Synthesis and base pairing properties of DNA-RNA heteroduplex containing 5-hydroxyuridine
2009
Cui, Song (Kyungpook National University, Daegu, Republic of Korea) | Kim, Y.H. (Kyungpook National University, Daegu, Republic of Korea) | Jin, Cheng-Hao (Sungkyunkwan University, Suwon, Republic of Korea) | Kim, S.K. (Kyungpook National University, Daegu, Republic of Korea) | Rhee, M.H. (Kyungpook National University, Daegu, Republic of Korea) | Kwon, O.S. (Kyungpook National University, Daegu, Republic of Korea) | Moon, B.J. (Kyungpook National University, Daegu, Republic of Korea), E-mail: [email protected]
5-Hydroxyuridine (5-OHU) is a major lesion of uridine and cytosine produced in RNA by various chemical oxidants. To elucidate its biochemical and biophysical effects on RNA replication, the site-specifically modified oligoribonucleotides containing 5-OHU were synthesized with C5-hydroxy-5'-O-DMTr-2'-TBDMS-uridine phosphoramidite using automated solid phase synthesis. The base-pairing properties of nucleotides opposite 5-OHU in 24 mer oligoribonulcleotides with dNTP were studied using three reverse transcriptases (Super-Script∨TMⅡ-, AMV-, MMLV-RT) in cDNA synthesis. Adenine as well as guanine was incorporated preferentially by all reverse transcriptases. In the UV-melting temperature experiment, the results from the relative stabilities of the base pairs were A : 5-OHU greater than G : 5-OHU greater than T : 5-OHU almost equal to C : 5-OHU. Circular Dichroism (CD) studies showed that DNA-RNA containing 5-OHU heteroduplexes exhibit a similar conformation between the A-type RNA and B-type DNA. These results suggest that 5-OHU from oxidative damage was mainly influenced by adenine mismatch.
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