METHODS FOR COVALENT LINKING OF OPTICAL REPORTERS

• US 20080312427A1
• Filed: 12/11/2007
• Published: 12/18/2008
• Est. Priority Date: 04/16/2003
• Status: Active Grant

• Current Assignee: APDN (B.V.I.) Inc. (Applied DNA Sciences Incorporated)
• Original Assignee: APDN (B.V.I.) Inc. (Applied DNA Sciences Incorporated)
• Inventors: Liang, Ming-Hwa, So, Stephane Shu Kin, Kwok, Thomas
• Granted Patent: US 8,124,333 B2
• Time in Patent Office: Days
• US Class Current: 536/25.32
• CPC Class Codes: C08K 9/12 Adsorbed ingredients , e.g. ingredients on carriers
• CPC Class Codes: C08L 5/00 Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
• CPC Class Codes: C09D 11/03 characterised by features other than the chemical nature of the binder
• CPC Class Codes: C09D 7/65 macromolecular C09D7/41-C09D7/48 take precedence

Abstract

A method to link a light emitting reporter to biomolecules with nucleotide oligomers is described. The light reporter particles are silylated and functionalized to produce a coated light reporter particle, prior to covalently linking the biomolecules to the light reporter particle. The light reporter particle generated by the methods of the invention can be excited by a light excitation source such as UV or IR light, and when the biomolecule is DNA, the attached DNA molecule(s) are detectable by amplification techniques such as PCR.

Claims

1. A method for linking nucleic acid oligomers of detectable sequence to an optical reporter particle, the method comprising:

introducing a plurality of reactive functional groups onto the optical reporter; and

covalently bonding the nucleic acid oligomers to the reactive functional groups.

2. The method of claim 1, further comprising determining at least a partial sequence of the nucleic acid oligomers covalently bound to the optical reporter.

3. The method of claim 1, wherein the optical reporter has a surface that is reactive to silylating agents.

4. The method of claim 3, wherein the introducing comprises:

treating the optical reporter with a mixture of

5. The method of claim 4, wherein the introducing further comprises:

treating the optical reporter with a linking agent of the formula

Y—

R²—

C(O)—

OR^d

wherein;

Y is a leaving group;

R² is C_1-8alkylene; and

R^d is hydrogen or C_1-6alkyl;

to form a plurality of groups of the formula

—

O—

R¹—

X—

R²—

C(O)—

OR^d

covalently bound to the surface of the optical reporter.

6. The method of claim 5, wherein the covalently bonding the nucleic acid oligomers to the reactive functional groups comprises:

treating the optical reporter with a functionalized nucleic acid oligomer reagent of the formula

HR^aN—

R³-DNA

wherein;

R³ is C_2-8alkylene; and

R^a is hydrogen or C_1-6alkyl;

to form a plurality of groups of the formula

—

O—

R¹—

X—

R²C(O)—

NR^a—

R³-DNA

covalently bound to the surface of the optical reporter.

7. The method of claim 6, wherein the covalently bonding the nucleic acid oligomers to the reactive functional groups comprises:

treating the optical reporter with an activating agent to form a plurality of groups of the formula

—

O—

R¹—

X—

R²—

C(O)—

Ag

covalently bound to the surface of the optical reporter, wherein Ag is an activating group; and

treating the optical reporter with a functionalized nucleic acid oligomer reagent of the formula

HR^aN—

R³-DNA

wherein R³ is C_2-8alkylene;

to form a plurality of groups of the formula

—

O—

R¹—

X—

R²C(O)—

NR^a—

R³-DNA

covalently bound to the surface of the optical reporter.

8. The method of claim 4, wherein R¹ is —

(CH₂)_t— and

wherein t is from 2 to 6.

9. The method of claim 8, wherein t is 3.

10. The method of claim 5, wherein R² is —

C(O)—

C_2-6alkylene-.

11. The method of claim 5, wherein R² is —

(CH₂)_u—

wherein u is from 1 to 6.

12. The method of claim 11, wherein u is 1.

13. The method of claim 10, wherein R² is —

C(O)—

(CH₂)_u—

wherein u is from 1 to 6.

14. The method of claim 13, wherein u is 2.

15. The method of claim 13, wherein u is 3.

16. The method of claim 5, wherein R is methyl or ethyl.

17. The method of claim 7, wherein R^a is hydrogen.

18. The method of claim 7, wherein R³ is —

(CH₂)_w—

wherein w is from 2 to 6.

19. The method of claim 7, wherein w is 6.

20. The method of claim 4, wherein the introducing further comprises:

treating the optical reporter with a linking agent of the formula

21. The method of claim 20, wherein the covalently bonding the nucleic acid oligomers to the reactive functional groups comprises:

treating the optical reporter with an activating agent to form a plurality of groups of the formula

—

O—

R¹—

X—

C(O)—

CH₂—

(CH₂)_s—

C(O)—

Ag

covalently bound to the surface of the optical reporter, wherein Ag is an activating group; and

treating the optical reporter with a functionalized nucleic acid oligomer reagent of the formula

HR^aN—

R³-DNA

wherein R³ is C_2-8alkylene;

to form a plurality of groups of the formula

—

O—

R¹—

X—

C(O)—

CH₂—

(CH₂)_s—

C(O)—

NR^a—

R³-DNA

22. The method of claim 4, wherein the introducing further comprises:

treating the optical reporter with an alpha haloacetic acid to form a plurality of groups of the formula

—

O—

R¹—

X—

CH₂—

C(O)—

OH

covalently bound to the optical reporter.

23. The method of claim 22, wherein the covalently bonding the nucleic acid oligomers to the reactive functional groups comprises:

treating the optical reporter with an activating agent to form a plurality of groups of the formula

—

O—

R¹—

X—

C(O)—

CH₂—

C(O)—

Ag

covalently bound to the surface of the optical reporter, wherein Ag is an activating group; and

treating the optical reporter with a functionalized nucleic acid oligomer reagent of the formula

HR^aN—

R³-DNA

wherein R³ is C_2-8alkylene;

to form a plurality of groups of the formula

—

O—

R¹—

X—

C(O)—

CH₂—

C(O)—

NR^a—

R³-DNA

24. A method for preparing DNA labeled-optical reporters of formula i

(UCP)-[O—

R¹—

X—

R²—

C(O)—

NR^a—

R³-DNA]_m  

 

i

wherein;

m is an integer greater than 1;

X—

is;

—

O—

;

—

S—

;

or —

NR^b—

, wherein R^b is hydrogen or C_1-6alkyl;

each R^a is C_1-6alkyl and may be the same or different at each occurrence;

R¹—

is C_2-8alkylene;

R² is C_1-8alkylene; and

R³ is C_2-8alkylene;

the method comprising;

reacting activated optical reporters of formula g

(UCP)-[O—

R¹—

X—

R²—

C(O)—

Ag]_m  

 

g;

wherein Ag is an activating group, with a nucleic acid oligomer reagent of the formula h;

HR^aN—

R³-DNA  

 

h

to form the DNA labeled-optical reporters of formula i.

25. The method of claim 24, further comprising:

reacting ester-functionalized optical reporters of formula f;

(UCP)-[O—

R¹—

X—

R²—

C(O)—

OR^d]_m  

 

f;

wherein Rd is hydrogen or C_1-6alkyl,

with an activating agent,

to form the activated optical reporters of formula g.

26. The method of claim 25, further comprising:

reacting functionalized optical reporters of formula d;

(UCP)-[O—

R¹—

X—

H]_m  

 

d;

with a reagent of formula e

Y—

R²—

C(O)—

OR^d  

 

e;

wherein Y is a leaving group

to form the functionalized optical reporters f.

27. The method of claim 26, further comprising:

reacting optical reporters a;

UCP  

 

a

with silylating agents b and c