摘要
用电泳迁移分析方法研究了２１ｎｔ脱氧寡核苷酸Ｇ３ＴＧ２ＴＧＴ２Ｇ５ＴＧ２ＴＧＴ（ＣＰ１）与１２９ｂｐ的乙肝病毒（ＨＢＶ）核衣壳启动子（Ｃｐ）片段内一位点结合形成的三链ＤＮＡ的特异性及稳定性．在克隆有ＨＢＶ基因组的质粒ｐＣＰ１０的酶切产物中，ＣＰ１仅与含Ｃｐ的１２９ｂｐ片段结合．在２０ｍｍｏｌ／ＬＭｇ２＋溶液中其解离常数（Ｋｄ）为１．４×１０－７ｍｏｌ／Ｌ．不同离子稳定三链ＤＮＡ的效果依次为ｓｐ４＋（精胺）＞Ｍｇ２＋＞Ｚｎ２＋＞Ｎａ＋＞Ｋ＋，离子之间存在相互竞争作用．比ＣＰ１多一误配碱基的脱氧寡核苷酸Ｇ２ＴＧ２ＴＧＴＧ３ＴＧ２ＴＧ２ＴＧ２Ｔ（ＣＰ２）在２０ｍｍｏｌ／ＬＭｇ２＋溶液中与Ｃｐ结合的Ｋｄ值约为ＣＰ１的１／７，而在６０ｍｍｏｌ／ＬＫ＋或５ｍｍｏｌ／ＬＺｎ２＋溶液中检测不到它与Ｃｐ的结合，这进一步显示了三链ＤＮＡ形成的特异性．细胞的生理离子浓度被认为是：Ｓｐ４＋１ｍｍｏｌ／Ｌ，Ｍｇ２＋１０ｍｍｏｌ／Ｌ，Ｋ＋１４０ｍｍｏｌ／Ｌ，因此，ＣＰ１在细胞内将能特异地与Ｃｐ结合并具有较好的稳定性．
The specificity and stability of triplex formation between a 21nt G rich oligodeoxyribonucleotide G 3TG 2TGT 2G 5TG 2TGT(CP1)and a single site within a 129 bp nucleocapsid promoter(Cp)fragment of hepatitis B virus(HBV)were investigated by electrophoretic mobility shift analysis.Among the products of plasmid pCP10 digested by StyⅠ and Rsa Ⅰ,which cloned with HBV genome,CP1 only binded to the 129 bp fragment that contains Cp,and the dissociation constant(Kd)was 1 4×10 -7 mol/L in a buffer containing 20 mmol/L Mg 2+ .The order of effectiveness of different cations on triplex formation was Sp 4+ (spermine)>Mg 2+ >Zn 2+ >Na + >K + .The competition effects between cations were observed.The oligodeoxyribonucleotide G 2TG 2TGTG 3TG 2TG 2TG 2T(CP2),which has a more mismatched base than CP1,binded to Cp with a K d one seventh of that of CP1 in a solution containing 20 mmol/L Mg 2+ .Furthermore,the binding of CP2 to Cp was not observed in 60 mmol/L K + or 5 mmol/L Zn 2+ .This further demonstrated the specificity of triplex formation.Considering the intracellular concentration of cations are Sp 4+ 1 mmol/L,Mg 2+ 10 mmol/L,K + 140 mmol/L,these data suggested that CP1 would bind to Cp specifically and remain with a good stability in cell.
