Monitoring ssDNA Secondary Structure Formation by Determining Topological State of DNA Catenanes
Xingguo Liang 1, Hieko Kuhn 2 and Maxim Frank-Kamenetskii 1*
1 Boston University
2 Boston Univeristy
* To whom correspondence should be addressed. E-mail: mfk{at}bu.edu.
Submitted on September 7, 2005
Revised on October 3, 2005
Accepted on 6 January 2006
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Abstract |
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Single-stranded DNA (ssDNA) has essential biological functions during DNA replication, recombination, repairing, and transcription. The structure of ssDNA must be better understood to elucidate its functions. However, the available data are too limited to give a clear picture of ssDNA due to the extremely capricious structure features of ssDNA. In this study, by forming DNA catenanes and determining their topology (the linking number, Lk) through the electrophoretic analysis, we demonstrate that the studies of catenanes formed from two ssDNA molecules can yield valuable new information about the ssDNA secondary structure. We construct catenanes out of two short (60/70 nt) ssDNA molecules by enzymatic cyclization of linear oligodeoxynucleotides. The secondary structure formed between the two DNA circles determines the topology (the Lk value) of the constructed DNA catenane. Thus, formation of the secondary structure is experimentally monitored by observing the changes of linking number with sequences and conditions. We found that the secondary structure of ssDNA is much easier to form than expected: the two strands in an internal loop in the folded ssDNA structure prefer to braid around each other rather than stay separately forming a loop, and a duplex containing only mismatched base pairs can form under physiological conditions.
Key Words:
DNA folding, DNA ligation, DNA topology, gel electrophoresis, linking number, mismatched duplex
