Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection.
Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection.
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转谷氨酰胺酶介导的 DNA-（酶）n 探针合成，用于高灵敏度 DNA 检测。
作者：
Momoko Kitaoka;Y. Tsuruda;Yukari Tanaka;M. Goto;Masayuki Mitsumori;Kounosuke Hayashi;Y. Hiraishi;K. Miyawaki;S. Noji;N. Kamiya
利用天然交联催化剂微生物转氨酶（MTG），探索了一种新的具有新结构的DNA-酶缀合物的合成策略。在脱氧尿苷三磷酸嘧啶的5位引入谷氨酰胺供体底物肽MTG，通过聚合酶链反应（PCR）制备具有多个谷氨酰胺供体位点的DNA链。通过基因工程将含有MTG反应性赖氨酸残基的底物肽融合到来自激烈火球菌的耐热碱性磷酸酶（Pyrococcus furiosus，PfuAP）的N末端。通过将引入到DNA模板上的MTG的底物部分与重组酶进行酶促结合，成功地获得了化学计量比为1：n的DNA-（酶）（n）缀合物。偶联物的酶/DNA比率随着DNA模板中苄氧基羰基-L-氨基甘氨酸（Z-QG）部分的增加而增加。在斑点杂交试验中验证了用信号传导酶修饰的新缀合物的潜在效用。在苛刻的杂交条件下，DNA-（酶）（n）探针可以清楚地检测具有互补序列的靶核酸的10（4）个拷贝，从而能够进行简单的检测程序，而不需要与常规的基于半抗原-抗体反应的DNA检测系统相关的繁琐的结合/游离过程。
A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.
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DOI：
10.1016/j.jinorgbio.2004.01.009
发表时间：
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期刊：
Journal of inorganic biochemistry.
影响因子：
--
作者：
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DOI：
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发表时间：
1982
影响因子：
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通讯作者：
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DOI：
10.1073/pnas.78.11.6633
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.80.13.4045
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
LEARY, JJ;BRIGATI, DJ;WARD, DC
通讯作者：
WARD, DC