Limits...
Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance.

Ritzefeld M, Sewald N - J Amino Acids (2012)

Bottom Line: In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides.Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published.Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany.

ABSTRACT
Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

No MeSH data available.


General principle of SPR. See text for details. n2 (refractive index of medium with lower refractive index), E (evanescent field amplitude), ksp (wavevector of surface plasmons), kx (wavevector of photon).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3303711&req=5

fig1: General principle of SPR. See text for details. n2 (refractive index of medium with lower refractive index), E (evanescent field amplitude), ksp (wavevector of surface plasmons), kx (wavevector of photon).

Mentions: In most practical applications of SPR, the Kretschmann-Reather ATR method that was already described in the last section is used. In this setup, a thin metal film (typically around 50 nm thick gold layer) is evaporated onto the glass prism and kept in direct contact with the medium of lower refractive index (n2) [10, 11]. In order to evaluate the interactions of a protein with a nucleic acid that results in the formation of a protein-DNA complex, one of the two interaction partners has to be immobilized on the surface of the conductor (“ligand” in Figure 1) [12]. In most cases, a sensor chip with preimmobilized streptavidin is used to immobilize biotinylated oligonucleotides (more details concerning immobilization will be discussed below). The other interaction partner (e.g., the protein = “analyte” in Figure 1) is injected into the running buffer that passes the surface at a constant flow. In Biacore instruments, the resulting change in concentration of molecules at the gold surface due to the formation of the protein-DNA complex is measured in resonance units (RUs) and can be described according to (1):(1)RU=n·X=[RII·c]·X=[(δnδc)ligand·c]·X.In this equation, n is the changing refractive index at the surface, X is a multiplier to convert n to RU, RII is the refractive index increment of the protein that is binding to the immobilized oligonucleotide, and c is the concentration of the protein. In general, 1000 RU correspond to a change in angle of 0.1°, or a protein concentration of 1 ng·mm−2 (alternatively 10 mg·mL−1) [13, 14]. One set of problems that is connected to the RII has to be mentioned when using the correlation of RU and protein concentration. The RII value of the molecules used is presumed to be in a range of ~0.18–0.19 mL·g−1. However, nonprotein molecules exhibit RII values beyond this range. In order to accurately perform an affinity ranking and correct stoichiometric measurements of small molecules the RU value has to be normalized for each measured compound [13]. Fortunately, the RII value is not important to get correct kinetic and thermodynamic results in simple protein-protein or protein-oligonucleotide interactions [15].


Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance.

Ritzefeld M, Sewald N - J Amino Acids (2012)

General principle of SPR. See text for details. n2 (refractive index of medium with lower refractive index), E (evanescent field amplitude), ksp (wavevector of surface plasmons), kx (wavevector of photon).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303711&req=5

fig1: General principle of SPR. See text for details. n2 (refractive index of medium with lower refractive index), E (evanescent field amplitude), ksp (wavevector of surface plasmons), kx (wavevector of photon).
Mentions: In most practical applications of SPR, the Kretschmann-Reather ATR method that was already described in the last section is used. In this setup, a thin metal film (typically around 50 nm thick gold layer) is evaporated onto the glass prism and kept in direct contact with the medium of lower refractive index (n2) [10, 11]. In order to evaluate the interactions of a protein with a nucleic acid that results in the formation of a protein-DNA complex, one of the two interaction partners has to be immobilized on the surface of the conductor (“ligand” in Figure 1) [12]. In most cases, a sensor chip with preimmobilized streptavidin is used to immobilize biotinylated oligonucleotides (more details concerning immobilization will be discussed below). The other interaction partner (e.g., the protein = “analyte” in Figure 1) is injected into the running buffer that passes the surface at a constant flow. In Biacore instruments, the resulting change in concentration of molecules at the gold surface due to the formation of the protein-DNA complex is measured in resonance units (RUs) and can be described according to (1):(1)RU=n·X=[RII·c]·X=[(δnδc)ligand·c]·X.In this equation, n is the changing refractive index at the surface, X is a multiplier to convert n to RU, RII is the refractive index increment of the protein that is binding to the immobilized oligonucleotide, and c is the concentration of the protein. In general, 1000 RU correspond to a change in angle of 0.1°, or a protein concentration of 1 ng·mm−2 (alternatively 10 mg·mL−1) [13, 14]. One set of problems that is connected to the RII has to be mentioned when using the correlation of RU and protein concentration. The RII value of the molecules used is presumed to be in a range of ~0.18–0.19 mL·g−1. However, nonprotein molecules exhibit RII values beyond this range. In order to accurately perform an affinity ranking and correct stoichiometric measurements of small molecules the RU value has to be normalized for each measured compound [13]. Fortunately, the RII value is not important to get correct kinetic and thermodynamic results in simple protein-protein or protein-oligonucleotide interactions [15].

Bottom Line: In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides.Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published.Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bielefeld University, P.O. Box 100131, 33501 Bielefeld, Germany.

ABSTRACT
Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

No MeSH data available.