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The addition of single-stranded binding protein (Promega) to the product mixture from the thermal denaturation of the oligomers 3 and subsequent electrophoretic analysis indicates the presence of free single-stranded DNA. Moreover, conjugates containing STV and one or more non-complementary single-stranded DNA molecules were determined to migrate as constituents of the diffuse background hand. The formation of the single-stranded species occurs due to the rapid cooling rates inhibiting quantitative intermolecular rehybridization.
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Circular dsDNA is widely distributed in procaryotic organisms (J. D. Watson, N. H. Hopkins, J. W. Roberts, J. A. Steitz, A. M. Weiner, Molecular Biology of the Gene, 4th Ed., Benjamin/Cummings, Menlo Park, CA. 1987). The length of these plasmids is typically greater than 1000 base pairs. Short circular dsDNA can be obtained by a ligation- closure reaction with a smallest size of about 100 base pairs (L. Ulanovsky, M. Bodner, E. N. Trifonov, M. Choder, Proc. Natl. Acad. Sci. USA 1986, 83, 862). The synthesis of a very small circle containing 42 base pairs was reported (M. Wolters, B. Wittig, Nucleic Acids Res 1989, 17, 5163). In contrast, DNA circles of any size can be obtained from single-stranded DNA (Review: E. T. Kool, Acc. Chem. Res. 1998, 31, 502).
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Temperature-dependent studies indicate that the displacement of the biotinylated dsDNA ligand in 3b by an 800-fold excess of D-biotin starts to occur by heating to about 70°C for two minutes. The displacement is half-completed at about 80°C. The denaturation of the dsDNA 1b occurs at 71 ±1°C and the preferred formation of the nanocircle 4b occurs at 79 ±2°C.
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Electrophoretic gel-shift analyses indicate that both low molecular weight biotin components, such as biotin-fluorescein conjugate, and biotinylated proteins, such as antibodies, bind to the nanocirclcs 4.
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