Hairpin Peptide Beacon: Dual-Labeled PNA-Peptide-Hybrids for Protein Detection

Citations Over TimeTop 12% of 2007 papers

Abstract

Probe molecules that report biomolecular interactions by changes of fluorescence properties are frequently used in drug screening, diagnostics, and cell biology. The predictability of nucleic acid−nucleic acid interaction has been a major driving force in the design of the powerful nucleic acid-based reporters that are available today. By comparison, the design of peptide-based probes that report on binding to (and dissociation from) specific protein targets by changes of fluorescence intensity is more challenging, owing to the lack of a universal peptide−protein recognition code. We herein introduce a new probe concept, in which protein binding is signaled by increases of fluorescence. Hairpin peptide beacons (HPB) are dual labeled peptide probes designed to form stable hairpins in the absence of a protein target. HPBs are designed in analogy to DNA-targeted molecular beacons and consist of a central, protein specific peptide sequence flanked by two DNA-analogous self-complementary peptide nucleic acid (PNA) arm segments. Binding of the target protein to the peptide sequence induces a structural reorganization and results in opening of the closed structure. The accompanying separation of the terminally appended chromophores leads to increases of fluorescence. The particular advantage of the hairpin peptide beacons is the generic design which should be applicable to any protein that interacts with a peptide while allowing almost unrestricted access of interesting fluorophores. It is shown that hairpin peptide beacons signal the presence of the SH2 domain of the Src-kinase by more than 10-fold increases of fluorescence. Furthermore, the HPB concept has been used in the design of a noncleavable probe that reports on the activity of the protease renin. It is suggested that the reversibility of fluorescence signaling mediated by noncleavable probes may enable the continuous monitoring of both increases and decreases of protease activity.

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