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US 10,309,959 B2Granted
Charged reactive oligomers
AssigneeLife Technologies Corporation
InventorsJames Stray, Shaheer H. Khan
Life of this patent
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Abstract
Methods, systems, compositions and kits are described for detecting cleaved glycans from a glycoconjugate. After labeling the glycan with a nucleic acid charged oligomer described herein, the labeled glycans can be separated under the influence of an electric field or based on their detectable tag and identified.
Claims11
A method of labeling a glycan on a biomolecule, comprising:
- (a) cleaving the glycan from the biomolecule generating a cleaved glycan;
- and
- (b) labeling the cleaved glycan with a nucleic acid oligomer to form a nucleic acid-charged glycan;
- wherein the nucleic acid oligomer comprises:
- i) a first site comprising an intrinsic charge and a reactive moiety, wherein the reactive moiety enables attachment of the nucleic acid oligomer to the glycan;
- and
- ii) a second site consisting of at least one detectable tag selected from:
4 more elements
A method for detecting a glycan on a biomolecule, comprising:
- (a) cleaving the glycan from the biomolecule generating a cleaved glycan;
- (b) labeling the cleaved glycan with a mobility modifier to form a charged glycan;
- and
- (c) detecting the charged glycan,
- wherein the mobility modifier comprises:
- i) a first site comprising an intrinsic charge and a reactive moiety, wherein the reactive moiety enables attachment of the nucleic acid oligomer to the glycan;
- and
- ii) a second site consisting of at least one detectable tag selected from:
5 more elements
A method of determining a glycan sequence, comprising:
- (a) separating a glycan from a cleaved glycan pool;
- (b) labeling the glycan with a nucleic acid oligomer to generate a nucleic acid labeled glycan;
- (c) making a plurality of aliquots of the nucleic acid labeled glycan and treating each aliquot with a distinct enzyme mixture generating an enzyme-treated aliquot with a variable, truncated glycan in each aliquot, wherein each distinct enzyme mixture comprises at least one, different, linkage-specific exoglycosidase enzyme;
- (d) resolving the plurality of variable, truncated glycans from step (c) by a suitable separation means, to generate a first set of characteristic mobility shift profiles;
- (e) optionally, sequentially repeating the enzyme treatment of one or more selected, enzyme-treated aliquot(s) of step (c) with a plurality of distinct enzyme mixtures, until the truncated glycan can no longer be digested, wherein each enzyme treatment generates a plurality of characteristic mobility shift profiles;
- and
- (f) determining the glycan sequence by analyzing the sequential and characteristic mobility shift profiles and mapping the profiles to the linkage-specific exoglycosidases used during enzyme digestion,
- wherein the nucleic acid oligomer comprises:
- i) an intrinsic charge and a reactive moiety at a first site that enables attachment to the glycan;
- and
- ii) a second site consisting of at least one detectable tag selected from:
8 more elements
Dependencies are inferred from the claim text.