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Optical probes for real-time nucleic acid detection and gene expression analysis in living cells

Fluorescent ratiometric bimolecular beacons with spatiotemporal resolution

Problem:

Common gene expression analysis methods, including polymerase chain reaction, microarrays, and in situ hybridization rely on fixed or lysed cells, hindering their ability to provide spatiotemporal information. This led to the development of optical probes that enable the imaging of RNA in living cells, but most suffer from high levels of false-positive signals, due to non-specific interactions, nuclease-mediated degradation, and sequestration into the nucleus.

Solution:

Scientists in the Tsourkas Lab have designed a nucleic acid-based optical probe that, upon RNA hybridization, is capable of accurately detecting nucleic acids in living cells with high signal-to-background and little to no false-positive signals. Moreover, this ratiometric bimolecular beacon (RBMB) simultaneously measures endogenous mRNA and the efficiency of probe entry, removing cell-to-cell variability and allowing confident analysis of gene expression in individual cells.

These functional improvements over existing probes simplify protocols for quantifying gene expression and enhance the reliability of ratiometric measurements. The high signal-to-background of RBMBs also allows individual RNA transcripts to be imaged in single cells.

Advantages:

Little to no false-positives
Probe distributes throughout cytoplasm
Reporter and Reference labels at a fixed 1:1 ratio for improved quantification of mRNA detection over time
Greater accuracy and reproducibility than previous ratiometric methods using separate reference and target probes

Applications:

Simple, sensitive quantification of endogenous mRNA with spatiotemporal resolution in living cells
Diagnostic and therapeutic development screens to identify compounds that modify gene expression
Regulation/silencing of gene expression
Understand cell function and behavior underlying disease pathophysiology
Monitor transfection efficiency
Imaging/quantification of individual RNA transcripts in real-time.

Fluorescent images of MEF/3T3 cells after microporation over a time course with molecular beacons (MBs) or ratiometric bimolecular beacons (RBMBs). In (A), images of the RBMB reference dye, and in (B), images of reporter dye with RBMB in top row and conventional MB in bottom row. In (C), image of HT1080 cells after microporation with RBMBs. The hybridization of multiple RBMBs to individual RNA transcripts allows each transcript to appear as a bright fluorescent spot that can be followed in real-time.