DETECTION OF NUCLEIC-ACID HYBRIDIZATION BY NONRADIATIVE FLUORESCENCE RESONANCE ENERGY-TRANSFER
DETECTION OF NUCLEIC-ACID HYBRIDIZATION BY NONRADIATIVE FLUORESCENCE RESONANCE ENERGY-TRANSFER
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DOI：
10.1073/pnas.85.23.8790
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发表时间：
1988-12-01
影响因子：
11.1
通讯作者：
WOLF, DE
中科院分区：
文献类型：
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作者：
CARDULLO, RA;AGRAWAL, S;WOLF, DE
采用非辐射荧光共振能量转移技术研究了互补寡核苷酸的杂交。(i)将黄绿素（供体）和罗丹明（受体）共价连接到不同长度的互补寡脱氧核苷酸的5 "末端。互补的寡脱氧核苷酸杂交后，检测到的荧光素发射强度的降低和罗丹明发射强度的增强的能量转移。在所有情况下，在未标记的补体存在下，荧光素发射强度被猝灭约26%。5 ℃时的转移效率随着杂交体中供体和受体荧光团之间的距离从8个核苷酸增加到12个核苷酸再到16个核苷酸，C从0.50下降到0.22到0.04。将这些杂合体模拟为双螺旋表明，转移效率随着供体-受体间隔R的六次方的倒数而降低，如理论预测的，相应的R0为49埃。(ii)荧光共振能量转移用于研究两个荧光团标记的寡核苷酸杂交到一个较长的，未标记的寡脱氧核苷酸。制备两个12聚体，其与29聚体上由四个碱基分隔的两个相邻序列互补。用荧光素和罗丹明标记的两个12-mer的相邻5 "和3"末端显示出约1.5的转移效率。0.60在5.5度。当它们都与未标记的29-mer杂交时。(iii)一种嵌入染料，吖啶橙子，被用作供体荧光团的一个单一的罗丹明共价连接到一个寡脱氧核苷酸的5 "末端的12碱基对的杂交。在这些条件下，转移效率为0.47在5.5度。C.这些结果表明，荧光调制和非辐射荧光共振能量转移可以检测溶液中的核酸杂交。这些技术，随着进一步的发展，也可以证明用于检测和定量活细胞中的核酸杂交。
Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5'' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5.degree. C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 .ANG.. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5'' and 3'' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of .apprxeq. 0.60 at 5.degree. C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5'' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was .apprxeq. 0.47 at 5.degree. C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy tranfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.