Reversible fixing reagents and methods of use thereof
Inventors
Delaney, Joshua • Gohil, Shalini • Lowe, Adam • Luo, Yi • Walter, Dagmar
Assignees
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Publication Number
US-12566113-B2
Publication Date
2026-03-03
Expiration Date
Abstract
The present disclosure relates to compositions and methods for reversible fixation of biological samples using fixation reagents that form bis-carbamate crosslinks between amine-bearing moieties in biomolecules.
Core Innovation
The invention relates to a method for preparing a biological sample by contacting the sample with a first fixing reagent composition comprising a compound of formula (I) and a carrier. Formula (I) includes a Linker defined as a linear alkane moiety of 2-24 carbons, optionally including a disulfide bond or substituted with —OR, where R is selected from —H, —O(CO)CH3, and —O(CO)-imidazole, and m is 1 to 12.
The disclosed approach achieves reversible fixation through bis-imidazole-carboxylate / bis-carbamate crosslinking fixing reagents that crosslink biomolecules in the biological sample. Representative embodiments also describe bis-carbamate crosslinked biomolecules formed from amine-bearing biomolecule moieties via a Linker comprising an ethylene glycol moiety and/or a linear or branched alkane moiety of 2-24 carbons, with disulfide-containing crosslinked structures providing fixed biological samples with reversibly cleavable linkages.
The document further describes optional additional contacting steps for reversal and additional fixation, including lysis and unfixing agents that cleave carbamate and/or disulfide bonds and reverse paraformaldehyde fixation, together with optional second fixation with a paraformaldehyde-containing second fixing reagent. It also describes assay configurations using discrete partitions, lysis/unfixing and assay reagents, barcodes, support beads, and kit composition.
The invention also includes partitioned processing of fixed samples reversibly fixed with formula (I), where an unfixing agent can be used with discrete partitions such as droplets or microfluidic compartments for downstream cellular analyte processing. Co-partitioned barcoding supports are described, including barcode-carrying beads with nucleic acid barcodes and UMIs in droplet/microfluidic partition contexts.
Claims Coverage
The consolidated independent claim coverage centers on one method for preparing a biological sample. It specifies a first fixing reagent composition comprising a carrier and a compound of formula (I) with defined linker options and m=1 to 12. Dependent claims further refine the formula sub-family and add optional additional contacting steps with lysis/unfixing agents and/or a second paraformaldehyde fixing reagent.
Formula (I) reversible fixing with defined linker and m range
Contacting the biological sample with a first fixing reagent composition comprising a compound of formula (I), wherein the Linker is a linear alkane moiety of 2-24 carbons, optionally includes a disulfide bond or is substituted with —OR where R is selected from —H, —O(CO)CH3, and —O(CO)-imidazole, and m is 1 to 12.
First fixing reagent composition including a carrier
The first fixing reagent composition comprises the compound of formula (I) and a carrier.
Formula sub-family selection for compound of formula (I)
The compound of formula (I) is a compound of formula (II) with n being 1 to 13.
Optional lysis and unfixing
Further contacting the sample with a lysis agent and/or an unfixing agent where the lysis agent cleaves carbamate bonds using DETA, EDA, hydrazine monohydrate, a carboxyesterase, or combinations, and/or cleaves disulfide bonds using DTT, and the unfixing agent reverses paraformaldehyde fixation using compounds (7a)–(7o).
Optional second fixation with paraformaldehyde and specified formula compounds
Further contacting the sample with a second fixing reagent comprising paraformaldehyde and either a compound of formula (III) where n is 1 to 12, or a compound of formula (V) where R is selected from —H, —O(CO)CH3, and —O(CO)-imidazole.
The claims primarily cover contacting a biological sample with a first fixing reagent composition containing a carrier and a formula (I) fixing compound defined by specific linker options and m=1 to 12. The dependent features additionally cover selected formula sub-families, optional lysis/unfixing agents that cleave carbamate and/or disulfide bonds and reverse paraformaldehyde fixation, and optional second fixation using paraformaldehyde with specified formula compounds.
Stated Advantages
Enables reversible fixing/unfixing by cleavage of carbamate crosslinks using carbamate-cleaving agents and, for disulfide linkers, by disulfide cleavage followed by carbamate cleavage.
Allows partitioned processing of fixed samples in discrete partitions such as droplets or microfluidic compartments for downstream cellular analyte processing.
Enables co-partitioned barcoding supports using barcode-carrying beads with nucleic acid barcodes and UMIs in droplet/microfluidic partition contexts.
Enables RNA recovery and sequencing-quality results when fixed samples are processed in droplet assays with unfixing.
Documented Applications
Downstream cellular analyte processing using fixed biological samples in discrete partitions such as droplets or microfluidic compartments with co-partitioned barcoding supports (barcode-carrying beads with nucleic acid barcodes/UMIs).
Processing fixed biological samples in partition-based droplet assays using barcoding workflows with barcode beads and UMIs.
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