Methods, kits and compositions pertaining to PNA molecular beacons

• US 6,355,421 B1
• Filed: 10/27/1998
• Issued: 03/12/2002
• Est. Priority Date: 10/27/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boston Probes, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Coull, James M., Gildea, Brian D., Hyldig-Nielsen, Jens J.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/179,298
• Time in Patent Office: 1,232 Days
• Field of Search: 530/300, 530/333, 530/350, 435/6
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer

Abstract

This invention is directed to methods, kits and compositions pertaining to PNA Molecular Beacons. PNA Molecular Beacons comprise self-complementary arm segments and flexible linkages which promote intramolecular or intermolecular interactions. In the absence of a target sequence, PNA Molecular Beacons facilitate efficient energy transfer between the linked donor and acceptor moieties of the probe. Upon hybridization of the probe to a target sequence, there is a measurable change in at least one property of at least one donor or acceptor moiety of the probe which can be used to detect, identify or quantitate the target sequence in a sample.

Claims

1. A polymer of covalently linked subunits, said polymer comprising;

a) a non-polynucleotide probing nucleobase sequence that is complementary or substantially complementary to a target sequence;

b) a first arm segment and a second arm segment that are covalently linked to, or embedded within, the probing nucleobase sequence and wherein at least one of the first or second arm segments is covalently linked to the probing nucleobase sequence through a flexible linkage;

c) at least one linked donor moiety and at least one linked acceptor moiety, wherein said donor and acceptor moieties are covalently linked to the polymer at positions that are separated by at least a portion of the probing nlcleobase sequence.

2. The polymer of claim 1, wherein the polymer is a PNA.

35. The polymer of claim 1, wherein the probing nucleobase sequence is 5-30 subunits in length and the first arm segment is 2-5 subunits in length.

36. The polymer of claim 1, wherein each of the PNA subunits of the probing nucleobase sequence has the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl, Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin and fluorescein.

37. The polymer of claim 36, wherein each PNA subunit consists of a naturally occurring nucleobase attached to the aza nitrogen of the N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

38. The polymer of claim 1, wherein the flexible linkage consists of one or more linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

wherein,

each Y is selected from the group consisting of;

a single bond, —

(CW₂)_p—

, —

C(O)(CW₂)_p—

, —

C(S)(CW₂)_p— and

—

S(O₂)(CW₂)_p—

;

each Z is selected from the group consisting of;

NH, NR², S or O;

each W is independently selected from the group consisting of;

H, R², —

OR², F, Cl, Br, I;

wherein, each R² is independently selected from the group consisting of;

39. The polymer of claim 38, wherein the flexible linkage consists of two linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

, wherein, Y is —

C(O)(CW₂)_p—

, Z is NH, each W is H, m is 1, n is 2, o is 2 and p is 1.

40. The polymer of claim 1, wherein the donor moiety is a fluorophore.

41. The polymer of claim 40, wherein the fluorophore is selected from the group consisting of 5(6)-carboxyfluorescein, 5-(2′

-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), bodipy, rhodamine, Cy2, Cy3, Cy 3.5, Cy5, Cy5.5 and texas red.

42. The polymer of claim 1, wherein the acceptor moiety is a quencher moiety.

43. The polymer of claim 42, wherein the quencher moiety is 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).

44. The polymer of claim 1, wherein the polymer is immobilized to a support.

47. The polymer of claim 44, wherein the support immobilized polymer is one polymer of an array of polymers.

3. A polymer of covalently linked subunits, said polymer comprising;

a) a first arm segment having a first and second end;

b) a non-polynucleotide probing nucleobase sequence having a first and second end, wherein the probing nucleobase sequence is complementary or substantially complementary to a target sequence;

c) a second arm segment that is embedded within the probing nulcleobase sequence and that is complementary or substantially complementary to the first arm segment;

d) a flexible linkage that covalently links the second end of the first arm segment to the second end of the probing nucleobase sequence;

e) a donor moiety covalently linked to the first end of one of either of the first arm segment or the probing nucleobase sequence; and

f) an acceptor moiety covalently linked to the first end of the other of either of the first arm segment or the probing nucleobase sequence.

4. The polymer of claim 3, wherein the probing nucleobase sequence is 5-30 subunits in length and the first arm segment is 2-5 subunits in length.

5. The polymer of claim 3, wherein each of the PNA subunits of the polymer has the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl, Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin and fluorescein.

6. The polymer of claim 3, wherein each PNA subunit consists of a naturally occurring nucleobase attached to the aza nitrogen of the N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

7. The polymer of claim 3, wherein the flexible linkage consists of one or more linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

wherein,

each Y is selected from the group consisting of;

a single bond, —

(CW₂)_p—

, —

C(O)(CW₂)_p—

, —

C(S)(CW₂)_p— and

—

S(O₂)(CW₂)_p—

;

each Z is selected from the group consisting of;

NH, NR², S or O;

each W is independently selected from the group consisting of;

H, R², —

OR², F, Cl, Br, I;

wherein, each R² is independently selected from the group consisting of;

8. The polymer of claim 7, wherein the flexible linkage consists of two linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

, wherein, Y is —

C(O)(CW₂)_p—

, Z is NH, each W is H, m is 1, n is 2, o is 2 and p is 1.

9. The polymer of claim 3, wherein the donor moiety is a fluorophore.

10. The polymer of claim 9, wherein the fluorophore is selected from the group consisting of 5(6)-carboxyfluorescein, 5-(2′

-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), bodipy, rhodamine, Cy2, Cy3, Cy 3.5, Cy5, Cy5.5 and texas red.

11. The polymer of claim 3, wherein the acceptor moiety is a quencher moiety.

12. The polymer of claim 11, wherein the quencher moiety is 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).

13. The polymer of claim 3, wherein one or more spacer moieties link one or both of the donor and acceptor moieties to the end of the polymer to which it is linked.

14. The spacer moiety of claim 13, wherein the spacer moiety comprises one or more linked amino acid moieties.

15. The spacer moiety of claim 13, wherein the spacer moiety consists of one or more linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

, wherein,

each Y is selected from the group consisting of;

a single bond, —

(CW₂)_p—

, —

C(O)(CW₂)_p—

, —

C(S)(CW₂)_p— and

—

S(O₂)(CW₂)_p—

;

each Z is selected from the group consisting of;

NH, NR², S or O;

each W is independently selected from the group consisting of;

H, R², —

OR², F, Cl, Br, I;

wherein, each R² is independently selected from the group consisting of;

16. The polymer of claim 3, wherein the polymer is continuous from the N-terminus to the C-terminus and the first arm segment is oriented toward the N-terminus of the polymer and the probing nucleobase sequence is oriented toward the C-terminus of the polymer.

17. The polymer of claim 3, wherein the polymer is immobilized to a support.

45. The polymer of claim 17, wherein the support immobilized polymer is one polymer of an array of polymers.

18. A polymer of covalently linked subunits, said polymer comprising:

a) a non-polynucleotide probing nucleobase sequence having a first and second end, wherein the probing nucleobase sequence is complementary or substantially complementary to a target sequence;

b) a first arm segment having a first and second end;

c) a second arm segment comprising a first and second end, wherein, at least a portion of the nucleobases sequence of the second arm segment is complementary to the nucleobase sequence of the first arm segment;

d) a first flexible linkage that covalently links the second end of the first arm segment to either of the first or second end of the probing nucleobase sequence;

e) a second linkage that covalently links the second end of the second arm segment to the other of either of the first or second end of the probing nucleobase sequence;

f) a donor moiety covalently linked to the first end of one of either of the first or second arm segments; and

g) an acceptor moiety covalently linked to the first end of the other of either of the first or the second arm segments.

19. The polymer of claim 18, wherein the second linkage consists of a single bond.

20. The polymer of claim 18, wherein the second linkage is a second flexible linkage.

21. The polymer of claim 18, wherein the probing nucleobase sequence is 5-30 subunits in length and each of the arm segments is independently 2-5 subunits in length.

22. The polymer of claim 18, wherein each of the PNA subunits of the polymer has the formula:

wherein,

each J is the same or different and is selected from the group consisting of;

H, R¹, OR¹, SR¹, NHR¹, NR¹₂, F, Cl, Br and I;

each K is the same or different and is selected from the group consisting of;

O, S, NH and NR¹;

each R¹ is the same or different and is an alkyl group having one to five carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group;

each A is selected from the group consisting of a single bond, a group of the formula;

—

(CJ₂)_s— and

a group of the formula;

—

(CJ₂)_sC(O)—

wherein, J is defined above and each s is an integer from one to five;

each t is 1 or 2;

each u is 1 or 2; and

each L is the same or different and is independently selected from the group consisting of J, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties, biotin and fluorescein.

23. The polymer of claim 22, wherein each PNA subunit consists of a naturally occurring nucleobase attached to the aza nitrogen of the N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.

24. The polymer of claim 18, wherein the first and second arm sequences are of equal subunit length and the nucleobase sequences of the first and second arm sequences are perfectly complementary.

25. The polymer of claim 18, wherein each of the first or second flexible linkages independently consist of one or more linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

, wherein,

each Y is selected from the group consisting of;

a single bond, —

(CW₂)_p—

, —

C(O)(CW₂)_p—

, —

C(S)(CW₂)_p— and

—

S(O₂)(CW₂)_p—

;

each Z is selected from the group consisting of;

NH, NR², S or O;

each W is independently selected from the group consisting of;

H, R², —

OR², F, Cl, Br, I;

wherein, each R² is independently selected from the group consisting of;

26. The polymer of claim 25, wherein each of the first or second flexible linkages independently consists of one or more linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

, wherein, Y is —

C(O)(CW₂)_p—

, Z is NH, each W is H, m is 1, n is 2, o is 2 and p is 1.

27. The polymer of claim 18, wherein the donor moiety is a fluorophore.

28. The polymer of claim 18, wherein the fluorophore is selected from the group consisting of 5(6)-carboxyfluorescein, 5-(2′

-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), bodipy, rhodamine, Cy2, Cy3, Cy 3.5, Cy5, Cy5.5 and texas red.

29. The polymer of claim 18, wherein the acceptor moiety is a quencher moiety.

30. The polymer of claim 18, wherein the quencher moiety is 4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).

31. The polymer of claim 18, wherein one or more spacer moieties link one or both of the donor and acceptor moieties to the first end of the arm segment.

32. The polymer of claim 31, wherein the spacer moiety consists of one or more amino acid moieties.

33. The polymer of claim 31, wherein the one or more spacer moieties consists of one or more linked compounds having the formula:

—

Y—

(O_m—

(CW₂)_n)_o—

Z—

, wherein,

each Y is selected from the group consisting of;

a single bond, —

(CW₂)_p—

, —

C(O)(CW₂)_p—

, —

C(S)(CW₂)_p— and

—

S(O₂)(CW₂)_p—

;

each Z is selected from the group consisting of;

NH, NR², S or O;

each W is independently selected from the group consisting of;

H, R², —

OR², F, Cl, Br, I;

wherein, each R² is independently selected from the group consisting of;

34. The polymer of claim 18, wherein the polymer is immobilized to a support.

46. The polymer of claim 24, wherein the support immobilized polymer is one polymer of an array of polymers.