Structure of Telomeric DNA, RNA and DNA-RNA Hybrid Oligonucleotides by Nondenaturing Polyacrylamide Gel Electrophoresis

국문 초록 (Abstract)

Tetrahymena human의 Telomere 염기배열을 가지는 DNA, RNA 및 DNA-RNA hybrid oligonucleotide를 다섯 종류 화학 합성하여 전기영동의 gel mobility shift 실험, Tm 측정 및 DMS methylation 반응으로 그들의 구조를 구명하였다. 합성한 oligonucleotide(T1~5) 의 염기배열은 다음과 같다. T1 : d$d(T_2G_4)_4$, T2 : $d(T_2AG_3)_4$, T3: $h(U_2dG_4)_4$, T4 : $h(T_2rG_4)_4$, T5: $r(U_2G_4)_4$. 이들에 대한 nondenaturing PAGE에서 gel mobility 실험결과 T1과 T2는 size marker DNA 24mer보다 훨씬 빠른 gel mobility를 나타내었고, Tm 측정 및 gel mobility 실험결과 T1은 T2보다 안정한 구조를 취하는 것을 확인하였다. 또한, DMS methylation 실험결과 5'말단의 dG가 methylation반응에 sensitive한 것을 확인하였다. 이상의 결과로서 T1 및 T2 oligoneotide는 용액 중에서 Hoogsteen 염기대를 형성하는 Telmere 구조를 취하는 것을 확인하였고. T1 oligoneotide의 염기배열에서 dT를 rU로 치환한 T3는 T1과 동일구조를 취하는 것이 확인되었으나, dG가 rG로 치환된 T4는 T1과는 다른 gel mobility를 나타내었지만, T5와는 동일한 gel mobility를 나타내었다. 이상의 결과로서 분자내 수소결합으로 Hairpin 구조를 취하는 Telomere 구조형성에 가장 중요한 염기는 deoxyguanosine임을 알았다.

다국어 초록 (Multilingual Abstract)

Five kinds of DNA, RNA and DNA-RNA hybrid oligonucleotides (T1~5) containing Tetrahymena and human telomeric sequences were synthesized. T1 had the sequence $d(T_2G_4)_4$. T2, T3, T4 and T5 were $d(T_2AG_3)_4$, $h(U_2dG_4)_4$, $h(T_2rG_4)_4$ and $r(U_2G_4)_4$, respectively. By nondenaturing PAGE, T1 and T2 showed faster gel mobility compared to size marker DNA 24mer in the presence of 50 mM NaCl at low temperatures. T1 showed a more stable telomeric structure than T2 in a Tm analysis experiment and gel mobility shift experiment. We have tested the susceptibility of dGs in telomeric structure to methylation by DMS. At low temperatures, 5'-terminal dG residue of T1 oligomer was hypersensitive to methylation, while other three dGs were resistant. These results suggest that T1 and T2 oligonucleotides form a telomeric structure containing Hoogsteen base pairs. T3 oligomer $h(U_2dG_4)_4$, where dT residues were substituted by rU, had the same mobility as T1 in nondenaturing PAGE. But T4 oligomer $h(U_2dG_4)_4$, where dG residues were substituted by rG, showed a different gel mobility compared with T1. T5 oligomer$r(U_2G_4)_4$ which contained the ribonucleosides only migrated to the same position as T4. These results show that telomeric DNA structure is dependent on dG to form intramolecular hydrogen bonding in the hairpin structure.