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Member
CepheidCepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Cepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Publication Number
US-11807643-B2
Publication Date
2023-11-07
Expiration Date
Abstract
Automated oligonucleotide synthesis-compatible fluorescent dye phosphoramidite compounds, solid supports, and labeled polynucleotides incorporating the compounds are provided. The compounds allow universal incorporation of the fluorescent label into any position of the polynucleotide.
Core Innovation
Automated oligonucleotide synthesis-compatible fluorescent dye phosphoramidite compounds, solid supports, and labeled polynucleotides incorporating the compounds are provided. The compounds allow universal incorporation of the fluorescent label into any position of the polynucleotide.
Fluorescent dye-labeled polynucleotides are typically prepared by post-synthetic conjugation which suffers from low conjugation yields and the need for additional purification. Few phosphoramidite derivatives of fluorescent dyes are commercially available that are compatible with automated phosphoramidite oligonucleotide synthesis and that allow incorporation of a fluorescent dye moiety at any position of a synthetic polynucleotide, and a need exists for dyes that are compatible with automated phosphoramidite oligonucleotide synthesis, can be incorporated into any position of a polynucleotide, and provide a high endpoint fluorescence signal in PCR applications.
Claims Coverage
Independent claims include five inventive features corresponding to a fluorescent dye compound of Formula I, a labeled polynucleotide prepared by automated phosphoramidite synthesis, a method for preparing a labeled conjugate, an automated molecular diagnostics cartridge comprising the labeled polynucleotide, and a repeated compound claim of Formula I.
Fluorescent dye compound of Formula I
A compound represented by Formula I, or a stereoisomer, a salt, or a tautomer thereof, wherein X is H, halogen, or an optionally substituted C1-C5 alkyl; R1, R2, R3, and R4 are independently H or optionally substituted C1-C6 alkyl; R5, R6, R7, R8, and R9 are independently H, halogen, or optionally substituted C1-C6 alkyl; L1 is an optionally substituted C2-C10 alkylene or optionally substituted C2-C50 heteroalkylene; L2 and L3 are independently an optionally substituted C2-C10 alkylene or optionally substituted C2-C30 heteroalkylene; Q1 is a hydroxyl protecting group; Z is CH, N, NHC(O)N, or OC(O)N; Y is OH, OP(OCH2CH2CN)NR10R11, or a solid support; and R10 and R11 are independently optionally substituted C1-C6 alkyl.
Labeled polynucleotide prepared by automated phosphoramidite synthesis
A labeled polynucleotide prepared using automated phosphoramidite synthesis formed from the compound of Formula I, wherein the labeled polynucleotide comprises the residue of a compound of the disclosure and can further comprise a fluorescence quencher, a BHQ type fluorescence quencher, and can be attached to a solid support such as a controlled pore glass bead, polystyrene bead, magnetic bead, or microwell plate.
Method for preparing a labeled conjugate via covalent attachment
A method for preparing a labeled conjugate of a biomolecule, a synthetic polymer, or a solid support comprising contacting the biomolecule, the synthetic polymer, or the solid support with a compound of Formula I wherein Y is OP(OCH2CH2CN)NR10R11 and R10 and R11 are independently optionally substituted C1-C6 alkyl, in a suitable solvent under conditions sufficient to covalently attach the compound to the biomolecule, the synthetic polymer, or the solid support thereby forming the labeled conjugate; embodiments specify the ligand can be a polynucleotide and that the conditions can be automated phosphoramidite oligonucleotide synthesis conditions.
Automated molecular diagnostics cartridge comprising labeled polynucleotide
An automated molecular diagnostics cartridge comprising the labeled polynucleotide prepared using automated phosphoramidite synthesis, wherein the cartridge can be a PCR diagnostic cartridge.
Repeated compound claim of Formula I
A compound represented by Formula I (reiterated), including salts, stereoisomers, and tautomers, with the same structural definitions and optional embodiments as provided for the fluorescent dye compound of Formula I.
The independent claims cover fluorescent dye compounds of Formula I with defined substituents and linkers, labeled polynucleotides prepared by automated phosphoramidite synthesis incorporating those compounds, a method for covalent attachment of the compounds to ligands, automated molecular diagnostics cartridges comprising the labeled polynucleotides, and a reiterated compound claim of Formula I.
Stated Advantages
Compatibility with automated phosphoramidite oligonucleotide synthesis.
Ability to be incorporated into any position of a synthetic polynucleotide (universal incorporation).
Provide a high endpoint fluorescence signal in PCR applications.
Documented Applications
Use in PCR (including real-time PCR and 5'-nuclease PCR) for detection and quantification of target DNA sequences.
Use in sequencing.
Preparation of fluorescent dye-labeled polynucleotides via automated phosphoramidite oligonucleotide synthesis.
Inclusion in PCR diagnostic kits and automated molecular diagnostics cartridges (e.g., a PCR diagnostic cartridge).
Labeling of ligands including biomolecules (e.g., polynucleotides, oligonucleotides, proteins, antibodies, peptides, polysaccharides), synthetic polymers, and solid supports.
Use in labeled probes such as 5'-nuclease PCR probes and other probe formats employing a fluorophore and a fluorescence quencher (FRET-based probe detection).
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