alkyl chain to the 5' terminus, which subsequently improved the limit of detection to 1 nM, an improvement by 1 order of magnitude. This was extended to a 53-bp PCR amplicon by modifying one primer with the hydrophobic moiety, which resulted in a 16% increase in signal intensity. We capitalized on this result to determine allele frequencies from pooled DNA for single-nucleotide polymorphisms down to 1%.
Phosphorescence and optical detection of magnetic resonance (ODMR) is used to study the excited triplet state of 4',6-diamidino-2-phenyl indole (DAPI) and its complexes with the oligonucleotides [d(CGACGTCG)](2) and [d(GGCCAATTGG)](2), where binding occurs by intercalation between GC base pairs and by minor groove insertion, respectively. Weaker binding of DAPI to phosphate is also detected, and the triplet state of this complex is characterized. Intercalation with [d(CGACGTCG)](2) produces a phosphorescence redshift, while groove binding with [d(GGCCAATTGG)](2) leads to a blueshift. Both binding modes give rise to a small decrease in the zero-field splitting (zfs) of the DAPI triplet state. The largest redshift and zfs decrease are found for the phosphate complex. The phosphorescence lifetimes are shorter by an order of magnitude than that of indole or tryptophan as expected for the lower triplet state energy, E(00), of DAPI. The lifetimes agree well with a correlation with E(00) introduced by Siebrand [Siebrand, W. (1966) J. Chem. Phys. 44, 4055-4057] except for the [d(GGCCAATTGG)](2) minor groove complex with a lifetime that is about 20% too long. The longer lifetime is attributed to distortion of the amidino groups in this complex, resulting in less efficient intersystem crossing.