Seryl-Histidine as an Alternative DNA Nicking Agent in Nick Translation Yields Superior DNA Probes and Hybridizations Yunsheng Li, a Scott Hatfield, a Jing Li, a Mark McMills, b,c Yufen Zhao d and Xiaozhuo Chen a,b,c,e, * a Edison Biotechnology Institute, Ohio University, Athens, OH 45701, USA b Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA c The Molecular and Cellular Biology Program, Ohio University, Athens, OH 45701, USA d Department of Chemistry, Tsinghua University, Beijing 100084, China e Department of Biomedical Sciences of School of Osteopathic Medicine, Ohio University, Athens, OH 45701, USA Received 21 June 2001; accepted 7 September 2001 Abstract—Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme’s high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3 0 hydroxyl and 5 0 phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less frag- mentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA. # 2002 Elsevier Science Ltd. All rights reserved. Introduction Nucleic acid hybridization is an important method for analyzing specific DNA or RNA sequences, in which a targeted sequence is detected by binding a com- plementary labeled nucleic acid probe. 1 7 A common way of labeling nucleic acid probes, with either radio- isotope labeled nucleotides or otherwise, is nick trans- lation. 8 11 This method typically employs DNase I as the DNA nicking agent to generate single-stranded breaks in double-stranded DNA, exposing new 5 0 - phosphate groups and 3 0 -hydroxyl groups (Fig. 1). The nicks are subsequently translated along the DNA in the 5 0 -3 0 direction as DNA Polymerase I uses the newly generated 3 0 -hydroxyl groups to introduce labeled deoxy-nucleotides into the nicked DNA while removing native nucleotides ahead of the nick. 8 11 Ideally, nicking of the DNA will be sufficiently extensive to allow max- imal labeling without occurring so frequently that proximal nicks on opposite strands result in excessive fragmentation of the DNA. Because DNase I is an effi- cient enzyme that has an intrinsic tendency to nick both strands at the same base-pairs and severely fragment the substrate DNA, the nicking reaction is usually per- formed at a temperature significantly lower than room temperature and with very low DNase I concentrations in the presence of Mg 2+ to reduce DNA fragmentation. 10 12 Even with such precautions, DNase I frequently makes nicks at corresponding positions on both strands, fragmenting the DNA substrate and leav- ing a significant proportion of the DNA probes in 0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(01)00339-X Bioorganic & Medicinal Chemistry 10 (2002) 667–673 *Corresponding author at: Edison Biotechnology Institute, 109 Konneker Research Center, Ohio University, the Ridges, Athens, OH 45701, USA. Tel.: +1-740-593-4713; fax: +1-740-593-4795; e-mail: chenx@ohiou.edu