An improved measurement of dsDNA elasticity using AFM.
The mechanical properties of a small fragment (30 bp) of an individual double-stranded deoxyribonucleic acid (dsDNA) in water have been investigated by atomic force microscopy (AFM). We have stretched three systems including ssDNA, double-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3'-end and thiolated at the 5'-end, this was reversed for the other complementary strand) and single-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3'-end and thiolated at the 5'-end, whereas the other complementary strand was biotinylated at only the 5'-end). The achieved thiolation and biotinylation were to bind ds- or ssDNA to the gold surface and streptavidin-coated AFM tip, respectively. Analysis of the force versus displacement (F-D) curves from tip-DNA-substrate systems shows that the pull-off length (L(o)) and stretch length (delta) from the double-fixed system were shorter than those observed in the ssDNA and the single-fixed system. The obtained stretch force (F(st)) from the single-fixed dsDNA was much greater than that from the ssDNA even though it was about 10 pN greater than the one obtained in the double-fixed system. As a result, the Young's modulus of the double-fixed dsDNA was greater than that of the single-fixed dsDNA and the ssDNA. A more reliable stiffness of the dsDNA was observed via the double-fixed system, since there is no effect of the unpaired molecules during stretching, which always occurred in the single-fixed system. The unpaired molecules were also observed by comparing the stiffness of ssDNA and single-fixed dsDNA in which the end of one strand was left free.
Nguyen
Thi-Huong
TH
Nano-Bio Research Center, Korea Institute of Science and Technology, Seoul, Korea.
Lee
Sang-Myung
SM
Na
Kyounghwan
K
Yang
Sungwook
S
Kim
Jinseok
J
Yoon
Eui-Sung
ES
D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D055119:Elastic Modulus; D006046:Gold / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods*; D016011:Normal Distribution; D019809:Streptavidin / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004277:DNA, Single-Stranded; D013438:Sulfhydryl Compounds; D001710:Biotin; D006046:Gold; D004247:DNA; D019809:Streptavidin