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Publication Number
US-12497653-B2
Publication Date
2025-12-16
Expiration Date
Abstract
In some aspects disclosed herein are methods and compositions for detecting analytes such as a plurality of nucleic acid molecules in a biological sample, said method comprising generating and analyzing a first detectable signal and a second detectable signal corresponding to a first target sequence and a second target sequence, respectively. The first and second target sequences can be present and/or expressed at different levels in the biological sample.
Core Innovation
A method for analyzing a biological sample includes contacting the biological sample with an encoding probe or probe set that comprises a first hybridization region complementary to a first target sequence and one or more first barcode regions, and contacting the biological sample with a circular probe or circularizable probe or probe set that comprises a second hybridization region complementary to a second target sequence and one or more second barcode regions. The method includes contacting the biological sample with a first detectable probe that binds to the first barcode regions and detecting a first signal associated with the first detectable probe.
The method also generates a rolling circle amplification (RCA) product in the biological sample using the circular probe or a circularized probe generated from the circularizable probe or probe set as a template. A second detectable probe binds to the RCA product at a sequence in the one or more second barcode regions or a complement thereof, and detecting a second signal associated with the second detectable probe yields detection of the second target sequence or a portion thereof.
The described approach supports detecting the first target sequence or a portion thereof and the second target sequence or a portion thereof in the same biological sample. The disclosed approach is also framed in a context of multiplexed analysis with consecutive hybridization cycles and fluorescence imaging, followed by signal removal, and combining sensitivity from an encoding probe format with brightness from an RCA product to mitigate sensitivity and optical overcrowding and dropout.
Claims Coverage
The document includes one independent method claim that covers analyzing a biological sample using two target-specific barcode readouts, where one target is detected via encoding probe barcodes and a second target is detected via barcodes present in or associated with an RCA product. The independent claim relies on multiple inventive features that jointly enable detection of two different target sequences, or portions thereof, in the same sample.
Dual target encoding with first barcode-region detection and RCA product detection
Contacting the biological sample with an encoding probe or probe set having a first hybridization region complementary to a first target sequence and one or more first barcode regions, and contacting with a circular probe or circularizable probe or probe set having a second hybridization region complementary to a second target sequence and one or more second barcode regions.
Direct or indirect barcode binding for first detectable signal detection
Contacting the biological sample with a first detectable probe that directly or indirectly binds to a sequence in the one or more first barcode regions and detecting a first signal associated with the first detectable probe.
Rolling circle amplification of a second template to create an RCA product
Generating a rolling circle amplification (RCA) product in the biological sample using the circular probe or a circularized probe generated from the circularizable probe or probe set as a template.
Detecting a second signal via second detectable probe binding to RCA-product barcodes
Contacting the biological sample with a second detectable probe that directly or indirectly binds to the RCA product at a sequence in the one or more second barcode regions or a complement thereof, and detecting a second signal associated with the second detectable probe.
Detecting both target sequences in the same biological sample
Detecting the first target sequence or a portion thereof and the second target sequence or a portion thereof in the biological sample based on the first and second signals.
Across the independent claim, the inventive coverage is directed to the combined use of an encoding probe with first barcode regions for a first detectable signal and a circular or circularizable probe with second barcode regions that generates an RCA product for a second detectable signal, thereby enabling detection of two target sequences or portions thereof in the same biological sample using distinct barcode-associated readouts.
Stated Advantages
Combining smFISH-style sensitivity with RCA brightness to mitigate sensitivity and optical overcrowding and dropout.
Documented Applications
In situ detection of two targets in the same sample using distinct readouts based on first barcode regions and RCA product-associated second barcode regions.
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