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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.


Addition of antisense RNA to the chimeric oligonucleotide consisting of DNA and RNA, results in the hybridization of the antisense strand and its complementary RNA sequence. RNase H only recognizes RNA-DNA heteroduplexes and cleaves the corresponding RNA strand. The resulting chimeric fragments end up in the next flow cell (flow cell 2). Due to the complementarity between the immobilized single stranded DNA in flow cell 2 and the DNA of the chimeric oligonucleotide fragments, both strands hybridize and induce a response in flow cell 2.
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fig9: Addition of antisense RNA to the chimeric oligonucleotide consisting of DNA and RNA, results in the hybridization of the antisense strand and its complementary RNA sequence. RNase H only recognizes RNA-DNA heteroduplexes and cleaves the corresponding RNA strand. The resulting chimeric fragments end up in the next flow cell (flow cell 2). Due to the complementarity between the immobilized single stranded DNA in flow cell 2 and the DNA of the chimeric oligonucleotide fragments, both strands hybridize and induce a response in flow cell 2.

Mentions: In the first step of the dual assay, biotinylated chimeric oligonucleotides that consist of an RNA sequence and a short DNA strand ligated to its 3′-end were immobilized on a streptavidin sensor chip (cf. Figure 9 left). An antisense oligonucleotide, complementary to the ribonucleotide sequence of the immobilized molecule, was injected and a heteroduplex was formed (cf. Figure 9 middle). In the following step, RNase H was added. The enzyme recognizes the heteroduplex consisting of the RNA sequence and the antisense strand and cleaves the RNA part. The produced fragments were released into the solution and hybridize with complementary oligonucleotides immobilized in the following flow cell (cf. Figure 9 right). The DNA fragment of the chimeric DNA molecule was necessary, to enhance the SPR response in the first flow cell and to facilitate the specific hybridization with the immobilized ligands in the second flow cell. This method has the potential to screen the properties of antisense oligonucleotides containing chemical modifications.


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

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

Addition of antisense RNA to the chimeric oligonucleotide consisting of DNA and RNA, results in the hybridization of the antisense strand and its complementary RNA sequence. RNase H only recognizes RNA-DNA heteroduplexes and cleaves the corresponding RNA strand. The resulting chimeric fragments end up in the next flow cell (flow cell 2). Due to the complementarity between the immobilized single stranded DNA in flow cell 2 and the DNA of the chimeric oligonucleotide fragments, both strands hybridize and induce a response in flow cell 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Addition of antisense RNA to the chimeric oligonucleotide consisting of DNA and RNA, results in the hybridization of the antisense strand and its complementary RNA sequence. RNase H only recognizes RNA-DNA heteroduplexes and cleaves the corresponding RNA strand. The resulting chimeric fragments end up in the next flow cell (flow cell 2). Due to the complementarity between the immobilized single stranded DNA in flow cell 2 and the DNA of the chimeric oligonucleotide fragments, both strands hybridize and induce a response in flow cell 2.
Mentions: In the first step of the dual assay, biotinylated chimeric oligonucleotides that consist of an RNA sequence and a short DNA strand ligated to its 3′-end were immobilized on a streptavidin sensor chip (cf. Figure 9 left). An antisense oligonucleotide, complementary to the ribonucleotide sequence of the immobilized molecule, was injected and a heteroduplex was formed (cf. Figure 9 middle). In the following step, RNase H was added. The enzyme recognizes the heteroduplex consisting of the RNA sequence and the antisense strand and cleaves the RNA part. The produced fragments were released into the solution and hybridize with complementary oligonucleotides immobilized in the following flow cell (cf. Figure 9 right). The DNA fragment of the chimeric DNA molecule was necessary, to enhance the SPR response in the first flow cell and to facilitate the specific hybridization with the immobilized ligands in the second flow cell. This method has the potential to screen the properties of antisense oligonucleotides containing chemical modifications.

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.