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Nucleic Acids Research 2005 33(11):e97; doi:10.1093/nar/gni096
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Published online 16 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Real-time investigation of nucleic acids phosphorylation process using molecular beacons

Zhiwen Tang, Kemin Wang*, Weihong Tan, Changbei Ma, Jun Li, Lingfeng Liu, Qiuping Guo and Xiangxian Meng

Biomedical Engineering Center, State Key Laboratory of Chemo/Biosensing and Chemometrics, Institute of Biological Technology, College of Chemistry and Chemical Engineering, Hunan University Changsha 410082, People's Republic of China

*To whom correspondence should be addressed. Tel: +86 731 8821566; Fax: +86 731 8821566; Email: kmwang{at}hnu.cn

Received September 20, 2004. Revised January 29, 2005. Accepted May 29, 2005.

Phosphorylation of nucleic acids is an indispensable process to repair strand interruption of nucleic acids. We have studied the process of phosphorylation using molecular beacon (MB) DNA probes in real-time and with high selectivity. The MB employed in this method is devised to sense the product of a ‘phosphorylation–ligation’ coupled enzyme reaction. Compared with the current assays, this novel method is convenient, fast, selective, highly sensitive and capable of real-time monitoring in a homogenous solution. The preference of T4 polynucleotide kinase (T4 PNK) has been investigated using this approach. The results revealed that a single-stranded oligonucleotide containing guanine at the 5' termini is most preferred, while those utilizing cytosine in this location are least preferred. The preference of (T)9 was reduced greatly when phosphoryl was modified at the 5' end, implying that T4 PNK could discern the phosphorylated/unphosphorylated oligonucleotides. The increase of oligonucleotide DNA length leads to an enhancement in preference. A fast and accurate method for assaying the kinase activity of T4 PNK has been developed with a wide linear detection range from 0.002 to 4.0 U/ml in 3 min. The effects of certain factors, such as NTP, ADP, (NH4)2SO4 and Na2HPO4, on phosphorylation have been investigated. This novel approach enables us to investigate the interactions between proteins and nucleic acids in a homogenous solution, such as those found in DNA repair or in drug development.


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