Using DNA-binding proteins as an analytical tool.
We propose that DNA-binding proteins can be used as highly efficient and versatile tools in analyses of DNA, RNA, and proteins. This work reports assays applying specific affinity probes: hybridization probes for analyses of DNA and RNA, and aptamer probes for analyses of proteins. Both types of probes are single-stranded DNA. In affinity analyses, in general, the probe (P) binds to a target molecule (T), and the amounts of the probe-target complex (P.T) and unbound P are determined. Distinguishing between P and P.T can be achieved by electrophoretic separation. If the electrophoretic mobilities of P and P.T are close in gel-free media, which is always the case for hybridization analyses, separation typically requires the use of a sieving matrix. Here we utilized a single-stranded DNA binding protein (SSB) to facilitate highly efficient gel-free separation of P and P.T in capillary electrophoresis (CE) for three types of targets: DNA, RNA, and proteins. When present in the CE run buffer, SSB binds differently to P and P.T. Due to this selective binding, SSB induces difference in electrophoretic mobilities of P and P.T in an SSB concentration-dependent fashion. The difference in the electrophoretic mobilities allows for affinity analyses of DNA, RNA, and proteins in gel-free CE. The large number of well-characterized DNA- and RNA-binding proteins and the diversity of their properties will allow researchers to design a comprehensive tool set for quantitative analyses of DNA, RNA, and proteins. Such analyses will facilitate identification of genomic DNA in ultra-small samples without error-prone and time-consuming PCR. They can also be used for monitoring gene expression at both mRNA and protein levels.
Berezovski
Maxim
M
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
Krylov
Sergey N
SN
D015342:DNA Probes / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D009693:Nucleic Acid Hybridization; D015347:RNA Probes / Q000737:chemistry* / Q000378:metabolism; D013917:Thrombin / Q000032:analysis / Q000378:metabolism
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D015347:RNA Probes; D013917:Thrombin