Kamakshaiah Charyulu Devarayapalli

Direct immobilization of DNA oligomers onto the amine-functionalized glass surface for DNA microarray fabrication through the activation-free reaction of oxanine

Oxanine having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. By the chemical synthesis, a nucleotide unit of oxanine (Oxa-N) was incorporated into the 5′-end of probe DNA with or without the -(CH2)n- spacers (n = 3 and 12) and found to immobilize the probe DNA covalently onto the NH2-functionalized glass slide by one-pot reaction, producing the high efficiency of the target hybridization. The methylene spacer, particularly the longer one, generated higher efficiency of the target recognition although there was little effect on the amount of the immobilized DNA oligomers. The post-spotting treatment was also carried out under the mild conditions (at 25 or 42°C) and the efficiencies of the immobilization and the target recognition were evaluated similarly, and analogous trends were obtained. It has also been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition. Immobilization of DNA oligomer by use of Oxa-N on the NH2-functionalized surface without any activation step would be employed as one of the advanced methods for generating DNA-conjugated solid surface.

Biophysical Stability and Enzymatic Recognition of Oxanine in Dna

Oxanine (Oxa), which is one of the major products generated from guanine by nitrosative oxidation and is as long-lived as Gua in DNA, has been thought to be one of the major causes for NO-induced DNA damage. In the present study, using several synthetic Oxa-containing oligodeoxynucleotides, biophysical stability and enzymatic recognition of Oxa was investigated in DNA strands. It was found that Oxa did not mediate marked distortion in the whole DNA structure although Oxa pairing with 4 normal bases decreased thermal stability of the DNA duplexes compared to Gua:Cyt base pair. Regarding the responses of the DNA-relevant enzymes to Oxa, it was determined that Oxa was recognized as Gua except that DNA polymerases incorporated Thy as well as Cyt opposite Oxa. These results imply that Oxa tends to behave as a kind of naturally occurring base, Gua and therefore, would be involved in the genotoxic and cytotoxic threats of NO in cellular system.

Quantitative analysis of specific target DNA oligomers using a DNA-immobilized packed-column system

Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on Tm values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar Tm values were separated and detected quantitatively by using a packed column carrying two different DNA probes.