Parallel proximity ligation event analysis
AssigneesPresident and Fellows of Harvard College, Massachusetts Institute of Technology
InventorsErez Lieberman, Andreas Gnirke, Daniel Aird
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Abstract
The present invention describes compositions and methods showing that the spatial proximity of intracellular components may be related to their ability to cooperate in intracellular biochemical reactions. In some embodiments, the present invention contemplates a variety of nucleic acid barcoded binding partners capable of determining the spatial proximity of intracellular components as determined by ligation of their respective nucleotide barcodes. As such, an intracellular component contact map may be constructed to fingerprint specific physiological and/or pharmacological intracellular conditions.
Claims24
A composition comprising a forked adapter molecule comprising a first end and a second end, wherein said first end comprises two single stranded portions and said second end is double stranded, wherein one of said two single stranded portions is attached to a single binding partner, wherein said second end comprises a component-specific oligonucleotide barcode duplex sequence wherein said barcode sequence comprises a nucleic acid sequence unique to said component-specific binding partner, wherein said molecule does not have a fluorescent reporter.
A composition comprising a nucleic acid sequence, wherein said nucleic acid sequence comprises a 3′-5′ first strand and a 5′-3′ second strand, wherein said first strand and said second strand join a first intracellular component-specific oligonucleotide barcode and a second intracellular component-specific oligonucleotide barcode thereby forming an asymmetric barcode region wherein said first strand 3′ end is attached to a single first binding partner having specific affinity for said first intracellular component and said second strand 3′ end is attached to a single second binding partner having specific affinity for said second intracellular component, wherein said asymmetric barcode region comprises a nucleic acid sequence unique to said first and second intracellular components.
A kit, comprising:
- a) a first container comprising a plurality of forked adapter molecules comprising a first end and a second end, wherein said first end comprises two single stranded portions and said second end is double stranded, wherein one of said two single stranded portions is attached to a single binding partner, wherein said second end comprises a component-specific oligonucleotide barcode duplex sequence wherein said barcode sequence comprises a nucleic acid sequence unique to said component-specific binding partner, wherein said molecule does not have a fluorescent reporter.
- b) a second container comprising a solution capable of fixing a biological cell sample;
- c) a third container comprising buffers and reagents capable of supporting binding of said binding partner to intracellular components of said fixed biological cell sample;
- and
- d) a set of instructions describing:
- i) how to identify said intracellular components bound to said binding partners;
- and ii) construction of an intracellular component contact map.
A composition consisting of a first nucleic acid sequence comprising a first duplex region encoding a first barcode sequence unique to an attached first intracellular component binding partner, and a second nucleic acid sequence comprising a second duplex region encoding a second barcode sequence unique to an attached second intracellular component binding partner, and wherein said first and second binding partners have specific affinity for different intracellular components.
A forked adapter molecule consisting of a first end and a second end, wherein said first end has two single stranded portions and said second end is double stranded, wherein one of said two single stranded portions is attached to a single binding partner, said binding partner having a specific affinity for a single intracellular component, wherein said second end is attached to an intracellular component-specific oligonucleotide barcode duplex sequence wherein said barcode sequence is a nucleic acid sequence unique to said intracellular component.
A nucleic acid sequence consisting of a 3′-5′ first strand and a 5′-3′ second strand, wherein said first strand and said second strand join a first intracellular component-specific oligonucleotide barcode and a second intracellular component-specific oligonucleotide barcode thereby forming an asymmetric barcode duplex region wherein said first strand 3′ end is attached to a single first binding partner having specific affinity for said first intracellular component and said second strand 3′ end is attached to a single second binding partner having specific affinity for said second intracellular component, wherein said asymmetric barcode duplex region is a nucleic acid sequence unique to said first and second intracellular components.
Dependencies are inferred from the claim text.