PNA FIT-Probes for the Dual Color Imaging of Two Viral
mRNA Targets in Influenza H1N1 Infected Live Cells
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Abstract
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