Charged reactive oligomers

Patent metadata

Field	Value
Publication number	US-10309959-B2
Application number	US-201515532271-A
Country	US
Kind code	B2
Filing date	Dec 3, 2015
Priority date	Dec 3, 2014
Publication date	Jun 4, 2019
Grant date	Jun 4, 2019

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.

First claim

What is claimed is:

1. 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:

2. The method of labeling the glycan of claim 1 , further comprising detecting the nucleic acid-charged glycan.

3. The method of labeling a glycan in claim 2 , wherein said detection is selected from the group consisting of UV absorbance, fluorescence, visible light, chemiluminescence, conductance and an electrical signal.

4. The method of labeling a glycan of claim 1 wherein, the cleaved glycan of step (a) is separated in a charge differential field to generate a charged glycan, or wherein, the nucleic acid-charged glycan of step (b) is separated in a charge differential field and is identified by a hybridization step.

5. The method of labeling a glycan of claim 4 wherein, the charge differential field comprises an electric field, a magnetic field, a salt gradient, or, wherein said charged glycan has a negative charge.

6. The method of labeling a glycan of claim 1 , wherein said biomolecule is selected from the group consisting of a glycoprotein, a glycolipid, a proteoglycan, a phosphoprotein, a glycosaminoglycan, a phospholipid-protein containing a glycan core, a synthetic glycan, a native glycan, a derivatized glycan, or, wherein said nucleic acid oligomer comprises 1 to 20 nucleotides.

7. The method of labeling a glycan of claim 6 , wherein said nucleic acid oligomer is selected from the group consisting of 1 to 5 nucleotides, 1 to 8 nucleotides, 1 to 10 nucleotides, 1 to 15 nucleotides and 1 to 2 nucleotides.

8. The method of labeling a glycan of claim 1 , wherein said nucleic acid oligomer comprises a deoxyribonucleic acid or analogs thereof, a ribonucleic acid or analogs thereof, a locked nucleic acid (LNA) or analogs thereof, a protein nucleic acid (PNA), or a nucleic acid with a phosphorothionate linkage.

9. The method of labeling a glycan of claim 1 , wherein the first site comprises at least one of a nucleotide base, a 2′ sugar, a 3′ sugar, or a 5′ sugar.

10. 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:

11. 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:

Assignees

• Life Technologies Corp

Inventors

• Stray James
• Khan Shaheer H

Classifications

• G16C99/00

  Subject matter not provided for in other groups of this subclass · CPC title

• G01N2333/924

  acting on glycosyl compounds (3.2) · CPC title

• G01N33/58

  involving labelled substances (G01N33/53 takes precedence) · CPC title

• G01N27/447

  using electrophoresis · CPC title

• G01N33/533

  with fluorescent label · CPC title