Methods for covalent linking of optical reporters
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.
1. A method for linking nucleic acid oligomers of detectable sequence to an optical reporter particle, the method comprising:
introducing by covalent bonding, a plurality of reactive functional groups onto the optical reporter, said optical reporter being an up-converting phosphor containing particle; 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
wherein:
each R is C 1-6 alkyl and may be the same or different at each occurrence;
R 1 — is C 2-8 alkylene; and
X— is: —O—; —S—; or —NR b —, wherein R b is hydrogen or C 1-6 alkyl;
to form a plurality of groups of the formula
—O—R 1 —XH
covalently bound to the surface of the optical reporter.
5. The method of claim 4 , wherein the introducing further comprises:
treating the optical reporter with a linking agent of the formula
Y—R 2 —C(O)—OR d
wherein:
Y is a leaving group;
R 2 is C 1-8 alkylene; and
R d is hydrogen or C 1-6 alkyl;
to form a plurality of groups of the formula
—O—R 1 —X—R 2 —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 a N—R 3 -DNA
wherein: R 3 is C 2-8 alkylene; and
R a is hydrogen or C 1-6 alkyl;
to form a plurality of groups of the formula
—O—R 1 —X—R 2 —C(O)—NR a —R 3 -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—R 1 —X—R 2 —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 a N—R 3 -DNA
wherein R 3 is C 2-8 alkylene;
to form a plurality of groups of the formula
—O—R 1 —X—R 2 —C(O)—NR a —R 3 -DNA
covalently bound to the surface of the optical reporter.
8. The method of claim 4 , wherein R 1 is —(CH 2 ) 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 R 2 is —C(O)—C 2-6 alkylene-.
11. The method of claim 5 , wherein R 2 is —(CH 2 ) 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 R 2 is —C(O)—(CH 2 ) 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 R 3 is —(CH 2 ) 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
wherein s is 1 or 2;
to form a plurality of groups of the formula
—O—R 2 —X—C(O)—CH 2 —(CH 2 ) s —C(O)—OH
covalently bound to the surface of the optical reporter.
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—R 1 —X—C(O)—CH 2 —(CH 2 ) 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 a N—R 3 -DNA
wherein R 3 is C 2-8 alkylene;
to form a plurality of groups of the formula
—O—R 1 —X—C(O)—CH 2 —(CH 2 ) s —C(O)—NR a —R 3 -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—R 1 —X—CH 2 —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—R 1 —X—C(O)—CH 2 —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 a N—R 3 -DNA
wherein R 3 is C 2-8 alkylene;
to form a plurality of groups of the formula
—O—R 1 —X—C(O)—CH 2 —C(O)—NR a —R 3 -DNA
24. A method for preparing DNA labeled-optical reporters of formula i
(UCP)-[O—R 1 —X—R 2 —C(O)—NR a —R 3 -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 1-6 alkyl;
each R a is C 1-6 alkyl and may be the same or different at each occurrence;
R 1 — is C 2-8 alkylene;
R 2 is C 1-8 alkylene;
R 3 is C 2-8 alkylene; and
UCP is an up-converting phosphor particle
the method comprising:
reacting activated optical reporters of formula g.
(UCP)-[O—R 1 —X—R 2 —C(O)—Ag] m g;
wherein Ag is an activating group,
with a nucleic acid oligomer reagent of the formula h;
HR a N—R 3 -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—R 1 —X—R 2 —C(O)—OR d ] m f;
wherein R d is hydrogen or C 1-6 alkyl,
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—R 1 —X—H] m d;
with a reagent of formula e
Y—R 2 —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
wherein each R is C 1-6 alkyl and may be the same or different at each occurrence to form the functionalized optical reporters d.
28. The method of claim 7 , wherein the optical reporter is an upconverting phosphor.
29. The method of claim 5 , wherein Y is halo, R 2 is methylene, and R d is hydrogen.
30. A method for linking nucleic acid oligomers of detectable sequence to an optical reporter particle, the method comprising:
introducing by covalent bonding, a plurality of reactive functional groups onto the optical reporter, said optical reporter being an up-converting phosphor containing particle, said up-converting phosphor containing particle having a surface that is reactive to silylating agents,
treating the up-converting phosphor containing particle with a mixture of
wherein:
each R is C 1-6 alkyl and may be the same or different at each occurrence;
R 1 — is C 2-8 alkylene; and
X— is: —O—; —S—; or —NR b —, wherein R b is hydrogen or C 1-6 alkyl;
to form a plurality of groups of the formula
—O—R 1 —XH
covalently bound to the surface of the optical reporter,
treating the optical reporter with a linking agent of the formula
Y—R 2 —C(O)—OR d
wherein:
Y is a leaving group;
R 2 is C 1-8 alkylene; and
R d is hydrogen or C 1-6 alkyl;
to form a plurality of groups of the formula
—O—R 1 —X—R 2 —C(O)—OR d
covalently bound to the surface of the optical reporter,
covalently bonding the nucleic acid oligomers to the reactive functional groups covalently bound to the upconverting phosphor containing particle; and
determining at least a partial sequence of the nucleic acid oligomers covalently bound to the optical reporter.
31. The method of claim 30 , 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 a N—R 3 -DNA
wherein: R 3 is C 2-8 alkylene; and
R a is hydrogen or C 1-6 alkyl;
to form a plurality of groups of the formula
—O—R 1 —X—R 2 —C(O)—NR a —R 3 -DNA
covalently bound to the surface of the optical reporter.
32. The method of claim 31 , 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—R 1 —X—R 2 —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 a N—R 3 -DNA
wherein R 3 is C 2-8 alkylene;
to form a plurality of groups of the formula
—O—R 1 —X—R 2 —C(O)—NR a —R 3 -DNA
covalently bound to the surface of the optical reporter.
33. The method of claim 30 , wherein R 1 is —(CH 2 ) t — and wherein t is from 2 to 6.
34. The method of claim 33 , wherein t is 3.
35. The method of claim 30 , wherein R 2 is —C(O)—C 2-6 alkylene-.
36. The method of claim 30 , wherein R 2 is —(CH 2 ) u — wherein u is from 1 to 6.
37. The method of claim 36 , wherein u is 1.
38. The method of claim 35 , wherein R 2 is —C(O)—(CH 2 ) u — wherein u is from 1 to 6.
39. The method of claim 38 , wherein u is 2.
40. The method of claim 38 , wherein u is 3.
41. The method of claim 30 , wherein R is methyl or ethyl.
42. The method of claim 32 , wherein R a is hydrogen.
43. The method of claim 32 , wherein R 3 is —(CH 2 ) w — wherein w is from 2 to 6.
44. The method of claim 32 , wherein w is 6.
45. The method of claim 30 , wherein the introducing further comprises:
treating the optical reporter with an alpha haloacetic acid to form a plurality of groups of the formula
—O—R 1 —X—CH 2 —C(O)—OH
covalently bound to the optical reporter.
46. The method of claim 45 , 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—R 1 —X—C(O)—CH 2 —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 a N—R 3 -DNA
wherein R 3 is C 2-8 alkylene;
to form a plurality of groups of the formula
—O—R 1 —X—C(O)—CH 2 —C(O)—NR a —R 3 -DNA
47. The method of claim 32 , wherein the optical reporter is an upconverting phosphor.
48. The method of claim 30 , wherein Y is halo, R 2 is methylene, and R d is hydrogen.
49. The composition of claim 30 , wherein the nucleic acid oligomer is double stranded DNA.
50. The composition of claim 30 , wherein the nucleic acid oligomer is a DNA molecule having a length of between about 40 base pairs and about 1000 base pairs.