title: Real-time detection of transcription factors using target-converted helicase-dependent amplification assay with zero-background signal.
abstract: Highly sensitive detection of transcription factors is essential to the evaluation of cellular development and the disease state. However, so far most detection methods are usually laborious and time-consuming with a poor sensitivity. Here, we demonstrate a simple and ultrasensitive approach for transcription factor detection based on the target-converted helicase-dependent amplification assay. We employ a hairpin probe bearing the transcription factor binding site to convert the protein signal to the DNA signal, which can be further amplified by helicase-dependent amplification. With the digestion of excess probes by the exonucleases and the subsequent one primer-triggered high fidelity amplification, zero-background signal can be achieved in the absence of a transcription factor. This method exhibits excellent specificity and high sensitivity with the detection limit of 9.3× 10(-13) M and the detection range over 4 orders of magnitude, which is superior to most currently used approaches for transcription factor detection. Moreover, the proposed method has significant advantages of simple, rapid, and low cost without the need of any labeled DNA probes and might be extended to selectively detect various DNA-binding proteins by simply changing the binding-site sequences of hairpin probes.
authors: Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China.
authors: Anping
authors: A
authors: Cao
authors: Chun-yang
authors: CY
authors: Zhang
mesh_terms: D052160:Benzothiazoles; D001665:Binding Sites; D004265:DNA Helicases / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D003959:Diamines; D005090:Exodeoxyribonucleases / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D009930:Organic Chemicals / Q000737:chemistry; D011485:Protein Binding; D011804:Quinolines; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D052002:NF-kappa B p50 Subunit; D009930:Organic Chemicals; D011804:Quinolines; D011994:Recombinant Proteins; D014157:Transcription Factors; C098022:SYBR Green I; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C020133:exodeoxyribonuclease III; D004265:DNA Helicases