NOVEL METHOD FOR SEQUENCE DETERMINATION USING NMR

• US 20090045811A1
• Filed: 10/29/2008
• Published: 02/19/2009
• Est. Priority Date: 05/20/2002
• Status: Active Grant

• Current Assignee: Istituto Di Ricerche Chimiche E Biochimiche G Ronzoni, Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Venkataraman, Ganesh, SASISEKHARAN, RAM, Raman, Rahul, Torri, Giangiacomo, Casu, Benito, Guerrini, Marco
• Granted Patent: US 7,737,692 B2
• Time in Patent Office: Days
• US Class Current: 324/307
• CPC Class Codes: G01N 24/08 by using nuclear magnetic resonance G01N24/12 takes precedence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention relates to methods for analyzing polysaccharides. In particular, compositional and sequence information about the polysaccharides are derived. Some methods use NMR in conjunction with another experimental method, such as, capillary electrophoretic techniques for the analysis.

Claims

1. A method of generating a list of possible sequences of an oligosaccharide in a heparin sample comprising:

providing a heparin sample,

defining a set of properties of an oligosaccharide in the heparin sample by performing nuclear magnetic resonance (NMR) spectroscopy and a second experimental method, wherein the NMR spectroscopy provides a measurement of a first type of disaccharide linkage and the second experimental method provides a measurement of a second type of disaccharide linkage;

constructing a list of possible oligosaccharides sequences based on the set of properties of the oligosaccharide, to thereby generate a list of possible sequences of the oligosaccharide in the heparin sample.

2. The method of claim 1, wherein the NMR spectroscopy provides a determination of a sulfation pattern of the oligosaccharide.

3. The method of claim 1, wherein the NMR spectroscopy provides a determination of the reducing end and non-reducing end of the oligosaccharide.

4. The method of claim 1, wherein the NMR spectroscopy provides a measure of the monosaccharide composition of the oligosaccharide.

5. The method of claim 1, wherein the NMR is one-dimensional (1D) NMR.

6. The method of claim 1, wherein the NMR is two-dimensional (2D) NMR.

7. The method of claim 6, wherein the 2D NMR is Correlation Spectroscopy (COSY), Total Correlation Spectroscopy (TOCSY), Heteronuclear Single Quantum Correlation (HSQC), Double-Quantum Filtered Correlation Spectroscopy (DQF-COSY), Nuclear Overhauser Effect Spectroscopy (NOESY), Rotating-Frame NOE Spectroscopy (ROESY) or a combination thereof.

8. The method of claim 7, wherein the 2D NMR is COSY, TOCSY, HSQC, ROESY or a combination thereof.

9. The method of claim 1, wherein the NMR is performed on the oligosaccharide in fragmented form.

10. The method of claim 9, wherein the oligosaccharide is fragmented using enzymatic digestion.

11. The method of claim 10, wherein the enzymatic digestion is with a heparinase, a glucuronidase, a sulfatase, or a combination thereof.

12. The method of claim 5, wherein the 1D NMR is performed on the oligosaccharide in fragmented form.

13. The method of claim 12, wherein the oligosaccharide is fragmented using enzymatic digestion.

14. The method of claim 13, wherein the enzymatic digestion is with a heparinase, a glucuronidase, a sulfatase, or a combination thereof.

15. The methods of claim 10, wherein the enzymatic digestion is complete.

16. The method of claim 13, wherein the enzymatic digestion is complete.

17. The method of claim 1, wherein the NMR is performed on the oligosaccharide in its intact form.

18. The method of claim 6, wherein the 2D NMR is performed on the intact oligosaccharide.

19. The method of claim 1, wherein the second experimental method is capillary electrophoresis.

20. The method of claim 19, wherein the capillary electrophoresis is performed on an oligosaccharide in fragmented form.

21. The method of claim 20, wherein the oligosaccharide is fragmented using enzymatic digestion.

22. The method of claim 21, wherein the enzymatic digestion is with a heparinase, a glucuronidase, a sulfatase, or a combination thereof.

23. The method of claim 21, wherein the enzymatic digestion is complete.

24. The method of claim 1, wherein the second experimental method is high performance liquid chromatography.

25. The method of claim 24, wherein the high performance liquid chromatography is performed on an oligosaccharide in fragmented form.

26. The method of claim 25, wherein the oligosaccharide is fragmented using enzymatic digestion.

27. The method of claim 26, wherein the enzymatic digestion is with a heparinase, a glucuronidase, a sulfatase, or a combination thereof.

28. The method of claim 26, wherein the enzymatic digestion is complete.

29. The method of claim 1, wherein the heparin sample is a pig mucosal heparin sample.

30. The method of claim 1, wherein the heparin sample is a low molecular weight heparin sample.

31. The method of claim 1, further comprising a data structure which represents the properties as non-character values.

32. The method of claim 4, further comprising a data structure which represents the properties as non-character values wherein the data structure includes a value for each type of monosaccharide.

33. The method of claim 31, wherein the data structure includes a value for each type of disaccharide linkage.

34. The method of claim 31, wherein the values are binary.

35. The method of claim 11, wherein the heparinase is heparinase I, heparinase II, heparinase III or a combination thereof.

36. The method of claim 14, wherein the heparinase is heparinase I, heparinase II, heparinase III or a combination thereof.

37. The method of claim 22, wherein the heparinase is heparinase I, heparinase II, heparinase III or a combination thereof.

38. The method of claim 27, wherein the heparinase is heparinase I, heparinase II, heparinase III or a combination thereof.