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

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