Peptide Beacons:  A New Design for Polypeptide-Based Optical Biosensors

Abstract

Both epitope mapping and other in vitro selection techniques produce short polypeptides that tightly and specifically bind to any of a wide range of macromolecular targets. Here, we demonstrate a potentially general means of converting such polypeptides into optical biosensors. The sensing architecture we have developed, termed peptide beacons, is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed to a macromolecular target. Using this effect to segregate a long-lived fluorophore from an electron transfer based, contact quencher (both covalently attached to the peptide), we have produced a robust optical sensor for anti-HIV antibodies. The binding-induced segregation of the fluorophore-quencher pair produces a 6-fold increase in sensor emission, thus allowing us to readily detect as low as approximately 250 pM of the target antibody. Because the sensor is based on binding-induced folding and a visible-light fluorophore, it is sufficiently selective to work directly in complex, contaminant-ridden samples such as saliva and blood.

Antibody Detection by Using a FRET‐Based Protein Conformational Switch

“…The binding of this monoclonal antibody to its linear peptide epitope (EKIRLR) is well-characterized, and it was also employed in previous studies on peptide molecular beacons. [8,9] The first sensor design that we explored (AbSense1) incorporated a flexible linker containing 17 GlySerGly repeats, similar to the linker used in previous FRET sensor designs (Figure 1). [6,10,11] In addition to the previously reported S208F/V224L mutations, we also explored a sensor variant containing Q204F/V224L mutations on each of the two fluorescent domains (cerulean and citrine).…”

“…A second, more-rational approach is based on peptide epitopes that are sandwiched in between two synthetic fluorophores. [8,9,16] Antibody binding to these peptide molecular beacons induces an extension of the peptide chain, and results in significant changes in either pyrene excimer fluorescence or energy transfer between the donor and acceptor moieties. The sensor design presented here does not depend on binding-induced conformational changes in an epitope sequence, but instead makes use of the unique Y-shaped structure common to all antibodies.…”

RNA polymerase molecular beacon as tool for studies of RNA polymerase–promoter interactions

“…1C). By analogy with previously described molecular and peptide beacons assays [30,31], we named this assay “an RNAP beacon assay”.…”

Sensing the Whole Body and Clinical Diagnostics

“…Peptide beacons have started to be used for the detection of anti-HIV antibodies (Oh et al 2007 ). These peptides contain the sequence recognized by the antibodies and adopt different conformations in the free and antibody-bound forms, which infl uences the effect of bound quencher on the fl uorescence of dye bound in a different position in peptide sequence.…”