Single-Molecule DNA Biosensors for Protein and Ligand Detection

Autor: Konstantinos Lymperopoulos, Mike Heilemann, Achillefs N. Kapanidis, Joseph P. Torella, Seamus Holden, Robert Crawford, Ling Chin Hwang
Jazyk: angličtina
Rok vydání: 2010
Předmět:
Zdroj: Angewandte Chemie International Edition; Vol 49
Angewandte Chemie International Edition
ISSN: 1433-7851
DOI: 10.1002/anie.200904597
Popis: Transcription factors (TFs) are sequence-specific DNA-binding proteins that control much of gene expression. TFs are natural biosensors and switches, translating chemical and physical signals (temperature shifts, light exposure, chemical concentrations, redox status) into transcriptional changes by modulating the binding of RNA polymerase to promoter DNA. Since changes in TF levels underlie fundamental biological processes such as DNA repair and cell-cycle progression, alterations in the levels of active TFs both lead to and indicate disease; for example, mutations in transcription factor p53 contribute to the rapid growth of cancer cells and, owing to their prevalence (p53 is mutated in roughly 50% of all human tumors), they have served as cancer biomarkers. Thus, methods for the sensitive detection and quantitation of TFs provide both fundamental information about gene regulation and a platform for diagnostics. TF detection often involves gel-based assays and Western blotting; although helpful in characterizing TF–DNA interactions, these assays are tedious, expensive, and qualitative, and consume large quantities of sample. Enzyme-linked immunosorbent assays (ELISAs) are more sensitive and offer higher throughput, but they require many preparation and signal-amplification steps for the detection of lowabundance TFs. Amplification is also required in the proximity-based ligation assay, making it incompatible with TF detection in living cells and diagnostic settings that demand results within minutes. An additional TF detection assay is based on fluorescence resonance energy transfer (FRET) between two doublestranded DNA (dsDNA) fragments containing fluorescently labeled single-stranded complementary overhangs (“molecular beacons”). In the presence of TF, the DNAs associate, resulting in donor fluorophore quenching as a result of FRET. This assay still requires significant amounts of sample and cannot detect low-abundance TFs; and because of the short dynamic range of FRET (1–10 nm), it also requires close proximity among the fluorophore, the quencher, and the protein–DNA interface, increasing the likelihood of steric interference with protein–DNA binding and complicating sensor design. Moreover, placing the fluorophore and the quencher on either side of the protein-binding site (usually 15–30 base pairs (bp) in length) on DNA results in very low FRET signals for most TFs. Here, we use alternating-laser excitation (ALEX) spectroscopy to detect TFs and small molecules by means of the TF-dependent coincidence of fluorescently labeled DNA. Like the molecular-beacon assay, our method is based on TFdriven DNA association, is rapid, and requires no amplification. However, our assay can detect pm levels of TFs in small amounts of sample, and it is FRET-independent, bypassing the need to optimize fluorophore position or know the structural details of TF–DNA binding; this flexibility in labeling ensures unperturbed TF–DNA binding. Using ALEX, we demonstrate TF and small-molecule detection, assay multiplexing, and suitability for analysis of complex biological samples. In our assay (Figure 1a,b), the full DNA-binding site for a TF is split in two (as in Ref. [5]): the left half-site (H1) and the right half-site (H2). Each site contains half of the TF-binding determinants and short, complementary 3’-overhangs. H1 is labeled with a “green” fluorophore (“G”) to give half-site H1, whereas H2 is labeled with a spectrally distinct “red” fluorophore (“R”) to give H2. In the absence of TF and at DNA concentrations of roughly 10–100 pm, H1 and H2 diffuse independently and associate only transiently. In contrast, in the presence of a TF that binds to the fully assembled DNA site, H1 and H2 diffuse as a complex (H1TF-H2; Figure 1a, bottom). We detect TF-dependent DNA coincidence using ALEX spectroscopy, wherein single molecules are excited by two lasers in an alternating fashion, with each laser capable of directly exciting either a G or a R fluorophore. ALEX allows molecular sorting on two-dimensional histograms of apparent FRET efficiency E* (a fluorescence ratio that reports on interfluorophore proximity) and probe stoichiometry S (a fluorescence ratio that reports on molecular stoichiometry). A search for all R-labeled molecules (i.e., G–R molecules [*] Dr. K. Lymperopoulos, R. Crawford, J. P. Torella, Dr. M. Heilemann, Dr. L. C. Hwang, S. J. Holden, Dr. A. N. Kapanidis Biological Physics Research Group, Department of Physics University of Oxford, Clarendon Laboratory Parks Road, Oxford, OX1 3PU (United Kingdom) E-mail: a.kapanidis1@physics.ox.ac.uk Dr. K. Lymperopoulos Current address: BioQuant Institute, Cellnetworks Cluster Ruprecht-Karls Universit t Heidelberg 69120 Heidelberg (Germany)
Databáze: OpenAIRE
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