Reagentless, electrochemical approach for the specific detection of double- and single-stranded DNA binding proteins.
Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.
Ricci
Francesco
F
Dipartimento di Scienze e Tecnologie Chimiche, Universita di Roma Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy.
Bonham
Andrew J
AJ
Mason
Aaron C
AC
Reich
Norbert O
NO
Plaxco
Kevin W
KW
D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D004563:Electrochemistry; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D035181:TATA-Box Binding Protein / Q000032:analysis / Q000378:metabolism
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D035181:TATA-Box Binding Protein