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传统磁镊的测量精度受限于磁球的布朗涨落, 当磁力小于约10 pN时, 磁球的布朗涨落明显增大, 对应磁镊的空间分辨率显著下降. 为了提高传统磁镊在小力条件下的测量精度, 本文将全内反射荧光技术引入到磁镊技术中, 并建立相适应的磁球-手柄-荧光微球-待测生物分子单分子连接系统, 在小力条件下(小于10 pN)获得纳米量级的测量精度. 应用改进的磁镊对DNA发卡的折叠-去折叠态的转变过程进行了研究, 依据DNA发卡的折叠-去折叠态转变的性质对全内反射场的穿透深度进行了校正, 并结合实验结果对改进后的磁镊的测量精度进行分析. 观察了Bloom解旋酶的解旋动力学过程, 获得初步实验结果, 证实了改进的磁镊在单分子研究中的实用性.The resolution of conventional magnetic tweezers is limited by the Brown motion of magnetic beads. When the force is lower than ～10 pN, the resolution of magnetic tweezers decreases significantly because of the increased Brown motion. To improve the resolution of magnetic tweezers under low forces, we combine the total internal reflection fluorescence techniques with magnetic tweezers, and design a novel single molecule connection: magnetic bead-DNA linker-fluorescent bead-single molecule. With the improved magnetic tweezers, we study the folding dynamics of a DNA hairpin. The results reveal that a nanometer-scale resolution is obtained. By analyzing these results, we calibrate the penetration depth of the total internal reflection field. Finally, we investigate the unwinding dynamics of a BLM helicase core protein. Some preliminary results of the helicase unwinding experiments confirm the practicability of the improved magnetic tweezers in the field of single molecular research.
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Keywords:
- magnetic tweezers /
- total internal reflection fluorescence /
- DNA hairpin /
- helicase
[1] Smith S B, Finazi L, Bustamante C 1992 Science 258 1122
[2] Cluzel P, Lebrun A, Heller C, Lavery R, Viovy J, Chatenay C, Caron F 1996 Science 271 792
[3] Dessinges M N, Lionnet T, Xi X G, Bensimon D, Croquette V 2004 Proc. Natl. Acad. Sci. USA 101 6439
[4] Johnson D S, Bai L, Smith B Y, Patel S S, Wang M D 2007 Cell 129 1299
[5] Perkings T T, Li H W, Dalal R V, Gelles J, Block S M 2004 Biophys. J. 86 1640
[6] Marsden S, Nardelli M, Linder P, Mccarthy J E 2006 J. Mol. Biol. 361 327
[7] Myong S, Bruno M M, Pyle A M, Ha T 2007 Science 317 513
[8] Theissen B, Karow A R, Kohler J, Gubaev A, Klostermeier D 2008 Proc. Natl. Acad. Sci. USA 105 548
[9] Smith S B, Cui Y, Bustamante C 1996 Science 271 795
[10] Sun B, Wei K J, Zhang B, Zhang X H, Dou S X, Li M, Xi X G 2008 EMBO J. 27 3279
[11] Neuman K C, Nagy A 2008 Nat. Methods 5 491
[12] Marko J F, Siggia E D 1995 Macromolecules 28 8759
[13] Lionnet T, Spiering M M, Benkovic S J, Bensimon D, Croquette V 2007 Natl. Acad. Sci. USA 104 19790
[14] Ran S Y 2012 Acta Phys. Sin. 61 170503 (in Chinese) [冉诗勇 2012 物理学报 61 170503]
[15] Hohng S, Zhou R, Nahas M K, Yu J, Schulten K, Lilley D M, Ha T 2007 Science 318 279
[16] Lee M, Kim S H, Hong S C 2010 Proc. Natl. Acad. Sci. USA 107 4985
[17] Luzzietti N, Brutzer H, Klaue D, Schwarz F W, Staroske W, Clausing S, Seidel R 2010 Nucl. Acids Res. 39 e15
[18] Guo R B, Rigolet P, Zargarian L, Fermandjian S, Xi X G 2005 Nucl. Acids Res. 33 3109
[19] Yang Y, Dou S X, Xu Y N, Bazeille N, Wang P Y, Rigolet P, Xu H Q, Xi X G 2010 Biochemistry 49 656
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[1] Smith S B, Finazi L, Bustamante C 1992 Science 258 1122
[2] Cluzel P, Lebrun A, Heller C, Lavery R, Viovy J, Chatenay C, Caron F 1996 Science 271 792
[3] Dessinges M N, Lionnet T, Xi X G, Bensimon D, Croquette V 2004 Proc. Natl. Acad. Sci. USA 101 6439
[4] Johnson D S, Bai L, Smith B Y, Patel S S, Wang M D 2007 Cell 129 1299
[5] Perkings T T, Li H W, Dalal R V, Gelles J, Block S M 2004 Biophys. J. 86 1640
[6] Marsden S, Nardelli M, Linder P, Mccarthy J E 2006 J. Mol. Biol. 361 327
[7] Myong S, Bruno M M, Pyle A M, Ha T 2007 Science 317 513
[8] Theissen B, Karow A R, Kohler J, Gubaev A, Klostermeier D 2008 Proc. Natl. Acad. Sci. USA 105 548
[9] Smith S B, Cui Y, Bustamante C 1996 Science 271 795
[10] Sun B, Wei K J, Zhang B, Zhang X H, Dou S X, Li M, Xi X G 2008 EMBO J. 27 3279
[11] Neuman K C, Nagy A 2008 Nat. Methods 5 491
[12] Marko J F, Siggia E D 1995 Macromolecules 28 8759
[13] Lionnet T, Spiering M M, Benkovic S J, Bensimon D, Croquette V 2007 Natl. Acad. Sci. USA 104 19790
[14] Ran S Y 2012 Acta Phys. Sin. 61 170503 (in Chinese) [冉诗勇 2012 物理学报 61 170503]
[15] Hohng S, Zhou R, Nahas M K, Yu J, Schulten K, Lilley D M, Ha T 2007 Science 318 279
[16] Lee M, Kim S H, Hong S C 2010 Proc. Natl. Acad. Sci. USA 107 4985
[17] Luzzietti N, Brutzer H, Klaue D, Schwarz F W, Staroske W, Clausing S, Seidel R 2010 Nucl. Acids Res. 39 e15
[18] Guo R B, Rigolet P, Zargarian L, Fermandjian S, Xi X G 2005 Nucl. Acids Res. 33 3109
[19] Yang Y, Dou S X, Xu Y N, Bazeille N, Wang P Y, Rigolet P, Xu H Q, Xi X G 2010 Biochemistry 49 656
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