Methods and materials using signaling probes
Inventors
Shekdar, Kambiz • Sawchuk, Dennis J. • Montez, Jason M.
Assignees
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Publication Number
US-8916503-B2
Publication Date
2014-12-23
Expiration Date
Abstract
Methods of isolating cells or generating cell lines comprising the step of exposing the cells to signaling probes that produce a signal upon hybridization to a target sequence, as well as methods of quantifying the level of expression of an RNA of interest, methods for identifying genetic recombinational events in living cells and methods of generating a transgenic animal using the isolated cells. Methods for isolating a plurality of cells encoding a plurality of different RNAs associated with a same nucleic acid tag sequence, comprising the step of exposing the cells to a same signaling probe that produces a detectable signal upon hybridization to the same nucleic acid tag sequence, are also provided. Signaling probes and protease probes that form stem-loop structures, three-arm junction structures, and dumbbell structures may be used in the above methods.
Core Innovation
The invention relates to signaling probes, including fluorogenic, wavelength-shifting, and FRET-based signaling, and protease probes that generate detectable signals when hybridized to target nucleic acids. The disclosure provides probe systems that produce fluorescence upon hybridization to target nucleic acids, including in living cells, and enables detection using fluorescence at a selected wavelength.
A key problem addressed is isolating a plurality of cells from a heterogeneous population when only a subset expresses particular RNAs while other subsets do not. The disclosed approach solves this by introducing DNAs encoding different RNAs along with nucleic acid tag sequences, and then using signaling probes that hybridize to shared tag sequences so that fluorescence reports RNA expression differences between cell subsets.
In the disclosed isolation methods, cells receive a plurality of DNAs encoding a plurality of different RNAs, with each DNA encoding a nucleic acid tag sequence, wherein at least a subset encodes the same tag sequence. The cells are exposed to signaling probes that produce detectable signals upon hybridization to the tag sequence, and cells are isolated in a single application based on exhibiting at least one selected level of fluorescence at a selected wavelength.
Claims Coverage
The partial content provides two independent claims. Together, they cover isolating a plurality of cells using nucleic-acid tag sequences reported by signaling probes, including a shared-tag single-library scenario and a two-tag, two-library scenario with isolation based on fluorescence at a selected wavelength for each probe.
Single application isolation using shared tag hybridization fluorescence
A method of isolating a plurality of cells, wherein a subset expresses an RNA that is not expressed by another subset, by introducing a plurality of DNAs encoding different RNAs, each DNA further encoding a nucleic acid tag sequence with at least a subset encoding the same nucleic acid tag sequence; exposing the cells to a signaling probe that produces a detectable signal upon hybridization to said same nucleic acid tag sequence; and isolating, in a single application, cells that exhibit at least one selected level of fluorescence at a selected wavelength.
Single application isolation from two RNA expression libraries using two distinct tag hybridization signals
A method of isolating cells each expressing RNAs from two or more RNA expression libraries by introducing DNAs encoding a first RNA expression library with a first nucleic acid tag sequence and introducing DNAs encoding a second RNA expression library with a second nucleic acid tag sequence; exposing the cells to a first signaling probe and to a second signaling probe, each producing a detectable signal upon hybridization to the respective tag sequence; and isolating, in a single application, cells that exhibit at least one selected level of fluorescence at a selected wavelength for each of the first and second signaling probes.
Overall, the independent claims claim isolating cells using DNA/RNA constructs that include nucleic acid tag sequences, where signaling probes hybridize to the tag sequences to generate detectable fluorescence, and cell isolation is performed in a single application based on selected fluorescence levels at a selected wavelength, including a combined approach using two signaling probes for two distinct tag sequences from two RNA expression libraries.
Stated Advantages
Allows isolating, in a single application, cells that exhibit at least one selected level of fluorescence at a selected wavelength.
Enables isolation of cells from heterogeneous populations based on subset RNA expression differences reported via tag hybridization fluorescence.
Provides a single application workflow for isolating cells with criteria satisfied for each of two signaling probes corresponding to two tag sequences.
Documented Applications
Isolating cells expressing RNAs from heterogeneous cell populations where a subset expresses an RNA that is not expressed by another subset.
Isolating cells each expressing RNAs from two or more RNA expression libraries by co-introducing DNAs encoding first and second RNA expression libraries and using two signaling probes targeting first and second nucleic acid tag sequences.
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