Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein-DNA complexes.
Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA.
Ebenstein
Yuval
Y
Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, USA. uv@chem.ucla.edu
Gassman
Natalie
N
Kim
Soohong
S
Weiss
Shimon
S
D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D045663:Quantum Dots; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D002373:Cyclic AMP Receptor Protein; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases