Bioengineering and characterization of DNA-protein assemblies floating on supported membranes.
This chapter describes the design, practical construction, and characterization of P-DNA and their applications in building a new generation of DNA chips. P-DNAs are artificial covalent assemblies involving a histidine tag head able to bind to modified phospholipids, a core protein domain derived from cytochrome b5 by genetic engineering that features specific spectroscopic and electrochemical properties useful for detection, a synthetic linker acting as a spacer, and an oligonucleotide acting as a probe. P-DNA has the property of being able to efficiently self-associate to a supported bilayer including nickel-iminodiacetate-modified phospholipids. The construction of P-DNA and its interaction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using a Biacore system or equivalent. P-DNA chips feature unique properties including tunable surface density of probes; very low nonspecific interaction with external DNA; lateral mobility, minimizing-steric interaction; optimization of hybridization efficiency; and, potentially, recognition by multiple probes of a single target and perfectly defined and homogeneous structure, permitting high density up to a compact monolayer. Potential applications of this new device are multiple, including high-sensitivity and high-selectivity chips for DNA-DNA, DNA-RNA, or DNA-protein interactions.
Boireau
Wilfrid
W
Department LPMO Institut FEMTO-ST UMR 617U, Besançon, France.
Duncan
Anthony C
AC
Pompon
Denis
D
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000032:analysis; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D015202:Protein Engineering; D011506:Proteins / Q000032:analysis / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature; D013997:Time Factors
D011506:Proteins; D004247:DNA; D015786:Cytochromes b5