Selective Binders of the Tandem Src Homology 2 Domains in Syk and Zap70 Protein Kinases by DNA-Programmed Spatial Screening

Members of the Syk family of tyrosine kinases arrange Src homology 2 (SH2) domains in tandem to allow the firm binding of immunoreceptor tyrosine-based interaction motifs (ITAMs). While the advantages provided by the bivalency enhanced interactions are evident, the impact on binding specificity is less-clear. For example, the spleen tyrosine kinase (Syk) and the ζ-chain-associated protein kinase (ZAP-70) recognize the consensus sequence pYXXI/L­(X)_6–8 pYXXI/L with near-identical nanomolar affinity. The nondiscriminatory recognition, on the one hand, poses a specificity challenge for the design of subtype selective protein binders and, on the other hand, raises the question as to how differential activation of Syk and ZAP-70 is ensured when both kinases are co-expressed. Herein, we identified the criteria for the design of binders that specifically address either the Syk or the Zap-70 tSH2 domain. Our approach is based on DNA-programmed spatial screening. Tyrosine-phosphorylated peptides containing the pYXXI/L motif were attached to oligonucleotides and aligned in tandem on a DNA template by means of nucleic acid hybridization. The distance between the pYXXI/L motifs and the orientation of strands were varied. The exploration exposed remarkably different recognition characteristics. While Syk tSH2 has a rather broad substrate scope, ZAP-70 tSH2 required a proximal arrangement of the phosphotyrosine ligands in defined strand orientation. The spatial screen led to the design of mutually selective, DNA-free binders, which discriminate Zap-70 and Syk tSH2 by 1 order of magnitude in affinity

Quantum Dot-PNA Conjugates for Target-Catalyzed RNA Detection

Detection of pathogenic nucleic acids remains one of the most reliable approaches for the diagnosis of a broad range of diseases. Current PCR-based methods require experienced personnel and cannot be easily used for point-of-care diagnostics, making alternative strategies for the sensitive, reliable, and cost-efficient detection of pathogenic nucleic acids highly desirable. Here, we report an enzyme-free method for the fluorometric detection of RNA that relies on a target-induced fluorophore transfer onto a semiconductor quantum dot (QD), uses PNA probes as selective recognition elements and can be read out with simple and inexpensive equipment. For QD-PNA conjugates with optimized PNA content, limits of detection of dengue RNA in the range of 10 pM to 100 nM can be realized within 5 h in the presence of a high excess of noncomplementary RNA

Brightness Enhanced DNA FIT-Probes for Wash-Free RNA Imaging in Tissue

Fluorogenic oligonucleotides enable RNA imaging in cells and tissues. A high responsiveness of fluorescence is required when unbound probes cannot be washed away. Furthermore, emission should be bright in order to enable detection against autofluorescent background. The development of fluorescence-quenched hybridization probes has led to remarkable improvement of fluorescence responsiveness. Yet, comparably little attention has been paid to the brightness of smart probes. We describe hybridization probes that combine responsiveness with a high brightness of the measured signal. The method relies upon quencher-free DNA forced intercalation (FIT)-probes, in which two (or more) intercalator dyes of the thiazole orange (TO) family serve as nucleobase surrogates. Initial experiments on multi-TO-labeled probes led to improvements of responsiveness, but self-quenching limited their brightness. To enhance both brightness and responsiveness the highly responsive TO nucleoside was combined with the highly emissive oxazolopyridine analogue JO. Single-stranded TO/JO FIT-probes are dark. In the probe–target duplex, quenching caused by torsional twisting and dye–dye contact is prevented. The TO nucleoside appears to serve as a light collector that increases the extinction coefficient and transfers excitation energy to the JO emitter. This leads to very bright JO emission upon hybridization (<i>F</i>/<i>F</i>₀ = 23, brightness = 43 mL mol^–1 cm^–1 at λ_ex = 516 nm). TO/JO FIT-probes allowed the direct fluorescence microscopic imaging of <i>oskar</i> mRNA within a complex tissue. Of note, RNA imaging was feasible under wide-field excitation conditions. The described protocol enables rapid RNA imaging in tissue without the need for cutting-edge equipment, time-consuming washing, or signal amplification

DNA-Triggered Dye Transfer on a Quantum Dot

Nucleic acid-templated reactions are frequently explored tools in nucleic acid diagnosis. To enable a separation-free DNA detection, the reactive probe molecules require conjugation with reporter groups that provide measurable changes of an observable parameter upon reaction. A widely used, generic read-out method is based on fluorescence resonance energy transfer (FRET) between two appended dyes. Yet, spectral cross-talk usually limits the achievable enhancements of the FRET signal in DNA-directed chemistries. We describe a DNA-triggered transfer reaction which provides for strong increases of a fluorescent signal caused by FRET. The method may involve DNA- and PNA-based probes and is based upon a proximity-triggered transfer reaction which leads to the covalent fixation of a fluorescence dye on the surface of a quantum dot (QD). The transfer reaction brings the dye closer to the QD than hybridization alone. The resulting FRET signal is a specific monitor of the reaction and allows efficient discrimination of single base mismatched templates. Of note, the 35-fold increase of the FRET signal is measured at 310 nm apparent Stokes shift and turnover in template provides a means for signal amplification

Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization

Fluorogenic oligonucleotide probes facilitate the detection and localization of RNA targets within cells. However, quantitative measurements of mRNA abundance are difficult when fluorescence signaling is based on intensity changes because a high concentration of unbound probes cannot be distinguished from a low concentration of target-bound probes. Here, we introduce qFIT (<u>q</u>uantitative <u>f</u>orced <u>i</u>n<u>t</u>ercalation) probes that allow the detection both of probe–target complexes and of unbound probes on separate, independent channels. A surrogate nucleobase based on thiazole orange (TO) probes the hybridization status. The second channel involves a nonresponsive near-IR dye, which serves as a reporter of concentration. We show that the undesirable perturbation of the hybridization reporter TO is avoided when the near-IR dye Cy7 is connected by means of short triazole linkages in an ≥18 nucleotides distance. We used the qFIT probes to localize and quantify <i>oskar</i> mRNA in fixed egg chambers of wild-type and mutant <i>Drosophila melanogaster</i> by wash-free fluorescence <i>in situ</i> hybridization. The measurements revealed a relative 400-fold enrichment of <i>oskar</i> within a 3000 μm³ large volume at the posterior pole of stage 8–9 oocytes, which peaked at a remarkably high 1.8 μM local concentration inside 0.075 μm³ volume units. We discuss detection limits and show that the number of <i>oskar</i> mRNA molecules per oocyte is independent of the oocyte size, which suggests that the final levels are attained already during the onset of <i>oskar</i> localization at stage 8

Solid Phase Synthesis of Short Peptide-Based Multimetal Tags for Biomolecule Labeling

We describe an unprecedented solid phase peptide synthesis (SPPS) of short peptide-based multimetal tags designated as elemental tags for the quantification of biomolecules via inductively coupled plasma mass spectrometry (ICP-MS). The macrocyclic chelator 1,4,7,10-tetraazacyclododecane <i>N</i>,<i>N</i>′,<i>N</i>″,<i>N</i>‴-tetra acetic acid (DOTA) was attached to the side chain of <i>N</i>-α-(9-fluorenylmethoxycarbonyl)-l-lysine (Fmoc-Lys-OH) and metalated with a lanthanide to provide a building block for Fmoc-based SPPS. Thereby, in contrast to existing strategies for the synthesis of DOTA–peptide conjugates, an already metalated DOTA-amino acid was used as a building block for SPPS. The DOTA-lanthanide complex was stable throughout the whole SPPS, even during the final cleavage in concentrated trifluoroacetic acid. This indicates that the strategy to first metalate the Fmoc-Lys­(DOTA)-OH and to utilize the metal coordination to protect the carboxyl groups of DOTA offers an alternative to conventional synthetic routes using <i>tert</i>-butyl protected DOTA. Several small peptides containing up to four metal ions were synthesized, among them peptides carrying defined metal sequences consisting of two different lanthanides. The peptides were N-terminally maleimide-functionalized, thus introducing a moiety for conjugation to thiol-bearing biomolecules. The final objective of this work was the signal enhancement in ICP-MS-based DNA quantification assays. To evaluate the performance of the multimetal peptide tags in assay, they were applied to label thiol-modified 15mer DNA oligonucleotide probes. These served as reporter probes in a model sandwich-type hybridization assay. Thereby, we found that the ICP-MS signal increased linearly with the number of lanthanide ions attached to the reporter probe

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

Spatial Screening of Hemagglutinin on Influenza A Virus Particles: Sialyl-LacNAc Displays on DNA and PEG Scaffolds Reveal the Requirements for Bivalency Enhanced Interactions with Weak Monovalent Binders

Attachment of the Influenza A virus onto host cells involves multivalent interactions between virus surface hemagglutinin (HA) and sialoside-containing glyco ligands. Despite the development of extremely powerful multivalent binders of the Influenza virus and other viruses, comparably little is known about the optimal spacing of HA ligands, which ought to bridge binding sites within or across the trimeric HA molecules. To explore the criteria for ligand economical high affinity binding, we systematically probed distance–affinity relationships by means of two differently behaving scaffold types based on (i) flexible polyethylene glycol (PEG) conjugates and (ii) rigid self-assembled DNA·PNA complexes. The bivalent scaffolds presented two sialyl-LacNAc ligands in 23–101 Å distance. A combined analysis of binding by means of microscale thermophoresis measurements and statistical mechanics models exposed the inherent limitations of PEG-based spacers. Given the distance requirements of HA, the flexibility of PEG scaffolds is too high to raise the effective concentration of glyco ligands above a value that allows interactions with the low affinity binding site. By contrast, spatial screening with less flexible, self-assembled peptide nucleic acid (PNA)·DNA complexes uncovered a well-defined and, surprisingly, bimodal distance–affinity relationship for interactions of the Influenza A virus HA with bivalent displays of the natural sialyl-LacNAc ligand. Optimal constructs conferred 10³-fold binding enhancements with only two ligands. We discuss the existence of secondary binding sites and shine light on the preference for intramolecular rather than intermolecular recognition of HA trimers on the virus surface

Remote Control of Lipophilic Nucleic Acids Domain Partitioning by DNA Hybridization and Enzymatic Cleavage

Lateral partitioning of lipid-modified molecules between liquid-disordered (ld) and liquid-ordered (lo) domains depends on the type of lipid modification, presence of a spacer, membrane composition, and temperature. Here, we show that the lo domain partitioning of the palmitoylated peptide nucleic acid (PNA) can be influenced by formation of a four-component complex with the ld domain partitioning tocopherol-modified DNA: the PNA–DNA complex partitioned into the ld domains. Enzymatic cleavage of the DNA linker led to the disruption of the complex and restored the initial distribution of the lipophilic nucleic acids into the respective domains. This modular system offers strategies for dynamic functionalization of biomimetic surfaces, for example, in nanostructuring and regulation of enzyme catalysis, and it provides a tool to study the molecular basis of controlled reorganization of lipid-modified proteins in membranes, for example, during signal transduction

Remote Control of Lipophilic Nucleic Acids Domain Partitioning by DNA Hybridization and Enzymatic Cleavage

Lateral partitioning of lipid-modified molecules between liquid-disordered (ld) and liquid-ordered (lo) domains depends on the type of lipid modification, presence of a spacer, membrane composition, and temperature. Here, we show that the lo domain partitioning of the palmitoylated peptide nucleic acid (PNA) can be influenced by formation of a four-component complex with the ld domain partitioning tocopherol-modified DNA: the PNA–DNA complex partitioned into the ld domains. Enzymatic cleavage of the DNA linker led to the disruption of the complex and restored the initial distribution of the lipophilic nucleic acids into the respective domains. This modular system offers strategies for dynamic functionalization of biomimetic surfaces, for example, in nanostructuring and regulation of enzyme catalysis, and it provides a tool to study the molecular basis of controlled reorganization of lipid-modified proteins in membranes, for example, during signal transduction