Fluorescence correlation spectroscopy (FCS)-based characterisation of aptamer ligand interaction.
Fluorescence correlation spectroscopy (Bacia and Schwille (2007) Nat. Protoc. 2, 2842-2856) reveals molecular mobilities, enabling to identify molecular interactions based on a change of diffusion times (Rigler and Elson, (2001) Fluorescence Correlation Spectroscopy: Theory and Applications. Springer, Berlin; Haustein, and Schwille, (2004) Curr. Opin. Struct. Biol. 14, 531-540). This technique can be applied to determine the dissociation constant of a complex formed by a fluorescence-labelled target and its corresponding RNA aptamer selected via systematic evolution of ligands by exponential enrichment (SELEX) (Schürer, et al. (2001) Biol. Chem. 382, 47948). Here, an FCS titration experiment is described in detail, where the binding properties of tetramethylrhodamine (TMR) labelled Moenomycin A to its corresponding RNA aptamer were determined (Schürer, et al. (2001) Biol. Chem. 382, 47948).
Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.
Arne
A
Werner
Ulrich
U
Hahn
D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001455:Bambermycins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D012235:Rhodamines / Q000737:chemistry; D052156:SELEX Aptamer Technique / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D017186:Titrimetry
D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D008024:Ligands; D012235:Rhodamines; D001455:Bambermycins; C437243:moenomycin A; C005358:tetramethylrhodamine