Design Principles for SuCESsFul Biosensors: Specific Fluorophore/Analyte Binding and Minimization of Fluorophore/Scaffold Interactions.

Quantifying protein location and concentration is critical for understanding function in situ. Scaffold conjugated to environment-sensitive fluorophore (SuCESsFul) biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can, in principle, detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins, and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best-reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors.

Two-photon fluorescence spectroscopy and imaging of 4-dimethylaminonaphthalimide peptide and protein conjugates.

We report detailed photophysical studies on the two-photon fluorescence processes of the solvatochromic fluorophore 4-DMN as a conjugate of the calmodulin (CaM) and the associated CaM-binding peptide M13. Strong two-photon fluorescence enhancement has been observed which is associated with calcium binding. It is found that the two-photon absorption cross-section is strongly dependent on the local environment surrounding the 4-DMN fluorophore in the CaM conjugates, providing sensitivity between sites of fluorophore attachment. Utilizing time-resolved measurements, the emission dynamics of 4-DMN under various environmental (solvent) conditions are analyzed. In addition, anisotropy measurements reveal that the 4-DMN-S38C-CaM system has restricted rotation in the calcium-bound calmodulin. To establish the utility for cellular imaging, two-photon fluorescence microscopy studies were also carried out with the 4-DMN-modified M13 peptide in cells. Together, these studies provide strong evidence that 4-DMN is a useful probe in two-photon imaging, with advantageous properties for cellular experiments.

Chemical biology 2012: from drug targets to biological systems and back.

Multiple sites sharing a common target: This year's EMBO conference on chemical biology encouraged over 340 researchers to come to Heidelberg, Germany, and discuss the use of diverse chemical strategies and tools to investigate biological questions and better understand cellular processes.

FRET-capture: a sensitive method for the detection of dynamic protein interactions.

Caught in the act: The FRET-Capture approach exploits a bound solvatochromic fluorophore, 4-N,N-dimethylamino-1,8-naphthalimide, as a FRET donor in both inter- and intramolecular energy transfer. A unique feature of this method is the additional level of signal selectivity as the FRET signal is only turned on when the donor is specifically bound to the protein of interest, eliminating high background and false positive signals.

PNA FIT-probes for the dual color imaging of two viral mRNA targets in influenza H1N1 infected live cells.

Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.

Dual fluorophore PNA FIT-probes--extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA.

Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.

Total synthesis of proximicin A-C and synthesis of new furan-based DNA binding agents.

The total synthesis of the natural occurring polyamides proximicin A-C (3-5) has been accomplished. A short and efficient synthesis of a thus far unknown 4-amino-2-furan carboxylic acid was developed. Furthermore, this unique heterocyclic gamma-amino-acid was used for the synthesis of a new class of AT-selective DNA-binding agents derived from the natural products combining structural features of the proximicins with those from the known DNA-binding natural products netropsin (1) and distamycin (2).

Synthesis and evaluation of a netropsin-proximicin-hybrid library for DNA binding and cytotoxicity.

The proximicins A-C (1-3) are novel naturally occurring gamma-peptides with a hitherto unknown 2,4-disubstituted furan amino acid as a core structure. They show a moderate cytotoxic activity and induce upregulation of cell cycle regulating proteins (p53 and p21) and lead to cell cycle arrest in G0/G1-phase. Hybrid molecules combining structural motifs of the proximicins and of netropsin (4), a structurally related natural product, seem to have similar effects. Herein we describe the synthesis of a netropsin-proximicin-hybrid library and its evaluation regarding cytotoxicity and minor groove binding activity.

FIT-probes in real-time PCR.

Forced intercalation probes (FIT-probes) are peptide nucleic acid-based probes in which the thiazole orange dye replaces a canonical nucleobase. FIT-probes are used in homogenous DNA detection. The analysis is based on sequence-specific binding of the FIT-probe with DNA. Binding of the FIT-probe places thiazole orange in the interior of the formed duplex. The intercalation of thiazole orange between nucleobases of the formed probe-target duplex restricts the torsional flexibility of the two heterocyclic ring systems. As a result, FIT probes show strong enhancements of fluorescence upon hybridization. A remarkable attenuation of fluorescence is observed when forcing thiazole orange to intercalate next to a mismatched base pair. This base specificity of fluorescence signaling, which adds to the specificity of probe-target recognition, allows the detection of single base mutations even at non-stringent hybridization conditions. The performance of FIT-probes in real-time PCR is demonstrated in an assay for the SNPtyping of human H-ras. FIT-probe was added at the start of a real-time amplification containing the wild-type (G,G)-allele, mutant (T,T)-allele or heterozygous (G,T)-allele of the human H-ras gene. The identity of the target DNA is determined in real time due to significant differences in signal intensities.

FIT probes: peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery.

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.