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KMID : 0624620090420060373
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BMB Reports
2009 Volume.42 No. 6 p.373 ~ p.379
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Synthesis and base pairing properties of DNA-RNA heteroduplex containing 5-hydroxyuridine
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Cui Song
Rhee Man-Hee
Kim Sang-Kook
Kim Yong-Hoon
Jin Cheng-Hao
Kwon Oh-Shin
Moon Byung-Jo
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Abstract
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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’-ODMTr-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-ScriptTMII-, 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 ＞ G : 5-OHU ＞ T : 5-OHU ≒ 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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KEYWORD
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Base pairing, CD spectra, Melting temperature, Oligoribonucleotide, 5-Hydroxyuridine
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