Mobility-shift analysis with microfluidics chips

Abstract

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of protein-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

Design and analysis of nanoscale bioassemblies

“…The mechanism of action of these enzymes makes it possible to covalently trap them at multiple sites on duplex DNA structures to create ordered assemblies. Multiple methyltransferases have been targeted to a single DNA molecule by placing multiple recognition sites in linear (Figure 6A) or branched (Figure 6B) DNA structures (13,14,45). In addition, three different methyltransferases (M Hha I, M Eco RII, and M Msp I) have been targeted to their specific recognition sites on a single DNA structure (13,14).…”

“…Multiple methyltransferases have been targeted to a single DNA molecule by placing multiple recognition sites in linear (Figure 6A) or branched (Figure 6B) DNA structures (13,14,45). In addition, three different methyltransferases (M Hha I, M Eco RII, and M Msp I) have been targeted to their specific recognition sites on a single DNA structure (13,14). Methyltransferase peptide fusion proteins have been successfully placed on a DNA scaffold and detected by standard gel electrophoretic mobility shift assay (EMSA) of 32 P-labeled DNA (13,14), microfluidics chip-based EMSA (14), and immunologically with Western blot analysis using an antibody to the fusion peptide (13).…”

“…In addition, three different methyltransferases (M Hha I, M Eco RII, and M Msp I) have been targeted to their specific recognition sites on a single DNA structure (13,14). Methyltransferase peptide fusion proteins have been successfully placed on a DNA scaffold and detected by standard gel electrophoretic mobility shift assay (EMSA) of 32 P-labeled DNA (13,14), microfluidics chip-based EMSA (14), and immunologically with Western blot analysis using an antibody to the fusion peptide (13). Microfluidics analysis of the gel shift associated with a given SMA makes it especially easy to determine whether the selected target sites are completely occupied.…”

“…(B) Ordered multi-arm SMAs using DNA methyltransferase fusion proteins as a linker. DNA methyltransferase fusion proteins are covalently linked at methyltransferase recognition sites, containing 5-fluorocytosine along the arms of a Y-junction DNA, creating an ordered multi-arm SMA (14). …”

“…Progress in this area has been rapid in the past few years, with a variety of protein domains and oligodeoxynucleotides being adapted as components for self-assembling SMAs. In addition to the numerous oligodeoxynucleotides (1016) that have been employed in building these constructs, a variety of proteins including streptavidin (10,17), barnase and barstar (18), influenza virus matrix protein M1 (19), ribosomal protein L9 (19), bromoperoxidase (19), carboxylesterase (19), the bacterial DNA methyltransferases M Hha I (13), and M Eco RII (14) have been employed.…”