U.S. patent application number 09/888341 was filed with the patent office on 2003-02-20 for methods, kits and compositions pertaining to pna molecular beacons.
Invention is credited to Coull, James M., Gildea, Brian D., Hyldig-Nielsen, Jens J..
Application Number | 20030036059 09/888341 |
Document ID | / |
Family ID | 25501029 |
Filed Date | 2003-02-20 |
United States Patent
Application |
20030036059 |
Kind Code |
A1 |
Coull, James M. ; et
al. |
February 20, 2003 |
Methods, kits and compositions pertaining to PNA Molecular
Beacons
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.
Inventors: |
Coull, James M.; (Westford,
MA) ; Gildea, Brian D.; (Billerica, MA) ;
Hyldig-Nielsen, Jens J.; (Holliston, MA) |
Correspondence
Address: |
BOSTON PROBES INC.
Attn: Brian D. Gildea, Esq.
15 DeAngelo Drive
BEDFORD
MA
01730
US
|
Family ID: |
25501029 |
Appl. No.: |
09/888341 |
Filed: |
June 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09888341 |
Jun 22, 2001 |
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09179298 |
Oct 27, 1998 |
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6355421 |
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09179298 |
Oct 27, 1998 |
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08958532 |
Oct 27, 1997 |
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Current U.S.
Class: |
435/6.11 ;
530/350 |
Current CPC
Class: |
C12Q 1/6818
20130101 |
Class at
Publication: |
435/6 ;
530/350 |
International
Class: |
C12Q 001/68; C07K
014/00 |
Claims
We claim:
1. A polymer suitable for detecting, identifying or quantitating a
target sequence, said polymer comprising; a. a nucleobase sequence;
b. a first arm segment and a second arm segment, wherein at least
one of the first or second arm segments is linked to the 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 linked to the polymer at positions which
are at opposite ends of the nucleobase sequence.
2. The polymer of claim 1, wherein the nucleobase sequence is a
probing nucleobase sequence.
3. The polymer of claim 1, wherein the polymer is a PNA.
4. A polymer suitable for detecting, identifying or quantitating a
target sequence, said polymer comprising; a. a first arm segment
having a first and second end; b. a probing nucleobase sequence
which is complementary or substantially complementary to the target
sequence; c. a second arm segment which is embedded within the
probing nucleobase sequence and which is complementary or
substantially complementary to the first arm segment; d. a flexible
linkage which links the second end of the first arm segment to the
second end of the probing nucleobase sequence; e. a donor moiety
linked to the first end of one of either of the first arm segment
or the probing nucleobase sequence; and f. an acceptor moiety
linked to the first end of the other of either of the first arm
segment or the probing nucleobase sequence.
5. The polymer of claim 4, wherein the probing nucleobase sequence
is 5-30 subunits in length and the first arm segment is 2-5
subunits in length.
6. The polymer of claim 4, wherein each of the PNA subunits of the
polymer has the formula: 2wherein, each J is the same or different
and is selected from the group consisting of: H, R.sup.1, OR.sup.1,
SR.sup.1, NHR.sup.1, NR.sup.1.sub.2, F, Cl, Br and I; each K is the
same or different and is selected from the group consisting of: O,
S, NH and NR.sup.1; each R.sup.1 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; --(CJ.sub.2).sub.s-- and a group of the
formula; --(CJ.sub.2).sub.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.
7. The polymer of claim 4, 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.
8. The polymer of claim 4, wherein the flexible linkage consists of
one or more linked compounds having the formula:
--Y--(O.sub.m--(CW.sub.2).sub.n- ).sub.o--Z-- wherein, each Y is
selected from the group consisting of: a single bond,
--(CW.sub.2).sub.p--, --C(O)(CW.sub.2).sub.p--,
--C(S)(CW.sub.2).sub.p-- and --S(O.sub.2)(CW.sub.2).sub.p--; each Z
is selected from the group consisting of: NH, NR.sup.2, S or O;
each W is independently selected from the group consisting of: H,
R.sup.2, --OR.sup.2, F, Cl, Br, I; wherein, each R.sup.2 is
independently selected from the group consisting of: --CX.sub.3,
--CX.sub.2CX.sub.3, --CX.sub.2CX.sub.2CX.sub.3,
--CX.sub.2CX(CX.sub.3).sub.2, and --C(CX.sub.3).sub.3; each X is
independently selected from the group consisting of H, F, Cl, Br or
I; each m is independently 0 or 1; and each n, o and p are
independently integers from 0 to 10.
9. The polymer of claim 8, wherein the flexible linkage consists of
two linked compounds having the formula:
--Y--(O.sub.m--(CW.sub.2).sub.n).sub- .o--Z--, wherein, Y is
--C(O)(CW.sub.2).sub.p--, Z is NH, each W is H, m is 1, n is 2, o
is 2and p is 1.
10. The polymer of claim 4, wherein the donor moiety is a
fluorophore.
11. The polymer of claim 10, wherein the fluorophore is selected
from the group consisting of 5(6)-carboxyfluorescein and its
derivatives, 5-(2'-aminoethyl)-aminonaphthalene-1-sulfonic acid
(EDANS), bodipy and its derivatives, rhodamine and its derivatives,
Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, texas red and its derivatives.
12. The polymer of claim 4, wherein the acceptor moiety is a
quencher moiety.
13. The polymer of claim 12, wherein the quencher moiety is
4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).
14. The polymer of claim 4, 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.
15. The spacer moiety of claim 14, wherein the spacer moiety
comprises one or more linked amino acid moieties.
16. The spacer moiety of claim 14, wherein the spacer moiety
consists of one or more linked compounds having the formula:
--Y--(O.sub.m--(CW.sub.2- ).sub.n).sub.o--Z--, wherein, each Y is
selected from the group consisting of: a single bond,
--(CW.sub.2).sub.p--, --C(O)(CW.sub.2).sub.p--,
--C(S)(CW.sub.2).sub.p-- and --S(O.sub.2)(CW.sub.2).sub.p--; each Z
is selected from the group consisting of: NH, NR.sup.2, S or O;
each W is independently selected from the group consisting of: H,
R.sup.2, --OR.sup.2, F, Cl, Br, I; wherein, each R.sup.2 is
independently selected from the group consisting of: --CX.sub.3,
--CX.sub.2CX.sub.3, --CX.sub.2CX.sub.2CX.sub.3,
--CX.sub.2CX(CX.sub.3).sub.2, and --C(CX.sub.3).sub.3; each X is
independently selected from the group consisting of H, F, Cl, Br or
I; each m is independently 0 or 1; and each n, o and p are
independently integers from 0 to 10.
17. The polymer of claim 4, 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.
18. The polymer of claim 4, wherein the polymer is immobilized to a
support.
19. A polymer suitable for detecting, identifying or quantitating a
target sequence, said polymer comprising: a. a probing nucleobase
sequence having a first and second end and which is complementary
or substantially complementary to the 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 which 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 which 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
linked to the first end of one of either of the first or second arm
segments; and g. an acceptor moiety linked to the first end of the
other of either of the first or the second arm segments.
20. The polymer of claim 19, wherein the second linkage consists of
a single bond.
21. The polymer of claim 19, wherein the second linkage is a second
flexible linkage.
22. The polymer of claim 19, wherein the probing nucleobase
sequence is 5-30 subunits in length and each of the arm segments is
independently 2-5 subunits in length.
23. The polymer of claim 19, wherein each of the PNA subunits of
the polymer has the formula: 3wherein, each J is the same or
different and is selected from the group consisting of: H, R.sup.1,
OR.sup.1, SR.sup.1, NHR.sup.1, NR.sup.1.sub.2, F, Cl, Br and I;
each K is the same or different and is selected from the group
consisting of: O, S, NH and NR.sup.1; each R.sup.1 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;
--(CJ.sub.2).sub.s-- and a group of the formula;
--(CJ.sub.2).sub.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.
24. The polymer of claim 23, 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.
25. The polymer of claim 19, 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.
26. The polymer of claim 19, wherein each of the first or second
flexible linkages independently consist of one or more linked
compounds having the formula:
--Y--(O.sub.m--(CW.sub.2).sub.n).sub.o--Z--, wherein, each Y is
selected from the group consisting of: a single bond,
--(CW.sub.2).sub.p--, --C(O)(CW.sub.2).sub.p--,
--C(S)(CW.sub.2).sub.p-- and --S(O.sub.2)(CW.sub.2).sub.p--; each Z
is selected from the group consisting of: NH, NR.sup.2, S or O;
each W is independently selected from the group consisting of: H,
R.sup.2, --OR.sup.2, F, Cl, Br, I; wherein, each R.sup.2 is
independently selected from the group consisting of: --CX.sub.3,
--CX.sub.2CX.sub.3, --CX.sub.2CX.sub.2CX.sub.3,
--CX.sub.2CX(CX.sub.3).sub.2, and --C(CX.sub.3).sub.3; each X is
independently selected from the group consisting of H, F, Cl, Br or
I; each m is independently 0 or 1; and each n, o and p are
independently integers from 0 to 10.
27. The polymer of claim 26, wherein each of the first or second
flexible linkages independently consists of one or more linked
compounds having the formula:
--Y--(O.sub.m--(CW.sub.2).sub.n).sub.o--Z--, wherein, Y is
--C(O)(CW.sub.2).sub.p--, Z is NH, each W is H, m is 1, n is 2, o
is 2 and p is 1.
28. The polymer of claim 19, wherein the donor moiety is a
fluorophore.
29. The polymer of claim 19, wherein the fluorophore is selected
from the group consisting of 5(6)-carboxyfluorescein and its
derivatives, 5-(2'-aminoethyl)-aminonaphthalene-1-sulfonic acid
(EDANS), bodipy and its derivatives, rhodamine and its derivatives,
Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, texas red and its derivatives.
30. The polymer of claim 19, wherein the acceptor moiety is a
quencher moiety.
31. The polymer of claim 19, wherein the quencher moiety is
4-((-4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).
32. The polymer of claim 19, wherein one or more spacer moieties
link one or both of the donor and acceptor moieties to the first
end of the arm segment.
33. The polymer of claim 32, wherein the spacer moiety consists of
one or more amino acid moieties.
34. The polymer of claim 32, wherein the one or more spacer
moieties consists of one or more linked compounds having the
formula: --Y--(O.sub.m--(CW.sub.2).sub.n).sub.o--Z--, wherein, each
Y is selected from the group consisting of: a single bond,
--(CW.sub.2).sub.p--, --C(O)(CW.sub.2).sub.p--,
--C(S)(CW.sub.2).sub.p-- and --S(O.sub.2)(CW.sub.2).sub.p--; each Z
is selected from the group consisting of: NH, NR.sup.2, S or O;
each W is independently selected from the group consisting of: H,
R.sup.2, --OR.sup.2, F, Cl, Br, I; wherein, each R.sup.2 is
independently selected from the group consisting of: --CX.sub.3,
--CX.sub.2CX.sub.3, --CX.sub.2CX.sub.2CX.sub.3,
--CX.sub.2CX(CX.sub.3).sub.2, and --C(CX.sub.3).sub.3; each X is
independently selected from the group consisting of H, F, Cl, Br or
I; each m is independently 0 or 1; and each n, o and p are
independently integers from 0 to 10.
35. The polymer of claim 19, wherein the polymer is immobilized to
a support.
36. A method for the detection, identification or quantitation of a
target sequence, said method comprising the steps of: a. contacting
the sample with a polymer comprising: (i). a probing nucleobase
sequence; (ii). a first arm segment and a second arm segment,
wherein at least one of the first or second arm segments is linked
to the probing nucleobase sequence through a flexible linkage;
(iii). at least one linked donor moiety and at least one linked
acceptor moiety, wherein said donor and acceptor moieties are
linked to the polymer at positions which are at opposite ends of
the probing nucleobase sequence. b. detecting, identifying or
quantitating the hybridization of the polymer to the target
sequence, under suitable hybridization conditions, wherein the
presence, absence or amount of target sequence present in the
sample can be correlated with a change in detectable signal
associated with at least one donor or acceptor moiety of the
polymer.
37. A method for the detection, identification or quantitation of a
target sequence, said method comprising the steps of: a. contacting
the sample with a polymer comprising: (i). a first arm segment
having a first and second end; (ii). a probing nucleobase sequence
having a first and second end wherein the probing nucleobase
sequence is complementary or substantially complementary to the
target sequence; (iii). a second arm segment which is embedded
within the probing nucleobase sequence and which is complementary
or substantially complementary to the first arm segment; (iv). a
flexible linkage which links the second end of the first arm
segment to the second end of the probing nucleobase sequence; (v) a
donor moiety linked to the first end of one of either of the first
arm segment or the probing nucleobase sequence; (vi). an acceptor
moiety linked to the first end of the other of either of the first
arm segment or the probing nucleobase sequence; and b. detecting,
identifying or quantitating the hybridization of the polymer to the
target sequence, under suitable hybridization conditions, wherein
the presence, absence or amount of target sequence present in the
sample can be correlated with a change in detectable signal
associated with at least one donor or acceptor moiety of the
polymer.
38. The method of claim 37, wherein the method is used to detect
target sequence in a closed tube (homogeneous) assay.
39. The method of claim 38, wherein the method is used to detect a
nucleic acid comprising a target sequence wherein said nucleic acid
has been synthesized or amplified in a reaction occurring in the
closed tube (homogeneous) assay.
40. The method of claim 37, wherein the method is used to detect a
target sequence in a living cell or tissue.
41. The method of claim 37, wherein the sample is contacted with
one or more blocking probes to thereby suppress the binding of the
polymer to a non-target sequence.
42. The method of claim 37, wherein the method is used to detect,
identify, or quantitate the presence or amount of an organism or
virus in the sample.
43. The method of claim 37, wherein the method is used to detect,
identify, or quantitate the presence or amount of one or more
species of an organism in the sample.
44. The method of claim 37, wherein the method is used to determine
the effect of antimicrobial agents on the growth of one or more
microorganisms in the sample.
45. The method of claim 37, wherein the method is used to determine
the presence or amount of a taxonomic group of organisms in the
sample.
46. The method of claim 37, wherein the method is used to diagnose
a condition of medical interest.
47. The method of claim 37, wherein the target sequence is
immobilized to a surface.
48. The method of claim 37, wherein the polymer is immobilized to a
surface.
49. The method of claim 48, wherein the polymer is one of many
polymers which are immobilized on an array to which the sample is
contacted.
50. A method for the detection, identification or quantitation of a
target sequence in a sample, said method comprising the steps of:
a. contacting the sample with a polymer comprising: (i). a probing
nucleobase sequence having a first and second end wherein
nucleobase sequence is complementary or substantially complementary
to the target sequence; (ii). a first arm segment comprising a
first and second end; (iii). a second arm segment comprising a
first and second end, wherein, at least a portion of the
nucleobases of the second arm segment are complementary to the
nucleobase sequence of the first arm segment; (iv). a first
flexible linkage which links the second end of the first arm
segment to either of the first or second end of the probing
nucleobase sequence; (v). a second linkage which 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; (vi). a donor
moiety linked to the first end of one of either of the first or
second arm segments; and (vii). an acceptor moiety linked to the
first end of the other of either of the first or the second arm
segments; and b. detecting, identifying or quantitating the
hybridization of the polymer to the target sequence, under suitable
hybridization conditions, wherein the presence, absence or amount
of target sequence present in the sample can be correlated with a
change in detectable signal associated with at least one donor or
acceptor moiety of the polymer.
51. The method of claim 50, wherein the second linkage is a single
bond.
52. The method of claim 50, wherein the second linkage is a second
flexible linkage.
53. The method of claim 50, wherein the method is used to detect
target sequence in a closed tube (homogeneous) assay.
54. The method of claim 53, wherein the method is used to detect a
nucleic acid comprising a target sequence wherein said nucleic acid
has been synthesized or amplified in a reaction occurring in the
closed tube (homogeneous) assay.
55. The method of claim 50, wherein the method is used to detect a
target sequence in a living cell or tissue.
56. The method of claim 50, wherein the sample is contacted with
one or more blocking probes to thereby suppress the binding of the
polymer to a non-target sequence.
57. The method of claim 50, wherein the method is used to detect,
identify, or quantitate the presence or amount of an organism or
virus in the sample.
58. The method of claim 50, wherein the method is used to detect,
identify, or quantitate the presence or amount of one or more
species of an organism in the sample.
59. The method of claim 50, wherein the method is used to determine
the effect of antimicrobial agents on the growth of one or more
microorganisms in the sample.
60. The method of claim 50, wherein the method is used to determine
the presence or amount of a taxonomic group of organisms in the
sample.
61. The method of claim 50, wherein the method is used to diagnose
a condition of medical interest.
62. The method of claim 50, wherein the target sequence is
immobilized to a support.
63. The method of claim 50, wherein the polymer is immobilized to a
support.
64. The method of claim 64, wherein the polymer is one of many
polymers which are immobilized on an array to which the sample of
interest is contacted.
65. An array comprising at least two support bound polymers
suitable for detecting, identifying or quantitating a target
sequence, wherein each polymer comprises; a. a first arm segment of
PNA subunits having a first and second end; b. a probing nucleobase
sequence of PNA subunits having a first and second end wherein the
probing nucleobase sequence is complementary or substantially
complementary to the target sequence; c. a second arm segment of
PNA subunits which is embedded within the probing nucleobase
sequence and is complementary or substantially complementary to the
first arm segment; d. a flexible linkage which links the second end
of the first arm segment to the second end of the probing
nucleobase sequence; e. a donor moiety linked to the first end of
one of either of the first arm segment or the probing nucleobase
sequence; and f. an acceptor moiety linked to the first end of the
other of either of the first arm segment or the probing nucleobase
sequence.
66. An array comprising at least two support bound polymers
suitable for detecting, identifying or quantitating a target
sequence, each polymer comprising; a. a probing nucleobase sequence
having a first and second end, wherein the probing nucleobase
sequence is complementary or substantially complementary to the
target sequence; b. a first arm segment comprising a first and
second end; c. a second arm segment comprising a first and second
end, wherein, at least a portion of the nucleobases of the second
arm segment are complementary to the nucleobase sequence of the
first arm segment; d. a first flexible linkage which 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
which 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 linked to the first end of one of
either of the first or second arm segments; and g. an acceptor
moiety linked to the first end of the other of either of the first
or the second arm segments.
67. A kit suitable for performing an assay which detects the
presence, absence or amount of target sequence in a sample, wherein
said kit comprises: a. at least one polymer having: (i). a probing
nucleobase sequence; (ii). a first arm segment and a second arm
segment, wherein at least one of the first or second arm segments
is linked to the probing nucleobase sequence through a flexible
linkage; (iii). at least one linked donor moiety and at least one
linked acceptor moiety, wherein said donor and acceptor moieties
are linked to the polymer at positions which are at opposite ends
of the probing nucleobase sequence. b. other reagents or
compositions necessary to perform the assay.
68. The kit of claim 67, wherein the kit is used to detect
organisms in food, beverages, water, pharmaceutical products,
personal care products, dairy products or environmental
samples.
69. The kit of claim 67, wherein the kit is used to examine
clinical samples such as clinical specimens or equipment, fixtures
and products used to treat humans or animals.
Description
CROSS REFERENCE TO RELATED APPLICATIONS:
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/958532 filed on Oct. 27, 1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is related to the field of probe-based
nucleic acid sequence detection, analysis and quantitation. More
specifically, this invention relates to novel compositions and
methods pertaining to PNA Molecular Beacons.
[0004] 2. Description of the Related Art
[0005] Quenching of fluorescence signal can occur by either
Fluorescence Resonance Energy Transfer "FRET" (also known as
non-radiative energy transfer: See: Yaron et al., Analytical
Biochemistry 95: 228-235 (1979) at p. 232, col. 1, lns. 32-39) or
by non-FRET interactions (also known as radiationless energy
transfer; See: Yaron et al., Analytical Biochemistry 95 at p. 229,
col. 2, lns. 7-13). The critical distinguishing factor between FRET
and non-FRET quenching is that non-FRET quenching requires short
range interaction by "collision" or "contact" and therefore
requires no spectral overlap between the moieties of the donor and
acceptor pair (See: Yaron et al., Analytical Biochemistry 95 at p.
229, col. 1, lns. 22-42). Conversely, FRET quenching requires
spectral overlap between the donor and acceptor moieties and the
efficiency of quenching is directly proportional to the distance
between the donor and acceptor moieties of the FRET pair (See:
Yaron et al., Analytical Biochemistry 95 at p. 232, col. 1, ln. 46
to col. 2, ln. 29). Extensive reviews of the FRET phenomenon are
described in Clegg, R. M., Methods Enzymol., 221: 353-388 (1992)
and Selvin, P. R., Methods Enzymol., 246: 300-334 (1995). Yaron et
al. also suggested that the principles described therein might be
applied to the hydrolysis of oligonucleotides (See: Yaron et al.,
Analytical Biochemistry 95 at p. 234, col. 2, lns. 14-18).
[0006] The FRET phenomenon has been utilized for the direct
detection of nucleic acid target sequences without the requirement
that labeled nucleic acid hybridization probes or primers be
separated from the hybridization complex prior to detection (See:
Livak et al. U.S. Pat. No. 5,538,848). One method utilizing FRET to
analyze Polymerase Chain Reaction (PCR) amplified nucleic acid in a
closed tube format is commercially available from Perkin Elmer. The
TaqMan.TM. assay utilizes a nucleic acid hybridization probe which
is labeled with a fluorescent reporter and a quencher moiety in a
configuration which results in quenching of fluorescence in the
intact probe. During the PCR amplification, the probe sequence
specifically hybridizes to the amplified nucleic acid. When
hybridized, the exonuclease activity of the Taq polymerase degrades
the probe thereby eliminating the intramolecular quenching
maintained by the intact probe. Because the probe is designed to
hybridize specifically to the amplified nucleic acid, the increase
in fluorescence intensity of the sample, caused by enzymatic
degradation of the probe, can be correlated with the activity of
the amplification process.
[0007] Nonetheless, this method preferably requires that each of
the fluorophore and quencher moieties be located on the 3' and 5'
termini of the probe so that the optimal signal to noise ratio is
achieved (See: Nazarenko et al., Nucl. Acids Res. 25: 2516-2521
(1997) at p. 2516, col. 2, lns. 27-35). However, this orientation
necessarily results in less than optimal fluorescence quenching
because the fluorophore and quencher moieties are separated in
space and the transfer of energy is most efficient when they are
close. Consequently, the background emission from unhybridized
probe can be quite high in the TaqMan.TM. assay (See: Nazarenko et
al., Nucl. Acids Res. 25: at p. 2516, col. 2, lns. 3640).
[0008] The nucleic acid Molecular Beacon is another construct which
utilizes the FRET phenomenon to detect target nucleic acid
sequences (See: Tyagi et al. Nature Biotechnology, 14: 303-308
(1996). A nucleic acid Molecular Beacon comprises a probing
sequence embedded within two complementary arm sequences (See:
Tyagi et al, Nature Biotechnology, 14: at p. 303, col. 1, lns.
22-30). To each termini of the probing sequence is attached one of
either a fluorophore or quencher moiety. In the absence of the
nucleic acid target, the arm sequences anneal to each other to
thereby form a loop and hairpin stem structure which brings the
fluorophore and quencher together (See: Tyagi et al., Nature
Biotechnology, 14: at p. 304, col. 2, lns. 14-25). When contacted
with target nucleic acid, the complementary probing sequence and
target sequence will hybridize. Because the hairpin stem cannot
coexist with the rigid double helix that is formed upon
hybridization, the resulting conformational change forces the arm
sequences apart and causes the fluorophore and quencher to be
separated (See: Tyagi et al. Nature Biotechnology, 14: at p. 303,
col. 2, lns. 1-17). When the fluorophore and quencher are
separated, energy of the donor fluorophore does not transfer to the
acceptor moiety and the fluorescent signal is then detectable.
Since unhybridized "Molecular Beacons" are non-fluorescent, it is
not necessary that any excess probe be removed from an assay.
Consequently, Tyagi et al. state that Molecular Beacons can be used
for the detection of target nucleic acids in a homogeneous assay
and in living cells. (See: Tyagi et al., Nature Biotechnology, 14:
at p. 303, col. 2; lns. 15-77).
[0009] The arm sequences of the disclosed nucleic acid Molecular
Beacon constructs are unrelated to the probing sequence (See: Tyagi
et al., Nature Biotechnology, 14: at p. 303, col. 1; ln. 30).
Because the Tyagi et al. Molecular Beacons comprise nucleic acid
molecules, proper stem formation and stability is dependent upon
the length of the stem, the G:C content of the arm sequences, the
concentration of salt in which it is dissolved and the presence or
absence of magnesium in which the probe is dissolved (See: Tyagi et
al., Nature Biotechnology, 14: at p. 305, col. 1; lns. 1-16).
Furthermore, the Tyagi et al. nucleic acid Molecular Beacons are
susceptible to degradation by endonucleases and exonucleases.
[0010] Upon probe degradation, background fluorescent signal will
increase since the donor and acceptor moieties are no longer held
in close proximity. Therefore, assays utilizing enzymes known to
have nuclease activity, will exhibit a continuous increase in
background fluorescence as the nucleic acid Molecular Beacon is
degraded (See: FIG. 7 in Tyagi et al: the data associated with
(.smallcircle.) and (.quadrature.) demonstrates that the
fluorescent background, presumably caused by probe degradation,
increases with each amplification cycle.) Additionally, Molecular
Beacons will also, at least partially, be degraded in living cells
because cells contain active nuclease activity.
[0011] The constructs described by Tyagi et al. are more broadly
described in WO95/13399 (hereinafter referred to as "Tyagi2 et al."
except that Tyagi2 et al. also discloses that the nucleic acid
Molecular Beacon may also be bimolecular wherein they define
bimolecular as being unitary probes of the invention comprising two
molecules (e.g. oligonucleotides) wherein half, or roughly half, of
the target complement sequence, one member of the affinity pair and
one member of the label pair are present in each molecule (See:
Tyagi2 et al., p. 8, ln. 25 to p. 9, ln. 3). However, Tyagi2 et al.
specifically states that in designing a unitary probe for use in a
PCR reaction, one would naturally choose a target complement
sequence that is not complementary to one of the PCR primers (See:
Tyagi2 et al., p. 41, ln. 27). Assays of the invention include
real-time and end point detection of specific single-stranded or
double stranded products of nucleic acid synthesis reactions,
provided however that if unitary probes will be subjected to
melting or other denaturation, the probes must be unimolecular
(See: Tyagi2 et al., p. 37, lns. 1-9). Furthermore, Tyagi2 et al.
stipulate that although the unitary probes of the invention may be
used with amplification or other nucleic acid synthesis reactions,
bimolecular probes (as defined in Tyagi2 et al.) are not suitable
for use in any reaction (e.g. PCR) wherein the affinity pair would
be separated in a target-independent manner (See: Tyagi2 et al., p.
13, lns. 9-12). Neither Tyagi et al. nor Tyagi2 et al. disclose,
suggest or teach anything about PNA.
[0012] In a more recent disclosure, modified hairpin constructs
which are similar to the Tyagi et al. nucleic acid Molecular
Beacons, but which are suitable as primers for polymerase
extension, have been disclosed (See: Nazarenko et al., Nucleic
Acids Res. 25: 2516-2521(1997)). A method suitable for the direct
detection of PCR-amplified DNA in a closed system is also
disclosed. According to the method, the Nazarenko et al. primer
constructs are, by operation of the PCR process, incorporated into
the amplification product. Incorporation into a PCR amplified
product results in a change in configuration which separates the
donor and acceptor moieties. Consequently, increases in the
intensity of the fluorescent signal in the assay can be directly
correlated with the amount of primer incorporated into the PCR
amplified product. The authors conclude, this method is
particularly well suited to the analysis of PCR amplified nucleic
acid in a closed tube format.
[0013] Because they are nucleic acids, the Nazarenko et al. primer
constructs are admittedly subject to nuclease digestion thereby
causing an increase in background signal during the PCR process
(See: Nazarenko et al., Nucleic Acids Res. 25: at p. 2519, col. 1;
lns. 28-39). An additional disadvantage of this method is that the
Molecular Beacon like primer constructs must be linearized during
amplification (See: Nazarenko et al., Nucleic Acids Res. 25: at p.
2519, col. 1, lns. 7-8). Consequently, the polymerase must read
through and dissociate the stem of the hairpin modified Molecular
Beacon like primer construct if fluorescent signal is to be
generated. Nazarenko et al. does not suggest, teach or disclose
anything about PNA.
[0014] In still another application of FRET to target nucleic acid
sequence detection, doubly labeled fluorescent oligonucleotide
probes which have been rendered impervious to exonuclease digestion
have also been used to detect target nucleic acid sequences in PCR
reactions and in-situ PCR (See: Mayrand, U.S. Pat. No. 5,691,146).
The oligonucleotide probes of Mayrand comprise a fluorescer
(reporter) molecule attached to a first end of the oligonucleotide
and a quencher molecule attached to the opposite end of the
oligonucleotide (See: Mayrand, Abstract). Mayrand suggests that the
prior art teaches that the distance between the fluorophore and
quencher is an important feature which must be minimized and
consequently the preferred spacing between the reporter and
quencher moieties of a DNA probe should be 6-16 nucleotides (See:
col. 7, lns. 8-24). Mayrand, however teaches that the reporter
molecule and quencher moieties are preferably located at a distance
of 18 nucleotides (See: col. 3, Ins 35-36) or 20 bases (See: col.
7, lns. 25-46) to achieve the optimal signal to noise ratio.
Consequently, both Mayrand and the prior art cited therein teach
that the detectable properties of nucleic acid probes (DNA or RNA)
comprising a fluorophore and quencher exhibit a strong dependence
on probe length.
[0015] Resistance to nuclease digestion is also an important aspect
of the invention (See: U.S. Pat. No. 5,691,146 at col. 6, lns.
42-64) and therefore, Mayrand suggests that the 5' end of the
oligonucleotide may be rendered impervious to nuclease digestion by
including one or more modified internucleotide linkages onto the 5'
end of the oligonucleotide probe (See: U.S. Pat. No. 5,691,146 at
col. 6, lns. 45-50). Furthermore, Mayrand suggests that a polyamide
nucleic acid (PNA) or peptide can be used as a nuclease resistant
linkage to thereby modify the 5' end of the oligonucleotide probe
of the invention and render it impervious to nuclease digestion
(See: U.S. Pat. No. 5,691,146 at col. 6, lns. 53-64). Mayrand does
not however, disclose, suggest or teach that a PNA probe construct
might be a suitable substitute for the practice of the invention
despite having obvious knowledge of its existence. Furthermore,
Mayrand does not teach one of skill in the art how to prepare
and/or label a PNA with the fluorescer or quencher moieties.
[0016] The efficiency of energy transfer between donor and acceptor
moieties as they can be influenced by oligonucleotide length
(distance) has been further examined and particularly applied to
fluorescent nucleic acid sequencing applications (See: Mathies et
al., U.S. Pat. No. 5,707,804). Mathies et al. states that two
fluorophores will be joined by a backbone or chain where the
distance between the two fluorophores may be varied (See: U.S. Pat.
No. 5,707,804 at col. 4, lns. 1-3). Thus, the distance must be
chosen to provide energy transfer from the donor to the acceptor
through the well-known Foerster mechanism (See: U.S. Pat. No.
5,707,804 at col. 4, lns. 7-9). Preferably about 3-10 nucleosides
separate the fluorophores of a single stranded nucleic acid (See:
U.S. Pat. No. 5,707,804 at col. 7, lns. 21-25). Mathies et al. does
not suggest, teach or disclose anything about PNA.
[0017] From the analysis of DNA duplexes is has been observed that:
1: the efficiency of FET (or FRET as defined herein) appears to
depend somehow on the nucleobase sequence of the oligonucleotide;
2: donor fluorescence changes in a manner which suggests that
dye-DNA interactions affect the efficiency of FET; and 3: the
Forster equation does not quantitatively account for observed
energy transfer and therefore the length between donor and acceptor
moieties attached to oligonucleotides cannot be quantitated, though
it can be used qualitatively (See: Promisel et al., Biochemistry,
29: 9261-9268 (1990). Promisel et al. suggest that non-Forster
effects may account for some of their observed but otherwise
unexplainable results (See: Promisel et al., Biochemistry, 29: at
p. 9267, col. 1, ln. 43 to p. 9268, col. 1, ln. 13). The results of
Promisel et al. suggest that the FRET phenomena when utilized in
nucleic acids in not entirely predictable or well understood.
Promisel et al. does not suggest, teach or disclose anything about
PNA and, in fact, the manuscript predates the invention of PNA.
[0018] The background art thus far discussed does not disclose,
suggest or teach anything about PNA oligomers to which are directly
attached a pair of donor and acceptor moieties. In fact, the FRET
phenomenon as applied to the detection of nucleic acids, appears to
be confined to the preparation of constructs in which the portion
of the probe which is complementary to the target nucleic acid
sequence is itself comprised solely of nucleic acid.
[0019] FRET has also been utilized within the field of peptides.
(See: Yaron et al. Analytical Biochemistry 95 at p. 232, col. 2,
ln. 30 to p. 234, col. 1, ln. 30). Indeed, the use of suitably
labeled peptides as enzyme substrates appears to be the primary
utility for peptides which are labeled with donor and acceptor
pairs (See: Zimmerman et al., Analytical Biochemistry, 70: 258-262
(1976), Carmel et al., Eur. J. Biochem., 73: 617-625 (1977), Ng et
al., Analytical Biochemistry, 183: 50-56 (1989), Wang et al., Tett.
Lett., 31: 6493-6496 (1990) and Meldal et al., Analytical
Biochemistry, 195: 141-147 (1991). Early work suggested that
quenching efficiency of the donor and acceptor pair was dependent
on peptide length (See: Yaron et al., Analytical Biochemistry 95 at
p. 233, col. 2, lns. 36-40). However, the later work has suggested
that efficient quenching was not so dependent on peptide length
(See: Ng et al., Analytical Biochemistry, 183: at p. 54, col. 2, In
23 to p. 55, col. 1, ln. 12; Wang et al., Tett. Lett., 31 wherein
the peptide is eight amino acids in length; and Meldal et al.
Analytical Biochemistry, 195 at p. 144, col. 1, lns. 33-37). It was
suggested by Ng et al. that the observed quenching in long peptides
might occur by an as yet undetermined mechanism (See: Ng et al.,
Analytical Biochemistry 183 at p. 55, col. 1, ln 13 to col. 2, ln
7.)
[0020] Despite its name, peptide nucleic acid (PNA) is neither a
peptide. a nucleic acid nor is it even an acid. Peptide Nucleic
Acid (PNA) is a non-naturally occurring polyamide (pseudopeptide)
which can hybridize to nucleic acid (DNA and RNA) with sequence
specificity (See U.S. Pat. No. 5,539,082 and Egholm et al., Nature
365: 566-568 (1993)). PNAs are synthesized by adaptation of
standard peptide synthesis procedures in a format which is now
commercially available. (For a general review of the preparation of
PNA monomers and oligomers please see: Dueholm et al., New J.
Chem., 21: 19-31 (1997) or Hyrup et. al., Bioorganic & Med.
Chem. 4: 5-23 (1996)). Alternatively, labeled and unlabeled PNA
oligomers can be purchased (See: PerSeptive Biosystems Promotional
Literature: BioConcepts, Publication No. NL612, Practical PNA,
Review and Practical PNA, Vol. 1, Iss. 2)
[0021] Being non-naturally occurring molecules, PNAs are not known
to be substrates for the enzymes which are known to degrade
peptides or nucleic acids. Therefore, PNAs should be stable in
biological samples, as well as have a long shelf-life. Unlike
nucleic acid hybridization which is very dependent on ionic
strength, the hybridization of a PNA with a nucleic acid is fairly
independent of ionic strength and is favored at low ionic strength,
conditions which strongly disfavor the hybridization of nucleic
acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect
of ionic strength on the stability and conformation of PNA
complexes has been extensively investigated (Tomac et al., J. Am.
Chem. Soc. 118: 5544-5552 (1996)). Sequence discrimination is more
efficient for PNA recognizing DNA than for DNA recognizing DNA
(Egholm et al., Nature, at p. 566). However, the advantages in
point mutation discrimination with PNA probes, as compared with DNA
probes, in a hybridization assay appears to be somewhat sequence
dependent (Nielsen et al., Anti-Cancer Drug Design 8: 53-65,
(1993)). As an additional advantage, PNAs hybridize to nucleic acid
in both a parallel and antiparallel orientation, though the
antiparallel orientation is preferred (See: Egholm et al., Nature
at p. 566).
[0022] Despite the ability to hybridize to nucleic acid in a
sequence specific manner, there are many differences between PNA
probes and standard nucleic acid probes. These differences can be
conveniently broken down into biological, structural, and
physico-chemical differences. As discussed in more detail below,
these biological, structural, and physico-chemical differences may
lead to unpredictable results when attempting to use PNA probes in
applications were nucleic acids have typically been employed. This
non-equivalency of differing compositions is often observed in the
chemical arts.
[0023] With regard to biological differences, nucleic acids, are
biological materials that play a central role in the life of living
species as agents of genetic transmission and expression. Their in
vivo properties are fairly well understood. PNA, on the other hand
is recently developed totally artificial molecule, conceived in the
minds of chemists and made using synthetic organic chemistry. It
has no known biological function (i.e. native (unmodified) PNA is
not known to be a substrate for any polymerase, ligase, nuclease or
protease).
[0024] Structurally, PNA also differs dramatically from nucleic
acid. Although both can employ common nucleobases (A, C, G, T, and
U), the backbones of these molecules are structurally diverse. The
backbones of RNA and DNA are composed of repeating phosphodiester
ribose and 2-deoxyribose units. In contrast, the backbones of the
most common PNAs are composed on N-[2-(aminoethyl)]glycine
subunits. Additionally, in PNA the nucleobases are connected to the
backbone by an additional methylene carbonyl moiety.
[0025] PNA is not an acid and therefore contains no charged acidic
groups such as those present in DNA and RNA. Because they lack
formal charge, PNAs are generally more hydrophobic than their
equivalent nucleic acid molecules. The hydrophobic character of PNA
allows for the possibility of non-specific (hydrophobic/hydrophobic
interactions) interactions not observed with nucleic acids.
Further, PNA is achiral, providing it with the capability of
adopting structural conformations the equivalent of which do not
exist in the RNA/DNA realm.
[0026] The unique structural features of PNA result in a polymer
which is highly organized in solution, particularly for purine rich
polymers (See: Dueholm et al., New J. Chem., 21: 19-31 (1997) at p.
27, col. 2, lns. 6-30). Conversely, a single stranded nucleic acid
is a random coil which exhibits very little secondary structure.
Because PNA is highly organized, PNA should be more resistant to
adopting alternative secondary structures (e.g. a hairpin stem
and/or loop).
[0027] The physico/chemical differences between PNA and DNA or RNA
are also substantial. PNA binds to its complementary nucleic acid
more rapidly than nucleic acid probes bind to the same target
sequence. This behavior is believed to be, at least partially, due
to the fact that PNA lacks charge on its backbone. Additionally,
recent publications demonstrate that the incorporation of
positively charged groups into PNAs will improve the kinetics of
hybridization (See: Iyer et al., J. Biol. Chem. 270:14712-14717
(1995)). Because it lacks charge on the backbone, the stability of
the PNA/nucleic acid complex is higher than that of an analogous
DNA/DNA or RNA/DNA complex. In certain situations, PNA will form
highly stable triple helical complexes through a process called
"strand displacement". No equivalent strand displacement processes
or structures are known in the DNA/RNA world.
[0028] Recently, the "Hybridization based screening on peptide
nucleic acid (PNA) oligomer arrays" has been described wherein
arrays of some 1000 PNA oligomers of individual sequence were
synthesized on polymer membranes (See: Weiler et al., Nucl. Acids
Res. 25: 2792-2799(1997)). Arrays are generally used, in a single
assay, to generate affinity binding (hybridization) information
about a specific sequence or sample to numerous probes of defined
composition. Thus, PNA arrays may be useful in diagnostic
applications or for screening libraries of compounds for leads
which might exhibit therapeutic utility. However, Weiler et al.
note that the affinity and specificity of DNA hybridization to
immobilized PNA oligomers depended on hybridization conditions more
than was expected. Moreover, there was a tendency toward
non-specific binding at lower ionic strength. Furthermore, certain
very strong binding mismatches were identified which could not be
eliminated by more stringent washing conditions. These unexpected
results are illustrative of the lack of complete understanding of
these newly discovered molecules (i.e. PNA).
[0029] In summary, because PNAs hybridize to nucleic acids with
sequence specificity, PNAs are useful candidates for investigation
as substitute probes when developing probe-based hybridization
assays. However, PNA probes are not the equivalent of nucleic acid
probes in both structure or function. Consequently, the unique
biological, structural, and physico-chemical properties of PNA
requires that experimentation be performed to thereby examine
whether PNAs are suitable in applications where nucleic acid probes
are commonly utilized.
SUMMARY OF THE INVENTION
[0030] Tyagi et al. and Tyagi2 et al. disclose nucleic acid
Molecular Beacons which comprise a hairpin loop and stem to which
energy transfer donor and acceptor moieties are linked at opposite
ends of the nucleic acid polymer. Numerous PNA polymers were
examined in an attempt to prepare a PNA Molecular Beacon. The
applicant's have determined that all probes they examined, which
contained linked donor and acceptor moieties exhibited a low
inherent noise (background) and an increase in detectable signal
upon binding of the probe to a target sequence. Very surprisingly,
these characteristic properties of a nucleic acid Molecular Beacon
were observed whether or not the PNA oligomer possessed
self-complementary arm segments intended to form a PNA hairpin. For
example, PNA oligomers prepared as control samples which by design
did not possess any self-complementary arm segments suitable for
forming a hairpin exhibited a signal (PNA oligomer bound to target
sequence) to noise (no target sequence present) ratio which was
quite favorable as compared with probes comprising flexible
linkages and self-complementary arm segments.
[0031] Applicant's data further demonstrates that flexible linkages
inserted within the probe and shorter self-complementary arm
segments are a preferred embodiment since the signal to noise ratio
of probes of this embodiment compare well with the signal to noise
ratio published for nucleic acid hairpins (approximately 25 to 1).
The data compiled by applicants is inconclusive with respect to
whether or not the PNA Molecular Beacons they prepared which have
shorter arms segments (2-5 subunits in length) and one or more
flexible linkages exist as hairpins. However, applicant's data
demonstrates that probes with longer arm segments (e.g. 9 subunits)
do form a hairpin (See: Example 19 of this specification) and
unlabeled probes having arms segments as short as six subunits do
not exist primarily as a hairpin (See: Example 19 of this
specification). Furthermore, the signal to noise ratio for those
the probes having longer arm segments suitable for forming a
hairpin exhibited very poor a signal to noise ratios upon melting
of the hairpin or when in the presence of a complementary nucleic
acid. Consequently, embodiments having longer arm segments (e.g. 6
or more subunits) do not appear to be well suited for use in the
detection of nucleic acid targets.
[0032] The data compiled by applicant's demonstrates the
non-equivalence of structure and function of PNA as compared with
nucleic acids. Consequently, this invention pertains to methods,
kits and compositions pertaining to PNA Molecular Beacons. Though
we refer to the probes of this invention as PNA Molecular Beacons,
we do not mean to imply that they exist as hairpins since they may
well exist as aggregates, bimolecular constructs or as higher order
hybrids (e.g. multimers). Regardless of the nature of the secondary
structure, a PNA Molecular Beacon efficiently transfers energy
between donor and acceptor moieties linked to the probe in the
absence of target sequence. Upon hybridization of the probing
nucleobase sequence to a target sequence, the efficiency of energy
transfer between donor and acceptor moieties of a PNA Molecular
Beacon is altered such that detectable signal from at least one
linked moiety can be used to monitor or quantitate the occurrence
of the hybridization event.
[0033] At a minimum a PNA Molecular Beacon comprises a probing
nucleobase sequence, two arm segments, wherein at least one arm
segment is linked to the probe through a flexible linkage, at least
one linked donor moiety and at least one linked acceptor moiety.
The donor and acceptor moieties can be linked at any position
within the PNA Molecular Beacon provided that the point of
attachment of donor and acceptor moieties of a set are located at
opposite ends of the probing nucleobase sequence.
[0034] The probing nucleobase sequence is designed to hybridize to
at least a portion of a target sequence. The first and second arm
segments of the PNA Molecular Beacon provide for intramolecular or
intermolecular interactions which stabilize secondary structures,
dimers and/or multimers which when formed stabilize the rate of
energy transfer between donor and acceptor moieties of the
unhybridized PNA Molecular Beacon. Without intending to be bound to
this hypothesis, it is believed that the flexible linkages provide
flexibility and randomness to the otherwise highly structured PNA
oligomer thereby resulting in more efficient energy transfer of the
linked donor and acceptor moieties of the unhybridized PNA
Molecular Beacon as compared with probes of similar nucleobase
sequence which do not comprise flexible linkages.
[0035] In one preferred embodiment, this invention is directed to
PNA Molecular Beacons comprising an arm segment having a first and
second end. Additionally, there is also a probing nucleobase
sequence having a first and second end wherein, the probing
nucleobase sequence is complementary or substantially complementary
to the target sequence. There is also a second arm segment which is
embedded within the probing nucleobase sequence and is
complementary or substantially complementary to the first arm
segment. The polymer also comprises a flexible linkage which links
the second end of the first arm segment to the second end of the
probing nucleobase sequence. A donor moiety is linked to the first
end of one of either of the first arm segment or the probing
nucleobase sequence; and an acceptor moiety is linked to the first
end of the other of either of the first arm segment or the probing
nucleobase sequence.
[0036] In still another preferred embodiment, this invention is
directed to PNA Molecular Beacons comprising a probing nucleobase
sequence having a first and second end, wherein, the probing
nucleobase sequence is complementary or substantially complementary
to the target sequence. There is also a first arm segment
comprising a first and second end and a second arm segment
comprising a first and second end, wherein, at least a portion of
the nucleobases of the second arm segment are complementary to the
nucleobase sequence to the first arm segment. The polymer also
comprises a first flexible linkage which links the second end of
the first arm segment to either of the first or second end of the
probing nucleobase sequence. There is a second linkage which 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. A donor
moiety is linked to the first end of one of either of the first or
second arm segments; and an acceptor moiety is linked to the first
end of the other of either of the first or the second arm
segments.
[0037] In one preferred embodiment, this invention is related to a
method for the detection, identification or quantitation of a
target sequence in a sample. The method comprises contacting the
sample with a PNA Molecular Beacon and then detecting, identifying
or quantitating the change in detectable signal associated with at
least one donor or acceptor moiety of the probe whereby the change
in detectable signal is used to determine the presence, absence or
amount of target sequence present in the sample of interest. The
measurable change in detectable signal of at least one donor or
acceptor moiety of the probe can be used to determine the presence,
absence or amount of target sequence present in the sample of
interest since applicant's have demonstrated that the efficiency of
energy transfer between donor and acceptor moieties is altered by
hybridization of the PNA Molecular Beacon to the intended target
sequence, under suitable hybridization conditions. Accurate
quantitation can be achieved by correcting for signal generated by
any unhybridized PNA Molecular Beacon. Consequently, the PNA
Molecular Beacons of this invention are particularly well suited
for the detection, identification or quantitation of target
sequences in closed tube assays. Because PNAs are not known to be
degraded by enzymes, PNA Molecular Beacons are also particularly
well suited for detection, identification or quantitation of target
sequences in cells, tissues or organisms, whether living or
not.
[0038] In still another embodiment, this invention is related to
kits suitable for performing an assay which detects the presence,
absence or number of a target sequences in a sample. The kits of
this invention comprise one or more PNA Molecular Beacons and other
reagents or compositions which are selected to perform an assay or
otherwise simplify the performance of an assay.
[0039] In yet another embodiment, this invention is also directed
to an array comprising two or more support bound PNA Molecular
Beacons suitable for detecting, identifying or quantitating a
target sequence of interest. Arrays of PNA Molecular Beacons are
convenient because they provide a means to rapidly interrogate
numerous samples for the presence of one or more target sequences
of interest in real time without using a secondary detection
system.
[0040] The methods, kits and compositions of this invention are
particularly useful for the detection of target sequences of
organisms which may be found in food, beverages, water,
pharmaceutical products, personal care products, dairy products or
environmental samples. The analysis of preferred beverages include
soda, bottled water, fruit juice, beer, wine or liquor products.
Additionally, the methods, kits and compositions will be
particularly useful for the analysis of raw materials, equipment,
products or processes used to manufacture or store food, beverages,
water, pharmaceutical products, personal care products dairy
products or environmental samples.
[0041] Whether support bound or in solution, the methods, kits and
compositions of this invention are particularly useful for the
rapid, sensitive, reliable and versatile detection of target
sequences which are particular to organisms which might be found in
clinical environments. Consequently, the methods, kits and
compositions of this invention will be particularly useful for the
analysis of clinical specimens or equipment, fixtures or products
used to treat humans or animals. For example, the assay may be used
to detect a target sequence which is specific for a genetically
based disease or is specific for a predisposition to a genetically
based disease. Non-limiting examples of diseases include,
.beta.-Thalassemia, sickle cell anemia, Factor-V Leiden, cystic
fibrosis and cancer related targets such as p53, p10, BRC-1 and
BRC-2.
[0042] In still another embodiment, the target sequence may be
related to a chromosomal DNA, wherein the detection, identification
or quantitation of the target sequence can be used in relation to
forensic techniques such as prenatal screening, paternity testing,
identity confirmation or crime investigation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure A is an illustration of several possible hairpin
configurations of a PNA Molecular Beacon.
[0044] FIG. 1 is a graphical illustration of experimental data.
[0045] FIG. 2 is a graphical illustration of experimental data.
[0046] FIG. 3 is a graphical illustration of experimental data.
[0047] FIG. 4A is an overlay of normalized fluorescence vs.
temperature and absorbance vs. temperature plots for a labeled
PNA/PNA bimolecular duplex.
[0048] FIG. 4B is an overlay of normalized fluorescence vs.
temperature and absorbance vs. temperature plots for a labeled
unimolecular PNA probe comprising a flexible linkage.
[0049] FIG. 4C is an overlay of normalized fluorescence vs.
temperature and absorbance vs. temperature plots for a labeled
unimolecular PNA probe which is continuous from the N- to
C-terminus.
[0050] FIG. 5 is a graphical representation of comparative
fluorescent melting signal to noise ratios.
[0051] FIG. 6 is an overlay of normalized absorbance vs.
temperature plots for three similar PNA unimolecular probes.
[0052] FIG. 7A is a graphical illustration of data for PNA probes
which exhibit a Type A Fluorescent Thermal Profile.
[0053] FIGS. 7B1, 7B2 and 7B3 are graphical illustrations of data
for PNA probes which exhibit a Type B Fluorescent Thermal
Profile.
[0054] FIG. 7C is a graphical illustration of data for PNA probes
which exhibit a Type C Fluorescent Thermal Profile.
[0055] FIGS. 8A1, 8A2 and 8A3 are a graphical illustration of data
for PNA probes which exhibit a Type A Hybridization Profile.
[0056] FIG. 8B is a graphical illustration of data for PNA probes
which exhibit a Type B Hybridization Profile.
[0057] FIG. 8C is a graphical illustration of data for PNA probes
which exhibit a Type C Hybridization Profile.
[0058] FIG. 9 is an overlay of normalized fluorescence vs.
temperature and absorbance vs. temperature plots for a the labeled
unimolecular PNA probe 0.001.
[0059] FIG. 10 is a graphical illustration of signal to noise data
obtained by Hybridization analysis of PNA oligomers listed in Table
1.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0060] 1. Definitions:
[0061] a. As used herein, the term "nucleobase" shall include those
naturally occurring and those non-naturally occurring heterocyclic
moieties commonly known to those who utilize nucleic acid
technology or utilize peptide nucleic acid technology to thereby
generate polymers which can sequence specifically bind to nucleic
acids.
[0062] b. As used herein, the term "nucleobase sequence" is any
segment of a polymer which comprises nucleobase containing
subunits. Non-limiting examples of suitable polymers or polymers
segments include oligonucleotides, oligoribonucleotides, peptide
nucleic acids and analogs or chimeras thereof.
[0063] c. As used herein, the term "target sequence" is any
sequence of nucleobases in a polymer which is sought to be
detected. The "target sequence" may comprise the entire polymer or
may be a subsequence of the nucleobase sequence which is unique to
the polymer of interest. Without limitation, the polymer comprising
the "target sequence" may be a nucleic acid, a peptide nucleic
acid, a chimera, a linked polymer, a conjugate or any other polymer
comprising substituents (e.g. nucleobases) to which the PNA
Molecular Beacons of this invention may bind in a sequence specific
manner.
[0064] d. As used herein, the term "peptide nucleic acid" or "PNA"
shall be defined as any oligomer, linked polymer or chimeric
oligomer, comprising two or more PNA subunits (residues), including
any of the compounds referred to or claimed as peptide nucleic
acids in U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,
5,736,336, 5,773,571 or 5,786,571 (all of which are herein
incorporated by reference). The term "Peptide Nucleic Acid" or
"PNA" shall also apply to those nucleic acid mimics described in
the following publications: Diderichsen et al., Tett. Lett.
37:475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7:637-627
(1997); Jordan et al., Bioorg. Med. Chem. Lett. 7:687-690 (1997);
Krotz et al., Tett. Lett. 36:6941-6944 (1995); Lagriffoul et al.,
Bioorg. Med. Chem. Lett. 4:1081-1082 (1994); Lowe et al., J. Chem.
Soc. Perkin Trans. 1, (1997) 1:539-546; Lowe et al., J. Chem. Soc.
Perkin Trans. 11:547-554 (1997); Lowe et al., J. Chem. Soc. Perkin
Trans. 11:555-560 (1997); and Petersen et al., Bioorg. Med. Chem.
Lett. 6:793-796 (1996).
[0065] In preferred embodiments, a PNA is a polymer comprising two
or more PNA subunits of the formula: 1
[0066] wherein, each J is the same or different and is selected
from the group consisting of H, R.sup.1, OR.sup.1, SR.sup.1,
NHR.sup.1, NR.sup.1.sub.2, F, Cl, Br and I. Each K is the same or
different and is selected from the group consisting of O, S, NH and
NR.sup.1. Each R.sup.1 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; --(CJ.sub.2).sub.s-- and a group of the formula;
--(CJ.sub.2).sub.5C(O)--, wherein, J is defined above and each s is
an integer from one to five. The integer t is 1 or 2 and the
integer u is 1 or 2. 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. In the most preferred embodiment,
a PNA subunit consists of a naturally occurring or non-naturally
occurring nucleobase attached to the aza nitrogen of the
N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl
linkage.
[0067] 2. Detailed Description
[0068] I. General:
[0069] PNA Synthesis:
[0070] Methods for the chemical assembly of PNAs are well known
(See: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331,
5,736,336, 5,773,571 or 5,786,571 (all of which are herein
incorporated by reference). Chemicals and instrumentation for the
support bound automated chemical assembly of Peptide Nucleic Acids
are now commercially available. Chemical assembly of a PNA is
analogous to solid phase peptide synthesis, wherein at each cycle
of assembly the oligomer possesses a reactive alkyl amino terminus
which is condensed with the next synthon to be added to the growing
polymer. Because standard peptide chemistry is utilized, natural
and non-natural amino acids are routinely incorporated into a PNA
oligomer. Because a PNA is a polyamide, it has a C-terminus
(carboxyl terminus) and an N-terminus (amino terminus). For the
purposes of the design of a hybridization probe suitable for
antiparallel binding to the target sequence (the preferred
orientation), the N-terminus of the probing nucleobase sequence of
the PNA probe is the equivalent of the 5'-hydroxyl terminus of an
equivalent DNA or RNA oligonucleotide.
[0071] Labels:
[0072] The labels attached to the PNA Molecular Beacons of this
invention comprise a set (hereinafter "Beacon Set(s)") of energy
transfer moieties comprising at least one energy donor and at least
one energy acceptor moiety. Typically, the Beacon Set will include
a single donor moiety and a single acceptor moiety. Nevertheless, a
Beacon Set may contain more than one donor moiety and/or more than
one acceptor moiety. The donor and acceptor moieties operate such
that one or more acceptor moieties accepts energy transferred from
the one or more donor moieties or otherwise quench signal from the
donor moiety or moieties. The energy transfer moieties of this
invention operate by both FRET and non-FRET but preferably do not
involve electron transfer.
[0073] Preferably the donor moiety is a fluorophore. Preferred
fluorophores are derivatives of fluorescein, derivatives of bodipy,
5-(2'-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS),
derivatives of rhodamine, Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, texas red
and its derivatives. Though the previously listed fluorophores
might also operate as acceptors, preferably, the acceptor moiety is
a quencher moiety. Preferably, the quencher moiety is a
non-fluorescent aromatic or heteroaromatic moiety. The preferred
quencher moiety is 4-((-4-(dimethylamino)phenyl)azo) benzoic acid
(dabcyl).
[0074] Transfer of energy may occur through collision of the
closely associated moieties of a Beacon Set or through a
nonradiative process such as fluorescence resonance energy transfer
(FRET). For FRET to occur, transfer of energy between donor and
acceptor moieties of a Beacon Set requires that the moieties be
close in space and that the emission spectrum of a donor(s) have
substantial overlap with the absorption spectrum of the acceptor(s)
(See: Yaron et al. Analytical Biochemistry, 95: 228-235 (1979) and
particularly page 232, col. 1 through page 234, col. 1).
Alternatively, collision mediated (radiationless) energy transfer
may occur between very closely associated donor and acceptor
moieties whether or not the emission spectrum of a donor
moiety(ies) has a substantial overlap with the absorption spectrum
of the acceptor moiety(ies) (See: Yaron et al., Analytical
Biochemistry, 95: 228-235 (1979) and particularly page 229, col. 1
through page 232, col. 1). This process is referred to as
intramolecular collision since it is believed that quenching is
caused by the direct contact of the donor and acceptor moieties
(See: Yaron et al.). As applicant's have demonstrated, the donor
and acceptor moieties attached to the PNA Molecular Beacons of this
invention need not have a substantial overlap between the emission
of the donor moieties and the absorbance of the acceptor moieties.
Without intending to be bound to this hypothesis, this data
suggests that collision or contact operates as the primary mode of
quenching in PNA Molecular Beacons.
[0075] Detecting Energy Transfer:
[0076] Because the efficiency of both collision mediated and
nonradiative transfer of energy between the donor and acceptor
moieties of a Beacon Set is directly dependent on the proximity of
the donor and acceptor moieties, detection of hybrid formation of a
PNA Molecular Beacon with a target sequence can be monitored by
measuring at least one physical property of at least one member of
the Beacon Set which is detectably different when the hybridization
complex is formed as compared with when the PNA Molecular Beacon
exists in the absence of target sequence. We refer to this
phenomenon as the self-indicating property of PNA Molecular
Beacons. This change in detectable signal results from the change
in efficiency of energy transfer between the donor and acceptor
upon hybridization of the PNA Molecular Beacon to a target
sequence. Preferably, the means of detection will involve measuring
fluorescence of a donor or acceptor fluorophore of a Beacon Set.
Most preferably, the Beacon Set will comprise at least one donor
fluorophore and at least one acceptor quencher such that the
fluorescence of the donor fluorophore is will be used to detect,
identify or quantitate hybridization.
[0077] PNA Labeling:
[0078] Chemical labeling of a PNA is analogous to peptide labeling.
Because the synthetic chemistry of assembly is essentially the
same, any method commonly used to label a peptide may be used to
label a PNA. Typically, the N-terminus of the polymer is labeled by
reaction with a moiety having a carboxylic acid group or activated
carboxylic acid group. One or more spacer moieties can optionally
be introduced between the labeling moiety and the probing
nucleobase sequence of the oligomer. Generally, the spacer moiety
is incorporated prior to performing the labeling reaction. However,
the spacer may be embedded within the label and thereby be
incorporated during the labeling reaction.
[0079] Typically the C-terminal end of the probing nucleobase
sequence is labeled by first condensing a labeled moiety with the
support upon which the PNA is to be assembled. Next, the first
synthon of the probing nucleobase sequence can be condensed with
the labeled moiety. Alternatively, one or more spacer moieties can
be introduced between the labeled moiety and the oligomer (e.g.
8-amino-3,6-dioxaoctanoic acid). Once the PNA Molecular Beacon is
completely assembled and labeled, it is cleaved from the support
deprotected and purified using standard methodologies.
[0080] The labeled moiety could be a lysine derivative wherein the
.epsilon.-amino group is modified with a donor or acceptor moiety.
For example the label could be a fluorophore such as
5(6)-carboxyfluorescein or a quencher moiety such as
4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl). Condensation
of the lysine derivative with the synthesis support would be
accomplished using standard condensation (peptide) chemistry. The
.alpha.-amino group of the lysine derivative would then be
deprotected and the probing nucleobase sequence assembly initiated
by condensation of the first PNA synthon with the .alpha.-amino
group of the lysine amino acid. As discussed above, a spacer moiety
could optionally be inserted between the lysine amino acid and the
first PNA synthon by condensing a suitable spacer (e.g.
Fmoc-8-amino-3,6-dioxaoctanoic acid) with the lysine amino acid
prior to condensation of the first PNA synthon of the probing
nucleobase sequence.
[0081] Alternatively, a functional group on the assembled, or
partially assembled, polymer is labeled with a donor or acceptor
moiety while it is still support bound. This method requires that
an appropriate protecting group be incorporated into the oligomer
to thereby yield a reactive functional to which the donor or
acceptor moiety is linked but has the advantage that the label
(e.g. dabcyl or a fluorophore) can be attached to any position
within the polymer including within the probing nucleobase
sequence. For example, the .epsilon.-amino group of a lysine could
be protected with a 4-methyl-triphenylmethyl (Mtt), a
4-methoxy-triphenylmethyl (MMT) or a 4,4'-dimethoxytriphenylmethyl
(DMT) protecting group. The Mtt, MMT or DMT groups can be removed
from PNA (assembled using commercially available Fmoc PNA monomers
and polystyrene support having a PAL linker; PerSeptive Biosystems,
Inc., Framingham, Mass.) by treatment of the resin under mildly
acidic conditions. Consequently, the donor or acceptor moiety can
then be condensed with the .epsilon.-amino group of the lysine
amino acid. After complete assembly and labeling, the polymer is
then cleaved from the support, deprotected and purified using well
known methodologies.
[0082] By still another method, the donor or acceptor moiety is
attached to the polymer after it is fully assembled and cleaved
from the support. This method is preferable where the label is
incompatible with the cleavage, deprotection or purification
regimes commonly used to manufacture the oligomer. By this method,
the PNA will generally be labeled in solution by the reaction of a
functional group on the polymer and a functional group on the
label. Those of ordinary skill in the art will recognize that the
composition of the coupling solution will depend on the nature of
oligomer and the donor or acceptor moiety. The solution may
comprise organic solvent, water or any combination thereof.
Generally, the organic solvent will be a polar non-nucleophilic
solvent. Non limiting examples of suitable organic solvents include
acetonitrile, tetrahydrofuran, dioxane, methyl sulfoxide and
N,N'-dimethylformamide.
[0083] Generally the functional group on the polymer to be labeled
will be an amine and the functional group on the label will be a
carboxylic acid or activated carboxylic acid. Non-limiting examples
of activated carboxylic acid functional groups include
N-hydroxysuccinimidyl esters. In aqueous solutions, the carboxylic
acid group of either of the PNA or label (depending on the nature
of the components chosen) can be activated with a water soluble
carbodiimide. The reagent, 1-(3-dimethylaminopropyl)-
-3-ethylcarbodiimide hydrochloride (EDC), is a commercially
available reagent sold specifically for aqueous amide forming
condensation reactions.
[0084] Generally, the pH of aqueous solutions will be modulated
with a buffer during the condensation reaction. Preferably, the pH
during the condensation is in the range of 4-10. When an arylamine
is condensed with the carboxylic acid, preferably the pH is in the
range of 4-7. When an alkylamine is condensed with a carboxylic
acid, preferably the pH is in the range of 7-10. Generally, the
basicity of non-aqueous reactions will be modulated by the addition
of non-nucleophilic organic bases. Non-limiting examples of
suitable bases include N-methylmorpholine, triethylamine and
N,N-diisopropylethylamine. Alternatively, the pH is modulated using
biological buffers such as N-[2-hydroxyethyl]piperazine-N-
'-[2-ethanesulfonic acid (HEPES) or 4-morpholineethane-sulfonic
acid (MES) or inorganic buffers such as sodium bicarbonate.
[0085] Spacer/Flexible Linker Moieties:
[0086] Spacers are typically used to minimize the adverse effects
that bulky labeling reagents might have on hybridization properties
of PNA Molecular Beacons. Flexible linkers typically induce
flexibility and randomness into the PNA Molecular Beacon or
otherwise link two or more nucleobase sequences of a probe.
Preferred spacer/flexible linker moieties for probes of this
invention consist of one or more aminoalkyl carboxylic acids (e.g.
aminocaproic acid) the side chain of an amino acid (e.g. the side
chain of lysine or ornithine) natural amino acids (e.g. glycine),
aminooxyalkylacids (e.g. 8-amino-3,6-dioxaoctanoic acid), alkyl
diacids (e.g. succinic acid) or alkyloxy diacids (e.g. diglycolic
acid). The spacer/linker moieties may also be designed to enhance
the solubility of the PNA Molecular Beacon.
[0087] Preferably, a spacer/linker moiety comprises one or more
linked compounds having the formula:
--Y--(O.sub.m--(CW.sub.2).sub.n).sub.o--Z--- . The group Y is a
single bond or a group having the formula selected from the group
consisting of: --(CW.sub.2).sub.p--,--C(O)(CW.sub.2).sub.p--,
--C(S)(CW.sub.2).sub.p--and --S(O.sub.2)(CW.sub.2).sub.p. The group
Z has the formula NH, NR.sup.2, S or O. Each W is independently H,
R.sup.2, --OR.sup.2, F, Cl, Br or I; wherein, each R.sup.2 is
independently selected from the group consisting of: --CX.sub.3,
--CX.sub.2CX.sub.3, --CX.sub.2CX.sub.2CX.sub.3,
--CX.sub.2CX(CX.sub.3).sub.2, and --C(CX.sub.3).sub.3. Each X is
independently H, F, Cl, Br or I. Each m is independently 0 or 1.
Each n, o and p are independently integers from 0 to 10. In the
most preferred embodiment, the spacer/flexible linker comprises two
linked 8-amino-3,6-dioxaoctanoic acid moieties. Consequently, Y is
--C(O)(CW.sub.2).sub.p--, Z is NH, each W is H, m is 1, n is 2, o
is 2and p is 1.
[0088] Chimeric Oligomer:
[0089] A chimeric oligomer comprises two or more linked subunits
which are selected from different classes of subunits. For example,
a PNA/DNA chimera would comprise at least two PNA subunits linked
to at least one 2'-deoxyribonucleic acid subunit (For methods and
compositions related to PNA/DNA chimera preparation See:
WO96/40709). The component subunits of the chimeric oligomers are
selected from the group consisting of PNA subunits, DNA subunits,
RNA subunits and analogues thereof.
[0090] Linked Polymer:
[0091] A linked polymer comprises two or more nucleobase sequences
which are linked by a linker. The nucleobase sequences which are
linked to form the linked polymer are selected from the group
consisting of an oligodeoxynucleotide, an oligoribonucleotide, a
peptide nucleic acid and a chimeric oligomer. The PNA probes of
this invention include linked polymers wherein the probing
nucleobase sequence is linked to one or more additional
oligodeoxynucleotide, oligoribonucleotide, peptide nucleic add or
chimeric oligomers.
[0092] Hybridization Conditions/Stringency:
[0093] Those of ordinary skill in the art of nucleic acid
hybridization will recognize that factors commonly used to impose
or control stringency of hybridization include formamide
concentration (or other chemical denaturant reagent), salt
concentration (i.e., ionic strength), hybridization temperature,
detergent concentration, pH and the presence or absence of
chaotropes. Optimal stringency for a probing nucleobase
sequence/target sequence combination is often found by the well
known technique of fixing several of the aforementioned stringency
factors and then determining the effect of varying a single
stringency factor. The same stringency factors can be modulated to
thereby control the stringency of hybridization of PNA Molecular
Beacons to target sequences, except that the hybridization of a PNA
is fairly independent of ionic strength. Optimal stringency for an
assay may be experimentally determined by examination of each
stringency factor until the desired degree of discrimination is
achieved.
[0094] Probing Nucleobase Sequence:
[0095] The probing nucleobase sequence of a PNA Molecular Beacon is
the sequence recognition portion of the construct. Therefore, the
probing nucleobase sequence is designed to hybridize to at least a
portion of the target sequence. Preferably the probing nucleobase
sequence hybridizes to the entire target sequence. The probing
nucleobase sequence is a non-polynucleotide and preferably the
probing nucleobase sequence is composed exclusively of PNA
subunits. The subunit length of the probing nucleobase sequence
will therefore generally be chosen such that a stable complex is
formed between the PNA Molecular Beacon and the target sequence
sought to be detected, under suitable hybridization conditions. The
probing nucleobase sequence of a PNA oligomer, suitable for the
practice of this invention, will generally have a length of between
5 and 30 PNA subunits. Preferably, the probing nucleobase sequence
will be 8 to 18 subunits in length. Most preferably, the probing
nucleobase sequence will be 11-17 subunits in length.
[0096] The probing nucleobase sequence of a PNA Molecular Beacons
will generally have a nucleobase sequence which is complementary to
the target sequence. Alternatively, a substantially complementary
probing sequence might be used since it has been demonstrated that
greater sequence discrimination can be obtained when utilizing
probes wherein there exists a single point mutation (base mismatch)
between the probing nucleobase sequence and the target sequence
(See: Guo et al., Nature Biotechnology 15: 331-335 (1997), Guo et
al., WO97/46711; and Guo et al., U.S. Pat. No. 5,780,233, herein
incorporated by reference).
[0097] Arm Segments
[0098] The arm segments of the PNA Molecular Beacon are designed to
anneal to each other and thereby stabilize the interactions which
fix the energy transfer of linked donor and acceptor moieties until
the PNA Molecular Beacon hybridizes to the target sequence. The arm
segments may be of different lengths, but, are preferably the same
length. The preferred length of the arm segments will depend on the
stability desired for the interactions. However, the arm segments
must not be so long that they prohibit hybridization to the target
sequence. Preferably, the arm-segments are 2-6 subunits in length
and most preferably the arm segments are 2-4 subunits in length
since applicant's data demonstrates that the highest signal to
noise ratios are obtained with PNA Molecular Beacons having arm
segments of 5 or less subunits. Preferably arm segments of a PNA
Molecular Beacon are comprised primarily of PNA subunits and
preferably comprised of only PNA subunits. However, salt pairs and
hydrophobic/hydrophobic interactions may contribute to the
stability of the interactions which fix the proximity of the donor
and acceptor moieties
[0099] In certain embodiments, both arm segments are external to
the probing nucleobase sequence (See: Figure A; Configuration III).
Alternatively, one arm segment may be embedded within a probing
nucleobase sequence (See: Figure A; Configurations I and II). When
one arm segment is embedded within the probing nucleobase sequence,
preferably the other arm segment is oriented to the N-terminus of
the PNA Molecular Beacon and the probing nucleobase sequence is
oriented toward the C-terminus of the PNA Molecular Beacon.
[0100] Flexible Linkages
[0101] The flexible linkages link one or more arm forming segments
to the PNA Molecular Beacon. Without intending to be bound to this
hypothesis, it is believed that flexible linkages provide
flexibility and randomness to the otherwise highly structured PNA
oligomer thereby resulting in more efficient energy transfer of the
linked donor and acceptor moieties of the unhybridized PNA
Molecular Beacon. The length and composition of the flexible
linkages will be judiciously chosen to facilitate intramolecular
interactions between functional groups of the polymer (e.g.
nucleobase-nucleobase interactions) which would otherwise not be
able to freely interact. Flexible linkages appear to produce PNA
Molecular Beacons which exhibit higher signal to noise ratios in
hybridization assays and a more reversible modulation of
fluorescent signal in response to thermal changes in environment as
compared with PNA Molecular Beacons which do not possess flexible
linkages. Thus, flexible linkages are an important feature of the
PNA Molecular Beacons of this invention.
[0102] Blocking Probes:
[0103] Blocking probes are PNA or nucleic acid probes which can be
used to suppress the binding of the probing nucleobase sequence of
a probe to a hybridization site which is unrelated or closely
related to the target sequence (See: Coull et al., PCT/US97/21845,
a.k.a. WO98/24933). Generally, the blocking probes suppress the
binding of the probing nucleobase sequence to closely related
non-target sequences because the blocking probe hybridizes to the
non-target sequence to form a more thermodynamically stable complex
than is formed by hybridization between the probing nucleobase
sequence and the non-target sequence. Thus, blocking probes are
typically unlabeled probes used in an assay to thereby suppress
non-specific signal. Because they are usually designed to hybridize
to closely related non-target sequence sequences, typically a set
of two or more blocking probes will be used in an assay to thereby
suppress non-specific signal from non-target sequences which could
be present and interfere with the performance of the assay.
[0104] II. Preferred Embodiments of the Invention:
[0105] PNA Molecular Beacons:
[0106] Tyagi et al. and Tyagi2 et al. disclose nucleic acid
Molecular Beacons which comprise a hairpin loop and stem to which
energy transfer donor and acceptor moieties are linked at opposite
ends of the nucleic acid polymer. Numerous PNA polymers were
examined in an attempt to prepare a PNA Molecular Beacon. The
applicant's have determined that all probes they examined, which
contained linked donor and acceptor moieties exhibited a low
inherent noise (background) and an increase in detectable signal
upon binding of the probe to a target sequence. Very surprisingly,
these characteristic properties of a nucleic acid Molecular Beacon
were observed whether or not the PNA oligomer possessed
self-complementary arm segments intended to form a PNA hairpin. For
example, PNA oligomers prepared as control samples which by design
did not possess any self-complementary arm segments suitable for
forming a hairpin exhibited a signal (PNA oligomer bound to target
sequence) to noise (no target sequence present) ratio which was
quite favorable as compared with probes comprising flexible
linkages and self-complementary arm segments.
[0107] Applicant's data further demonstrates that flexible linkages
inserted within the probe and shorter self-complementary arm
segments are a preferred embodiment since the signal to noise ratio
of probes of this embodiment compare well with the signal to noise
ratio published for nucleic acid hairpins (approximately 25 to 1).
The data compiled by applicants is inconclusive with respect to
whether or not the PNA Molecular Beacons they prepared which have
shorter arms segments (2-5 subunits in length) and one or more
flexible linkages exist as hairpins. However, applicant's data
demonstrates that probes with longer arm segments (e.g. 9 subunits)
do form a hairpin (See: Example 19 of this specification) and
unlabeled probes having arms segments as short as six subunits do
not exist primarily as a hairpin (See: Example 19 of this
specification). Furthermore, the signal to noise ratio for those
the probes having longer arm segments suitable for forming a
hairpin exhibited very poor a signal to noise ratios upon melting
of the hairpin or when in the presence of a complementary nucleic
acid. Consequently, embodiments having longer arm segments (e.g. 6
or more subunits) do not appear to be well suited for use in the
detection of nucleic acid targets.
[0108] This invention pertains to methods, kits and compositions
pertaining to PNA Molecular Beacons. Though we refer to the probes
of this invention as PNA Molecular Beacons, we do not mean to imply
that they exist as hairpins since they may well exist as
aggregates, bimolecular constructs or as higher order hybrids (e.g.
multimers). Regardless of the nature of the secondary structure, a
PNA Molecular Beacon efficiently transfers energy between donor and
acceptor moieties linked to the probe in the absence of target
sequence. Upon hybridization of the probing nucleobase sequence to
a target sequence, the efficiency of energy transfer between donor
and acceptor moieties of a PNA Molecular Beacon is altered such
that detectable signal from at least one linked moiety can be used
to monitor or quantitate the occurrence of the hybridization
event.
[0109] Generally, a PNA Molecular Beacon is a polymer suitable for
detecting, identifying or quantitating a target sequence. At a
minimum, a PNA Molecular Beacon comprises a probing nucleobase
sequence, two arm segments, wherein at least one arm segment is
linked to the probe through a flexible linkage, at least one linked
donor moiety and at least one linked acceptor moiety. The donor and
acceptor moieties can be linked at any position within the PNA
Molecular Beacon provided they are separated by at least a portion
of the probing nucleobase sequence. Preferably the donor and
acceptor moieties of a Beacon Set are located at opposite ends of
the probing nucleobase sequence and most preferably at the termini
of the PNA Molecular Beacon. The PNA Molecular Beacon is further
characterized in that the probe exhibits detectable change in at
least one property of at least one linked donor or acceptor moiety
which occurs upon hybridization to the target sequence under
suitable hybridization conditions.
[0110] In one preferred embodiment, this invention is directed to a
PNA Molecular Beacons comprising an arm segment having a first and
second end. Additionally, there is also a probing nucleobase
sequence having a first and second end wherein, the probing
nucleobase sequence is complementary or substantially complementary
to the target sequence. There is also a second arm segment which is
embedded within the probing nucleobase sequence and is
complementary or substantially complementary to the first arm
segment. The polymer also comprises a flexible linkage which links
the second end of the first arm segment to the second end of the
probing nucleobase sequence. A donor moiety is linked to the first
end of one of either of the first arm segment or the probing
nucleobase sequence; and an acceptor moiety is linked to the first
end of the other of either of the first arm segment or the probing
nucleobase sequence.
[0111] In still another preferred embodiment, this invention is
directed to a PNA Molecular Beacon comprising a probing nucleobase
sequence having a first and second end, wherein, the probing
nucleobase sequence is complementary or substantially complementary
to the target sequence. There is also a first arm segment
comprising a first and second end and a second arm segment
comprising a first and second end, wherein, at least a portion of
the nucleobases of the second arm segment are complementary to the
nucleobase sequence to the first arm segment. The polymer also
comprises a first flexible linkage which links the second end of
the first arm segment to either of the first or second end of the
probing nucleobase sequence. There is a second linkage which 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. A donor
moiety is linked to the first end of one of either of the first or
second arm segments; and an acceptor moiety is linked to the first
end of the other of either of the first or the second arm
segments.
[0112] Preferably, a PNA Molecular Beacons is assembled by stepwise
condensation of suitably protected amino acid moieties.
Consequently, the polymer is preferably continuous from the amino
to the carboxyl terminus. In the most preferred configuration, PNA
Molecular Beacons are continuous from the N-terminus to the
C-terminus wherein the first arm segment is oriented toward the
N-terminus and the probing nucleobase sequence is oriented toward
the C-terminus of the polymer. Additionally, the preferred PNA
Molecular Beacons comprise a probing nucleobase sequence which is
perfectly complementary to the target sequence and a first arm
segment which is perfectly complementary to the second arm
segment.
[0113] It is not a requirement that the PNA Molecular Beacons of
this invention form a hairpin. However, if hairpins are formed,
preferred embodiments of the PNA Molecular Beacons of this
invention can generally be represented in three configurations with
are illustrated in Figure A. In configuration I, the probing
nucleobase sequence is located at the carboxyl terminus of the
polymer. The probing nucleobase sequence is linked to the arm
forming segment through one or more flexible linker moieties. In
this embodiment, one of the two arm segments is embedded within the
probing nucleobase sequence. As illustrated, the donor and acceptor
moieties are located at opposite ends of the PNA Molecular Beacon
but either orientation of the labels is acceptable. This embodiment
of a Molecular Beacon is unique even in light of the nucleic acid
Molecular Beacons, because one of the two arm forming segments is
embedded within the probing segment. Minimization of sequence
length is preferred since it should reduce non-specific
interactions.
[0114] In configuration II, the positioning of the probing
nucleobase sequence and arm segments are inverted as compared with
configuration I. In this configuration the probing nucleobase
sequence is located at the amino terminus of the polymer and is
linked to an arm forming segment through one or more flexible
linker moieties. As illustrated, the donor and acceptor moieties
are located at opposite termini of the PNA Molecular Beacon but
either orientation of the labels is acceptable.
[0115] In configuration III, the entire probing nucleobase sequence
is external to the two arm forming segments. Thus, this embodiment
is more similar to the nucleic acid Molecular Beacons than is
either configuration I or II. Configuration III, however, differs
from nucleic acid Molecular Beacons in that it is comprised of PNA
subunits and also contains at least one flexible linkage separating
a probing nucleobase sequence and the arm segments.
[0116] Unique Features of PNA Molecular Beacons:
[0117] There are many differences between prior art nucleic acid
constructs and the PNA Molecular Beacons of this invention. For
example, nucleic acid constructs comprise a polynucleotide backbone
whereas the Linear Beacons of this invention comprise a probing
nucleobase sequence which is not a polynucleotide. Thus, PNA
Molecular Beacons which comprise PNA subunits exhibit all of the
favorable properties of PNA such as resistance to nuclease
degradation, salt independent sequence hybridization to
complementary nucleic acids and rapid hybridization kinetics. For
probes which do form hairpin stems, the Tm of the stem duplex is
substantially independent of the presence or absence of magnesium
and the ionic strength of the environment.
[0118] Additionally, several of the constructs designed by
applicants are PNA Molecular Beacons having arm segments which are
embedded within the probing nucleobase sequence. These unique
constructs are shorter than corresponding nucleic acid Molecular
Beacons. Shorter probes are less costly to synthesize, are
generally easier to purify and should exhibit few non-specific
interactions since they will comprise less nucleobase sequence
diversity.
[0119] Additionally, the constructs described herein comprise
flexible linkages which applicants have demonstrated to be a
preferred embodiment since a higher signal to noise ratio is
achieved as compared with PNA probes of similar subunit design
which do not comprise flexible linkages. Similarly, the preferred
PNA Molecular Beacons of this invention comprise short arm segments
since applicant's data demonstrates a clear inverse correlation
between arm length and signal to noise ratio. The preferred PNA
Molecular Beacons of this invention comprise arms sequences of five
or less, and more preferably three or less, subunits.
[0120] Probe Sets:
[0121] In another embodiment, this invention is directed to sets of
PNA Molecular Beacons suitable for detecting or identifying the
presence, absence or amount of two or more different target
sequences which might be present in a sample. The characteristics
of PNA Molecular Beacons suitable for the detection, identification
or quantitation of target sequences have been previously described
herein. The grouping of PNA Molecular Beacons within sets
characterized for specific detection of two or more target
sequences is a preferred embodiment of this invention.
[0122] Probe sets of this invention shall comprise at least one PNA
Molecular Beacon but need not comprise only PNA Molecular Beacons.
For example, probe sets of this invention may comprise mixtures of
PNA Molecular Beacons, other PNA probes and/or nucleic acid probes,
provided however that a set comprises at least one PNA Molecular
Beacon as described herein. In preferred embodiments, at least one
probe of the set is a blocking probe, as defined herein.
[0123] Immobilization of a PNA Molecular Beacon to a Surface:
[0124] One or more PNA Molecular Beacons may optionally be
immobilized to a surface. In one embodiment, the probe can be
immobilized to the surface using the well known process of
UV-crosslinking. Alternatively, the PNA oligomer is synthesized on
the surface in a manner suitable for deprotection but not cleavage
from the synthesis support.
[0125] Preferably, the probe is covalently linked to a surface by
the reaction of a suitable functional groups on the probe and
support. Functional groups such as amino groups, carboxylic acids
and thiols can be incorporated in a PNA Molecular Beacon by
extension of one of the termini with suitable protected moieties
(e.g. lysine, glutamic acid and cystine). When extending the
terminus, one functional group of a branched amino acid such as
lysine can be used to incorporate the donor or acceptor label at
the appropriate position in the polymer (See: Section entitled "PNA
Labeling") while the other functional group of the branch is used
to optionally further extend the polymer and immobilize it to a
surface.
[0126] Methods for the attachment of probes to surfaces generally
involve the reaction of a nucleophilic group, (e.g. an amine or
thiol) of the probe to be immobilized, with an electrophilic group
on the support to be modified. Alternatively, the nucleophile can
be present on the support and the electrophile (e.g. activated
carboxylic acid) present on the PNA Molecular Beacon. Because
native PNA possesses an amino terminus, a PNA will not necessarily
require modification to thereby immobilize it to a surface (See:
Lester et al., Poster entitled "PNA Array Technology").
[0127] Conditions suitable for the immobilization of a PNA to a
surface will generally be similar to those conditions suitable for
the labeling of a PNA (See: subheading "PNA Labeling"). The
immobilization reaction is essentially the equivalent of labeling
the PNA whereby the label is substituted with the surface to which
the PNA probe is to be covalently immobilized.
[0128] Numerous types of surfaces derivatized with amino groups,
carboxylic acid groups, isocyantes, isothiocyanates and malimide
groups are commercially available. Non-limiting examples of
suitable surfaces include membranes, glass, controlled pore glass,
polystyrene particles (beads), silica and gold nanoparticles.
[0129] When immobilized to a surface, energy transfer between
moieties of a Beacon Set will occur in the PNA Molecular Beacon.
Upon hybridization to a target sequence under suitable
hybridization conditions, the location on the surface where the PNA
Molecular Beacon (of known sequence) is attached will generate
detectable signal based on the measurable change in signal of at
least one member of the Beacon Set of the immobilized PNA Molecular
Beacon. Consequently, the intensity of the signal on the surface
can be used to detect, identify or quantitate the presence or
amount of a target sequence in a sample which contacts the surface
to which the PNA Molecular Beacon is immobilized. In a preferred
embodiment, detection of surface fluorescence will be used to
detect hybridization to a target sequence.
[0130] Detectable and Independently Detectable Moieties/Multiplex
Analysis:
[0131] In preferred embodiments of this invention, a multiplex
hybridization assay is performed. In a multiplex assay, numerous
conditions of interest are simultaneously examined. Multiplex
analysis relies on the ability to sort sample components or the
data associated therewith, during or after the assay is completed.
In preferred embodiments of the invention, distinct independently
detectable moieties are used to label the different PNA Molecular
Beacons of a set. The ability to differentiate between and/or
quantitate each of the independently detectable moieties provides
the means to multiplex a hybridization assay because the data which
correlates with the hybridization of each of the distinctly
(independently) labeled PNA Molecular Beacons to a target sequence
can be correlated with the presence, absence or quantity of the
target sequence sought to be detected in a sample. Consequently,
the multiplex assays of this invention may be used to
simultaneously detect the presence, absence or amount of one or
more target sequences which may be present in the same sample in
the same assay. Preferably, independently detectable fluorophores
will be used as the independently detectable moieties of a
multiplex assay using PNA Molecular Beacons. For example, two PNA
Molecular Beacons might be used to detect each of two different
target sequences wherein a fluorescein (green) labeled probe would
be used to detect the first of the two target sequences and a
rhodamine or Cy3 (red) labeled probe would be used to detect the
second of the two target sequences. Consequently, a green, a red or
a green and red signal in the assay would signify the presence of
the first, second and first and second target sequences,
respectively.
[0132] Arrays of PNA Molecular Beacons:
[0133] Arrays are surfaces to which two or more probes of interest
have been immobilized at predetermined locations. Arrays comprising
both nucleic acid and PNA probes have been described in the
literature. The probe sequences immobilized to the array are
judiciously chosen to interrogate a sample which may contain one or
more target sequences of interest. Because the location and
sequence of each probe is known, arrays are generally used to
simultaneously detect, identify or quantitate the presence or
amount of one or more target sequences in the sample. Thus, PNA
arrays may be useful in diagnostic applications or in screening
compounds for leads which might exhibit therapeutic utility.
[0134] For example, in a diagnostic assay a target sequence is
captured by the complementary probe on the array surface and then
the probe/target sequence complex is detected using a secondary
detection system. In one embodiment the probe/target sequence
complex is detected using a second probe which hybridizes to
another sequence of the target molecule of interest. In another
embodiment, a labeled antibody is used to detect, identify or
quantitate the presence of the probe/target sequence complex.
[0135] Since the composition of the PNA Molecular Beacon is known
at the location on the surface of the array (because the PNA was
synthesized or attached to this position in the array), the
composition of target sequence(s) can be directly detected,
identified or quantitated by determining the location of detectable
signal generated in the array. Because hybridization of the PNA
Molecular Beacon to a target sequence is self-indicating, no
secondary detection system is needed to analyze the array for
hybridization between the PNA Molecular Beacon and the target
sequence.
[0136] Arrays comprised of PNAs have the additional advantage that
PNAs are highly stable and should not be degraded by enzymes which
degrade nucleic acid. Therefore, PNA arrays should be reusable
provided the nucleic acid from one sample can be striped from the
array prior to introduction of the second sample. Upon stripping of
hybridized target sequences, signal on the array of PNA Molecular
Beacons should again become reduced to background. Because PNAs are
not degraded by heat or endonuclease and exonuclease activity,
arrays of PNA Molecular Beacon should be suitable for simple and
rapid regeneration by treatment with heat, nucleases or chemical
denaturants such as aqueous solutions containing formamide, urea
and/or sodium hydroxide.
[0137] Methods:
[0138] In yet another embodiment, this invention is directed to a
method for the detection, identification or quantitation of a
target sequence in a sample. The method comprises contacting the
sample with a PNA Molecular Beacon and then detecting, identifying
or quantitating the change in detectable signal associated with at
least one moiety of a Beacon Set whereby correlation between
detectable signal and hybridization is possible since PNA Molecular
Beacons are self-indicating. Because PNA Molecular Beacons are
self-indicating, this method is particularly well suited to
analysis performed in a closed tube assay (a.k.a. "homogeneous
assays"). By closed tube assay we mean that once the components of
the assay have been combined, there is no need to open the tube or
remove contents of the assay to determine the result. Since the
tube need not, and preferably will not, be opened to determine the
result, there must be some detectable or measurable change which
occurs and which can be observed or quantitated without opening the
tube or removing the contents of the assay. Thus, most closed tube
assays rely on a change in fluorescence which can be observed with
the eye or otherwise be detected and/or quantitated with a
fluorescence instrument which uses the tube as the sample holder.
Examples of such instruments include the Light Cycler from Idaho
Technologies and the Prism 7700 from Perkin Elmer.
[0139] Preferred closed tube assays of this invention comprise the
detection of nucleic acid target sequences which have been
synthesized or amplified by operation of the assay. Non-limiting
examples of preferred nucleic acid synthesis or nucleic acid
amplification reactions are Polymerase Chain Reaction (PCR), Ligase
Chain Reaction (LCR), Strand Displacement Amplification (SDA),
Transcription-Mediated Amplification (TMA), Rolling Circle
Amplification (RCA) and Q-beta replicase. The PNA Molecular Beacons
present in the closed tube assay will generate detectable signal in
response to target sequence production from the nucleic acid
synthesis or nucleic acid amplification reaction occurring in the
closed tube assay. In a most preferred embodiment, the assay is an
asymmetric PCR reaction.
[0140] Because the PNA Molecular Beacons of this invention can be
designed to be stable to the enzymes found in the cell, this method
is particularly well suited to detecting a target sequence in a
cell, tissue or organism, whether living or not. Thus, in preferred
embodiments, in-situ hybridization is used as the assay format for
detecting identifying or quantitating target organisms. Most
preferably, fluorescence in-situ hybridization (FISH or PNA-FISH)
is the assay format. Exemplary methods for performing PNA-FISH can
be found in: Thisted et al. Cell Vision, 3:358-363 (1996) or WIPO
Patent Application WO97/18325, herein incorporated by
reference.
[0141] Organisms which have been treated with the PNA Molecular
Beacons of this invention can be detected by several exemplary
methods. The cells can be fixed on slides and then visualized with
a microscope or laser scanning device. Alternatively, the cells can
be fixed and then analyzed in a flow cytometer (See for example:
Lansdorp et al.; WIPO Patent Application; WO97/14026). Slide
scanners and flow cytometers are particularly useful for rapidly
quantitating the number of target organisms present in a sample of
interest.
[0142] Because the method of this invention may be used in a
probe-based hybridization assay, this invention will find utility
in improving assays used to detect, identify of quantitate the
presence or amount of an organism or virus in a sample through the
detection of target sequences associated with the organism or
virus. (See: U.S. Pat. No. 5,641,631, entitled "Method for
detecting, identifying and quantitating organisms and viruses"
herein incorporated by reference). Similarly, this invention will
also find utility in an assay used in the detection, identification
or quantitation of one or more species of an organism in a sample
(See U.S. Pat. No. 5,288,611, entitled "Method for detecting,
identifying and quantitating organisms and viruses" herein
incorporated by reference). This invention will also find utility
in an assay used to determine the effect of antimicrobial agents on
the growth of one or more microorganisms in a sample (See: U.S.
Pat. No. 5,612,183, entitled "Method for determining the effect of
antimicrobial agents on growth using ribosomal nucleic acid subunit
subsequence specific probes" herein incorporated by reference).
This invention will also find utility in an assay used to determine
the presence or amount of a taxonomic group of organisms in a
sample (See: U.S. Pat. No. 5,601,984, entitled "Method for
detecting the presence of amount of a taxonomic group of organisms
using specific r-RNA subsequences as probes" herein incorporated by
reference).
[0143] When performing the method of this invention, it may be
preferable to use one or more unlabeled or independently detectable
probes in the assay to thereby suppress the binding of the PNA
Molecular Beacon to a non-target sequence. The presence of the
"blocking probe(s)" helps to increase the discrimination of the
assay and thereby improve reliability and sensitivity (signal to
noise ratio).
[0144] In certain embodiments of this invention, one target
sequence is immobilized to a surface by proper treatment of the
sample. Immobilization of the nucleic acid can be easily
accomplished by applying the sample to a membrane and then
UV-crosslinking. For example, the samples may be arranged in an
array so that the array can be sequentially interrogated with one
or more PNA Molecular Beacons to thereby determine whether each
sample contains one or more target sequence of interest.
[0145] In still another embodiment, the PNA Molecular Beacon is
immobilized to a support and the samples are sequentially
interrogated to thereby determine whether each sample contains a
target sequence of interest. In preferred embodiments, the PNA
Molecular Beacons are immobilized on an array which is contacted
with the sample of interest. Consequently, the sample can be
simultaneously analyzed for the presence and quantity of numerous
target sequences of interest wherein the composition of the PNA
Molecular Beacons are judiciously chosen and arranged at
predetermined locations on the surface so that the presence,
absence or amount of particular target sequences can be
unambiguously determined. Arrays of PNA Molecular Beacons are
particularly useful because no second detection system is required
since PNA Molecular Beacons are self-indicating. Consequently, this
invention is also directed to an array comprising two or more
support bound PNA Molecular Beacons suitable for detecting,
identifying or quantitating a target sequence of interest.
[0146] Kits:
[0147] In yet another embodiment, this invention is directed to
kits suitable for performing an assay which detects the presence,
absence or amount of one or more target sequence which may be
present in a sample. The characteristics of PNA Molecular Beacons
suitable for the detection, identification or quantitation of
amount of one or more target sequence have been previously
described herein. Furthermore, methods suitable for using the PNA
Molecular Beacon components of a kit to detect, identify or
quantitate one or more target sequence which may be present in a
sample have also been previously described herein.
[0148] The kits of this invention comprise one or more PNA
Molecular Beacons and other reagents or compositions which are
selected to perform an assay or otherwise simplify the performance
of an assay. Preferred kits contain sets of PNA Molecular Beacons,
wherein each of at least two PNA Molecular Beacons of the set are
used to distinctly detect and distinguish between the two or more
different target sequences which may be present in the sample.
Thus, the PNA Molecular Beacons of the set are preferably labeled
with independently detectable moieties so that each of the two or
more different target sequences can be individually detected,
identified or quantitated (a multiplex assay).
[0149] Exemplary Applications for Using the Invention:
[0150] Whether support bound or in solution, the methods, kits and
compositions of this invention are particularly useful for the
rapid, sensitive, reliable and versatile detection of target
sequences which are particular to organisms which might be found in
food, beverages, water, pharmaceutical products, personal care
products, dairy products or environmental samples. The analysis of
preferred beverages include soda, bottled water, fruit juice, beer,
wine or liquor products. Consequently, the methods, kits and
compositions of this invention will be particularly useful for the
analysis of raw materials, equipment, products or processes used to
manufacture or store food, beverages, water, pharmaceutical
products, personal care products, dairy products or environmental
samples.
[0151] Whether support bound or in solution, the methods, kits and
compositions of this invention are particularly useful for the
rapid, sensitive, reliable and versatile detection of target
sequences which are particular to organisms which might be found in
clinical environments. Consequently, the methods, kits and
compositions of this invention will be particularly useful for the
analysis of clinical specimens or equipment, fixtures or products
used to treat humans or animals. For example, the assay may be used
to detect a target sequence which is specific for a genetically
based disease or is specific for a predisposition to a genetically
based disease. Non-limiting examples of diseases include,
.beta.-Thalassemia, sickle cell anemia, Factor-V Leiden, cystic
fibrosis and cancer related targets such as p53, p10, BRC-1 and
BRC-2.
[0152] In still another embodiment, the target sequence may be
related to a chromosomal DNA, wherein the detection, identification
or quantitation of the target sequence can be used in relation to
forensic techniques such as prenatal screening, paternity testing,
identity confirmation or crime investigation.
EXAMPLES
[0153] This invention is now illustrated by the following examples
which are not intended to be limiting in any way.
Example 1
Synthesis of N--(Fmoc)-N-.epsilon.-(NH.sub.2)-L-Lysine-OH
[0154] To 20 mmol of
N-.alpha.-(Fmoc)-N-.epsilon.-(t-boc)-L-lysine-OH was added 60 mL of
2/1 dichloromethane (DCM)/trifluoroacetic acid (TFA). The solution
was allowed to stir until the tert-butyloxycarbonyl (t-boc) group
had completely been removed from the
N-.alpha.-(Fmoc)-N-.epsilon.-(- t-boc)-L-lysine-OH. The solution
was then evaporated to dryness and the residue redissolved in 15 mL
of DCM. An attempt was then made to precipitate the product by
dropwise addition of the solution to 350 mL of ethyl ether. Because
the product oiled out, the ethyl ether was decanted and the oil put
under high vacuum to yield a white foam. The white foam was
dissolved in 250 mL of water and the solution was neutralized to pH
4 by addition of saturated sodium phosphate (dibasic). A white
solid formed and was collected by vacuum filtration. The product
was dried in a vacuum oven at 35-40.degree. C. overnight. Yield
17.6 mmol, 88%.
Example 2
Synthesis of N-.alpha.-(Fmoc)-N-.epsilon.-(dabcyl)-L-Lysine-OH
[0155] To 1 mmol of
N-.alpha.-(Fmoc)-N-.epsilon.-(NH.sub.2)-L-Lysine-OH (Example 1) was
added 5 mL of N,N'-dimethylformamide (DMF) and 1.1 mmol of TFA.
This solution was allowed to stir until the amino acid had
completely dissolved.
[0156] To 1.1 mmol of 4-((4-(dimethylamino)phenyl)azo)benzoic acid,
succinimidyl ester (Dabcyl-NHS; Molecular Probes, P/N D-2245) was
added 4 mL of DMF and 5 mmol of diisopropylethylamine (DIEA). To
this stirring solution was added, dropwise, the
N-.alpha.-(Fmoc)-N-.epsilon.-(NH.sub.2)- -L-Lysine-OH solution
prepared as described above. The reaction was allowed to stir
overnight and was then worked up.
[0157] The solvent was vacuum evaporated and the residue
partitioned in 50 mL of DCM and 50 mL of 10% aqueous citric acid.
The layers were separated and the organic layer washed with aqueous
sodium bicarbonate and again with 10% aqueous citric acid. The
organic layer was then dried with sodium sulfate, filtered and
evaporated to an orange foam. The foam was crystallized from
acetonitrile (ACN) and the crystals collected by vacuum filtration.
Yield 0.52 mmol, 52%.
Example 3
Synthesis of
N-.alpha.-(Fmoc)-N-.epsilon.-(dabcyl)-L-Lysine-PAL-Peg/PS Synthesis
Support
[0158] The N-.alpha.-(Fmoc)-N-.epsilon.-(dabcyl)-L-Lysine-OH
(Example 2) was used to prepare a synthesis support useful for the
preparation of C-terminal dabcylated PNAs. The
fluorenylmethoxycarbonyl (Fmoc) group of 0.824 g of commercially
available Fmoc-PAL-Peg-PS synthesis support (PerSeptive Biosystems,
Inc.; P/N GEN913384) was removed by treatment, in a flow through
vessel, with 20% piperidine in DCM for 30 minutes. The support was
then washed with DCM. Finally, the support was washed with DMF and
dried with a flushing stream of argon.
[0159] A solution containing 0.302 g
N-.alpha.-(Fmoc)-N-.epsilon.-(dabcyl)- -L-Lysine-OH, 3.25 mL of
DMF, 0.173 g [O-(7-azabenzotriaol-1-yl)-1,1,3,3-t-
etramethyluronium hexafluorophosphate (HATU), 0.101 mL DIEA and
0.068 mL 2,6-lutidine was prepared by sequential combination of the
reagents. This solution was then added to the washed synthesis
support and allowed to react for 2 hours. The solution was then
flushed through the vessel with a stream of argon and the support
washed sequentially with DMF, DCM and DMF. The resin was then dried
with a stream of argon.
[0160] The support was the treated with 5 mL of standard
commercially available PNA capping reagent (PerSeptive Biosystems,
Inc., P/N GEN063102). The capping reagent was then flushed from the
vessel and the support was washed with DMF and DCM. The support was
then dried with a stream of argon. Finally, the synthesis support
was dried under high vacuum.
[0161] Final loading of the support was determined by analysis of
Fmoc loading of three samples of approximately 6-8 mg. Analysis
determined the loading to be approximately 0.145 mmol/g.
[0162] This synthesis support was packed into an empty PNA
synthesis column, as needed, and used to prepare PNA oligomers
having a C-terminal dabcyl quenching moiety attached to the PNA
oligomer through the e-amino group of the C-terminal L-lysine amino
acid.
Example 4
Synthesis of PNA
[0163] PNAs were synthesized using commercially available reagents
and instrumentation obtained from PerSeptive Biosystems, Inc.
Double couplings were routinely performed to improve the quality of
the crude product. PNAs possessing a C-terminal dabcyl moiety were
prepared by performing the synthesis using the dabcyl-lysine
modified synthesis support prepared as described in Example 3 or by
labeling the N-.epsilon.-amino group of the C-terminal lysine
residue while the PNA was still support bound as described in
Example 10. All PNAs possessing both an N-terminal fluorescein
moiety, as well as, a C-terminal dabcyl moiety were treated with
the appropriate labeling reagents and linkers (as required) prior
to cleavage from the synthesis support.
Example 5
Preferred Method for Removal of the Fmoc Protecting Group
[0164] The synthesis support was treated with a solution of 25%
piperidine in DMF for 5-15 minutes at room temperature. After
treatment, the synthesis support was washed and dried under high
vacuum. The support was then treated with the appropriate labeling
reagent and/or cleaved from the synthesis support.
Example 6
Synthesis of Fluorescein-O-Linker
[0165] To 7.5 mmol of
N-(tert-butyloxycarbonyl)-8-amino-3,6-dioxaoctanoic acid stirring
in 10 mL of DCM was added 50 mmol of TFA. The solution was stirred
at room temperature until the t-boc group was completely removed.
The solvent was then removed by vacuum evaporation and the product
was then resuspended in 10 mL of DCM.
[0166] To this stirring solution was added, dropwise, a solution
containing 7.5 mmol of Di-O-pivaloyl-5(6)-carboxyfluorescein-NHS
ester, 30 mmol of N-methylmorpholine (NMM) and 20 mL of DCM. The
reaction was allowed to run overnight and was then transferred to a
separatory funnel in the morning.
[0167] This organic solution was washed with aqueous 10% citric
acid two times and then dried with sodium sulfate, filtered and
evaporated to a brown foam. The product was column purified using
silica gel. A DCM mobile phase and stepwise methanol gradient was
used to elute the product from the stationary phase. Yield 2.8 g of
foam which was precipitated by dissolution in a minimal amount of
DCM and dropwise addition of that solution to hexane. Yield 2.32 g
white powder. The purity of the product was not suitable for
labeling so an additional reversed phase chromatographic separation
was performed on a sample of this material.
[0168] One gram of the precipitated product was dissolved in 30 mL
of a 50 mM aqueous triethylammonium acetate (pH 7) containing 40%
acetonitrile. This solution was then added to a pre-equilibrated 2
g Waters Sep-Pack Vac 12 cc tC18 cartridge (P/N WAT043380) in 10, 3
mL aliquots. After the addition of all loading solvent, two 3 mL
aliquots of 50 mM aqueous triethylammonium acetate (pH 7)
containing 40% acetonitrile was loaded as a first wash. Two 3 mL
aliquots of 50 mM aqueous triethylammonium acetate (pH 7)
containing 60% acetonitrile was then loaded as a second wash.
Finally, a single 3 mL aliquot of acetonitrile was used to elute
material remaining on the column. The eluent of each aliquot was
collected individually and analyzed by HPLC for purity. The
aliquots were vacuum evaporated and the mass of each determined.
Fractions of suitable purity were redissolved in DCM, the fractions
were combined and precipitated in hexane. Yield 0.232 g.
Example 7
General Procedure for N-terminal Labeling of Support Bound PNA with
Fluorescein-O-Linker
[0169] For N-terminal fluorescein labeling, the amino terminal
fluorenylmethoxycarbonyl (Fmoc) group of several of the fully
assembled PNA oligomers was removed by piperidine treatment and the
resin was washed and dried under vacuum. The resin was then treated
for 20-30 minutes with approximately 300 .mu.L of a solution
containing 0.07 M Fluorescein-O-Linker, 0.06 M (HATU), 0.067 M DIEA
and 0.1 M 2,6-lutidine. After treatment the resin was washed and
dried under high vacuum. The PNA oligomer was then cleaved,
deprotected and purified as described below.
Example 8
General Procedure for Labeling of Support Bound PNA with
5(6)carboxyfluorescein-NHS
[0170] This method was used as an alternative to the procedure
described in Example 7, for labeling PNAs with
5(6)-carboxyfluorescein. This procedure requires that the
N-terminus of the PNA oligomer be reacted with
Fmoc-8-amino-3,6-dioxaoctanoic acid prior to performing the
labeling reaction so that equivalent PNA constructs are prepared.
The amino terminal fluorenylmethoxycarbonyl (Fmoc) group of the
fully assembled PNA oligomer was removed by piperidine treatment
and the synthesis support was washed and dried under vacuum. The
synthesis support was then treated for 4-5 hours at 37.degree. C.
with approximately 300 .mu.L of a solution containing 0.1M
5(6)carboxyfluorescein-NHS (Molecular Probes, P/N C-1311), 0.3M
DIEA and 0.3M 2,6-lutidine. After treatment the synthesis support
was washed and dried under high vacuum. The PNA oligomer was then
cleaved, deprotected and purified as described below.
[0171] More preferably, the synthesis support was then treated for
2-5 hours at 30-37.degree. C. with approximately 250 .mu.L of a
solution containing 0.08M 5(6)carboxyfluorescein-NHS, 0.24M DIEA
and 0.24M 2,6-lutidine.
Example 9
General Procedure for Labeling of Support Bound PNA with
5(6)carboxyfluorescein
[0172] After proper reaction with linkers and removal of the
terminal amine protecting group, the resin was treated with 250
.mu.L of a solution containing 0.5M 5(6)carboxyfluorescein, 0.5M
N,N'-diisopropylcarbodiimide, 0,5M 1-hydroxy-7-azabenzotriazole
(HOAt) in DMF (See: Weber et al., Bioorganic & Medicinal
Chemistry Letters, 8: 597-600 (1998). After treatment the synthesis
support was washed and dried under high vacuum. The PNA oligomer
was then cleaved, deprotected and purified as described below.
[0173] Note on Fluorescein Labeling: The fluorescein labeled PNAs
described herein were prepared using several different procedures.
The different procedures have evolved to optimize fluorescein
labeling conditions. At this time we prefer to use the procedure of
Weber et al. for most fluorescein labeling operations.
Example 10
General Procedure for Dabcyl Labeling of the N-.epsilon.-amino
Group of Support Bound L-Lysine
[0174] This procedure was used as an alternative to using the
prederivatized support when preparing dabcylated PNAs. This
procedure has the advantage that the lysine moiety (and therefore
the attached dabcyl moiety) may be placed location in the polymer
including within the probing nucleobase sequence.
[0175] The resin (still in the synthesis column) was treated with
10 mL of a solution containing 1% trifluoroacetic acid, 5%
triisopropylsilane (TIS) in dichloromethane by passing the solution
through the column over a period of approximately 15 minutes. After
treatment, the synthesis support was washed with DMF. Prior to
treatment with labeling reagent the support was neutralized by
treatment with approximately 10 mL of a solution containing 5%
diisopropylethylamine in DMF. After treatment, the support was
treated with Dabcyl-NHS (as a substitute for
5(6)carboxyfluorescein-NHS in the procedure) essentially as
described in Example 8.
[0176] Note: This procedure was only performed on PNA prepared
using Fmoc-PAL-PEG/PS (PerSeptive P/N GEN913384). It was not
performed with the more acid labile Fmoc-XAL-PEG/PS (PerSeptive P/N
GEN913394).
Example 11
General Procedure for Cleavage. Deprotection and Purification
[0177] The synthesis support (Fmoc-PAL-PEG/PS; P/N GEN913384) was
removed from the synthesis cartridge, transferred to a Ultrafree
spin cartridge (Millipore Corp., P/N SE3P230J3) and treated with a
solution of TFA/m-cresol (either of 7/3 or 8/2 (preferred)) for 1-3
hours. The solution was spun through the support bed and again the
support was treated with a solution of TFA/m-cresol for 1-3 hours.
The solution was again spun through the support bed. The combined
eluents (TFA/m-cresol) were then precipitated by addition of
approximately 1 mL of diethyl ether. The precipitate was pelletized
by centrifugation. The pellet was then resuspended in ethyl ether
and pelletized two additional times. The dried pellet was then
resuspended in 20% aqueous acetonitrile (ACN) containing 0.1% TFA
(additional ACN was added as necessary to dissolve the pellet). The
product was analyzed and purified using reversed phase
chromatographic methods.
[0178] Note: Several PNAs were prepared using new product
Fmoc-XAL-PEG/PS synthesis support (P/N GEN 913394) available from
PerSeptive Biosystems, Inc. This support has the advantage that the
PNA can be removed more rapidly and under more mildly acid
conditions. For PNAs prepared with Fmoc-XAL-PEG/PS the support was
treated as described above except that a solution of TFA/m-cresol
9/1 was generally used for a period of 10-15 minutes (2x).
[0179] Experiment 12: Analysis and Purification of PNA
Oligomers
[0180] All PNA probes were analyzed and purified by reversed phase
HPLC. Probe composition was confirmed by comparison with
theoretical calculated masses. The crude products for PNA probes P3
and P4 (Table 5) were prepurified using anion exchange
chromatography prior to reversed phase HPLC purification. Anion
exchange chromatography generally improved the purity level to
better than 70 percent. Sephadex (Pharmacia Biotech) was used as
the stationary phase and the mobile phase was 10 mM sodium
hydroxide with a sodium chloride gradient.
[0181] HPLC Procedures:
[0182] Generally, two different high performance liquid
chromatography (HPLC) gradients were used to analyze and purify the
PNA oligomers (Gradients A & B). Preparative purifications were
scaled based on the analytical analysis conditions described in
Gradients A & B. Gradient B was developed because initial
purification using standard gradients (Gradient A) proved to be
less than satisfactory. The experimental conditions are as
described below except that some attempts were made to improve
purifications by the addition of 20% formamide to the running
buffers during some of the purifications. This procedure was
abandoned since it did not appear to produce any beneficial
results. Curiously however, careful review of the data suggested
that the HPLC artifacts previously thought to correlate with the
structure of certain probes (See: Provisional Patent Application
No. 60/063,283 filed on Oct. 27, 1997) was also found to correlate
with the presence of formamide during the purification. Therefore,
no correlation is now believed to exist between structure of the
PNA probe and the HPLC profiles observed for the purified
oligomers.
[0183] Gradients A & B
[0184] Buffer A=0.1% TFA in water.
[0185] Buffer B-0.1% TFA in acetonitrile.
[0186] Flow Rate: 0.2 mL/min.
[0187] Column Temperature: 60.degree. C.
[0188] Instrument: Water 2690 Alliance: Control by Waters
Millennium Software
[0189] Stationary Phase: Waters Delta Pak C18, 300 .ANG., 5 .mu.m,
2.times.150 mm (P/N WAT023650)
[0190] Detection at 260 nm.
1 Gradient Profile A Time (min.) Percent Buffer A Percent Buffer B
Curve 0.00 100 0 0 4.00 100 0 6 22.00 80 20 6 38.00 40 60 6 40.00
20 80 11
[0191]
2 Gradient Profile B Time (min.) Percent Buffer A Percent Buffer B
Curve 0.00 90 10 0 40.00 60 40 6 50.00 20 80 6
[0192] Mass analysis:
[0193] Samples were analyzed using a linear Voyager Delayed
Extraction Matrix Assisted Laser Desorption Ionization-Time Of
Flight (DE MALDI-TOF) Mass spectrometer (PerSeptive Biosystems,
Inc.). Sinipinic acid was used as the sample matrix and also used
as one point for calibration of the mass axis. Bovine insulin was
used as an internal calibration standard for the second calibration
point of the mass axis.
[0194] Samples were generally prepared for analysis by first
preparing a solution of sinipinic acid at a concentration of 10
mg/mL in a 1:2 mixture of acetonitrile and 0.1% aqueous
trifluoroacetic acid. Next, an insulin solution was prepared by
dissolving 1 mg of bovine insulin (Sigma) in 0.1% aqueous
trifluoroacetic acid. Finally, an insulin/matrix solution was then
prepared by mixing 9 parts of the sinipinic acid solution to 1 part
of the bovine insulin solution. Samples were prepared for analysis
by spotting 1 .mu.L of the insulin/matrix solution followed by
spotting 1 .mu.L of diluted sample (approximately 0.1 to 1 OD per
mL) onto the mass spectrometer target. The instrument target was
allowed to dry before being inserted into the mass
spectrometer.
[0195] Tables of PNA Oligomers Prepared for Study
3TABLE 1 Probes Prepared To Evaluate PNA Molecular Beacons
(Hairpins) Probe Desc. CODE.sup.1 PNA Probe Sequence N-terminal Arm
Forming Segments .001 5205
Flu-O-TGG-AGO-OAC-GCC-ACC-AGC-TCC-AK(dabcyl)-NH.sub.2 .007 5105
Flu-O-TGG-AGO-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH.sub.2 .010 5005
Flu-O-TGG-AGA-CGC-CAC-CAG-CTC-CAK(dabcyl)-NH.sub.2 .002 3203
Flu-O-TGG-OOA-CGC-CAC-CAG-CTC-CAK(dabcyl)-NH.sub.2 .008 3103
Flu-O-TGG-OAC-GCC-ACC-AGC-TCC-AK(dabcyl)-NH.sub.2 .009 4004.sup.2
Flu-O-TGG-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH.sub.2 C-terminal Arm
Forming Segments .018 7027
Flu-O-ACG-CCA-CCA-GCT-CCA-OO-GTG-GCG-T-K(dabcyl)-NH.sub.2 .011A
5025 Flu-O-ACG-CCA-CCA-GCT-CCA-OOC-GCG-TK(dabcyl)-NH.sub.2 .006
3023 Flu-O-ACG-CCA-CCA-GCT-CCA-OOC-GTK(dabcyL)-NH.sub.2 Probing
Sequence External to the Arm Sequences .017 5115
Flu-O-TAG-CAO-ACG-CCA-CCA-GCT-CCA-OTG-CTA-K(dabcyl)-NH.sub.2 .005
3113 Flu-O-TAG-O-ACG-CCA-CCA-GCT-CCA-O-CTA-K(dabcyl)-NH.sub.2
Control Probes; No Arm Forming Segments .003 0000
Flu-O-ACG-CCA-CCA-GCT-CCA-K(dabcyl)-NH.sub.2 .004 0110
Flu-OO-ACG-CCA-CCA-GCT-CCA-OK(dabcyl)-NH.sub.2
[0196] 1. The CODE is a simple means to determine the length of the
complementary nucleobases at the amine and carboxyl termini of the
PNA polymer and the number and location of any
8-amino-3,6-dioxaoctanoic acid flexible linker units. The probing
nucleobase sequence is the same for all probes listed in the table.
The first digit in the CODE represents the length of the N-terminal
arm segment which is complementary to the C-terminal arm segment.
The second digit in the CODE represents the number of flexible
linker units which link the N-terminal arm to the probing
nucleobase sequence. The third digit in the CODE represents the
number of flexible linker units which link the C-terminal arm to
the probing nucleobase sequence. The fourth digit in the CODE
represents the length of the C-terminal arm segment which is
complementary to the N-terminal arm segment. Consequently, the CODE
can be used to visually compare the general structure of the
different PNA oligomers listed in Table 1.
[0197] 2. A coincidental, 4 bp. overlap between the nucleobases at
the amine and carboxyl termini are present in this construct
instead of the directly comparable 3 bp. overlap.
[0198] 3. PNA sequences are written from the amine to the carboxyl
terminus. Abbreviations are: Flu=5-(6)-carboxyfluorescein,
dabcyl=4-((4-(dimethylamino)phenyl)azo)benzoic acid,
O=8-amino-3,6-dioxaoctanoic acid; K=the amino acid L-Lysine
Example 13
Synthesis of DNA Oligonucleotides Prepared for Study
[0199] For this study, biotin labeled DNA oligonucleotides suitable
as nucleic acids comprising a target sequence which are
complementary to the PNA probing sequence of the k-ras PNA probes
were either synthesized using commercially available reagents and
instrumentation or obtained from commercial vendors. All DNAs were
purified by conventional methods. The sequences of the DNA
oligonucleotides prepared for Examples 14-20 are illustrated in
Table 2. Methods and compositions for the synthesis and
purification of synthetic DNAs are well known to those of ordinary
skill in the art.
4TABLE 2 DNA Targets Description Target DNA Sequence wt k-ras
Biontin-GTG-GTA-GTT-GGA-GCT-GGT-GGC-GTA Seq. Id. No.1 mu k-ras
Biotin-GTG-GTA-GTT-GGA-GCT-TGT-GGC-GTA Seq. Id. No.2 Univ. Comp.
Biotin-ACT-CCT-ACG-GGA-GGC-AGC Seq. Id. No.3
[0200] The difference between the wild type (wt) and mutant (mu)
target sequences is only a single base (a G to T point mutation.
The position of the point mutation is depicted in bold underlined
script. These nucleic acid targets are illustrated from the 5' to
3' terminus.
[0201] Initial Experimental Analysis of PNA Molecular Beacons
[0202] In the initial experiments using a fluorescence detection
instrument and PNA oligomers 0.001 and 0.002, it was determined
that the PNA constructs have very little intrinsic fluorescence at
room temperature. However, upon hybridization of either PNA
oligomer to its complementary target sequence, an increasing
fluorescent signal was observed.
Example 14
Hybridization Experiments
[0203] Amounts of PNA oligomer and target DNA used in this
experiment are recorded in Table 3. The PNA oligomer and/or the
target DNA was mixed in 20 .mu.l of Hybridization Buffer (50 mM
Tris-HCl, pH 8.3; 100 mM NaCl) and heated to 95.degree. C. for 10
minutes. After cooling slowly to room temperature, the mixture was
diluted to a total volume of 4 mL (vol. needed in cuvette for
measurement). Control samples containing Hybridization Buffer (Hyb.
Buffer) and the individual DNA or PNA oligomers were also examined
under identical conditions. Additionally, a fluorescein labeled PNA
without quencher or arm forming segment was included
(Flu-OO-ACG-CCA-CCA-GCT-CC A-NH.sub.2; "F-PNA"). The experimental
measurements which were recorded are reproduced in Table 3.
[0204] With reference to Table 3, there was a low background
fluorescence from the individual components of the test system
(e.g. hybridization buffer, single stranded DNA, and PNA
oligomers). When target DNA and PNA oligomer was mixed and allowed
to hybridize, a significant increase in fluorescent signal was
detected. Moreover, the intensity of fluorescent signal varied as
the relative concentrations of the target DNA and PNA oligomer was
altered. Consequently, the data demonstrates that hybridization of
the PNA oligomers to the complementary DNA target generated very
intense fluorescent signal.
[0205] The signal obtained using PNA oligomer 0.002 (3 bp. stem)
was between 29 and 83% higher than the signal observed for the PNA
oligomer 0.001 (5 bp. stem) (Compare: data in rows 2,7 and 12 with
data in rows 1, 6 and 11). However, as demonstrated by the greater
fluorescent intensity observed for the control probe (F-PNA), the
presence of the quenching moiety attached to the PNA oligomer
results in a significant quenching effect (Compare: data in rows 3,
13 and 16 with data in rows 1-2, 6-7 and 11-12). Because the
fluorescence of the control probe F-PNA was so intense, it was
diluted to obtain fluorescent signal which was comparable in
intensity to the data obtained using a PNA oligomer having an
linked dabcyl quencher moiety. Specifically, the signal obtained
with the fluorescein labeled control PNA oligomer (F-PNA) was
approximately two to three times the greatest intensity of the
signal generated from the PNA oligomers containing a quencher
moiety.
5TABLE 3 Ex. L(490) Row No. Assay Components pmol 521 nm 1 wt k-ras
DNA/PNA .001 125/25 300 2 wt k-ras DNA/PNA .002 125/25 447 3 wt
k-ras DNA/F-PNA 12.5/2.5 87 4 PNA .001 25 1 5 PNA .002 25 1 6 wt
k-ras DNA/PNA .001 25/25 189 7 wt k-ras DNA/PNA .002 25/25 345 8 wt
k-ras DNA/F-PNA 12.5/12.5 423 9 PNA .001 25 1 10 PNA .002 25 1 11
wt k-ras DNA/PNA .001 25/125 353 12 wt k-ras DNA/PNA .002 25/125
455 13 wt k-ras DNA/F-PNA 2.5/12.5 373 14 PNA .001 125 25 15 PNA
.002 125 38 16 wt k-ras DNA/F-PNA 12.5/12.5 423 17 wt k-ras DNA 125
-3 18 Hybridization Buffer -- -5
Example 15
Titration of PNA Oligomer with Nucleic Acid Target
[0206] In another experiment, 50 pmol of PNA oligomers 0.001 and
0.002 were mixed with differing amounts of nucleic acid target
(0-200 pmol) in a total volume of 20 .mu.l of Hyb. Buffer. The
mixtures were then heated to 95.degree. C. for 10 minutes and
cooled slowly to ambient temperature. The samples were diluted into
a total volume of 4 mL and excitation/emission at 493/521.6 nm was
recorded using a RF-5000 spectrofluorophotometer (Shimadzu).
Results are illustrated graphically in FIG. 1.
[0207] With reference to FIG. 1, the fluorescent signal generated
from the sample continuously increased with the addition of target
sequence until a concentration of 40-60 pmol was present (50 pmol
PNA oligomer was used in the assay). There was no significant
increase in fluorescent signal as the amount of target sequence was
increased between 60-200 pmol. Consequently, the data demonstrates
that the signal generated in proportional to the amount of target
sequence added, thereby indicating that the production of the
signal was caused by the hybridization of the PNA oligomer to the
target nucleic acid.
Example 16
Kinetics of Hybridization for PNA Molecular Beacons
[0208] In this experiment, 100 pmol (5 .mu.L of 20 pmole/.mu.L) of
wt k-ras DNA (ssDNA oligonucleotide) was mixed with 4 mL
Hybridization Buffer in a cuvette and adjusted to ambient
temperature. Next, 50 pmol (2.5 .mu.L of 20 pmole/.mu.L) of PNA
oligomer 0.002, was added and the excitation/emission at 493/521.6
run was recorded RF-5000 spectrofluorophotometer (Shimadzu). The
data obtained is graphically illustrated in FIG. 2.
[0209] With reference to FIG. 2, the results demonstrate that the
PNA Molecular Beacon hybridizes to the target DNA present in the
sample to thereby generate a fluorescent signal with measurable
kinetics. The generation of fluorescent signal occurred with an
initial rate of 7.2 relative light units (rlu)/minute. After 120
minutes of hybridization the signal was 595 rlu. The kinetic
profile of the increase in fluorescent intensity is strongly
indicative of hybridization of the PNA oligomer to the target
nucleic acid.
Example 17
Hybridization Related to the Composition of the DNA Target
[0210] In this experiment, 50 pmol of either wt k-ras or mu k-ras
DNA, in hybridization buffer, was mixed with 50 pmol PNA Molecular
Beacon 0.001 in hybridization buffer. As a control, 50 pmol of a
totally unrelated target DNA oligonucleotide, mixed with 50 pmol of
PNA Molecular Beacon 0.001, was also examined (Univ. Comp., See:
Table 2). Excitation/emission at 493/521.6 run was recorded was
recorded RF-5000 spectrofluorophotometer (Shimadzu). The results
are presented graphically in FIG. 3.
[0211] With reference to FIG. 3, the increasing fluorescent
intensity observed when mixing PNA 0.001 and the complementary
target sequence demonstrates that the PNA Molecular Beacon
hybridized to its perfect complement wt k-ras DNA. The rate of
fluorescent signal generation and the maximal fluorescent signal
generated was significantly lower for the sample when the
non-complementary mu k-ras DNA (having a single point mutation as
compared with the wild type target) was substituted in the assay.
Furthermore, mixing a totally unrelated nucleic acid sequence
(Univ. Comp) with PNA Molecular Beacon 0.001 did not produce any
fluorescent signal, even after heating the sample to 95.degree. C.
Consequently, the data strongly demonstrates that the generation of
the fluorescent signal in the assay was directly related to the
sequence specific interactions between the PNA Molecular Beacon and
the nucleic acid present in the sample.
Example 18
Effect of Blocking Probes on Sequence Discrimination
[0212] This experiment was designed to increase discrimination of
the PNA oligomer 0.001 by introducing some unlabeled "blocking PNA"
into the hybridization mixture. This "blocking PNA" has a sequence
complementary to the mu k-ras DNA and should, therefore,
effectively compete with the labeled PNA oligomer 0.001 for binding
to the non-target mu k-ras DNA. The unlabeled "blocking PNA" had
the sequence:
[0213] Blocking PNA: H-ACG-CCA-CAA-GCT-CCA-NH.sub.2
[0214] In this experiment, 50 pmol of wt k-ras or mu k-ras DNA was
mixed with 50 pmol PNA oligomer 0.001. To one pair of reactions was
added 500 pmol "blocking PNA" (data in Table 4, row 2). The samples
were heated to 95.degree. C. and then cooled slowly to RT.
Excitation/emission at 493/521.6 nm were recorded. The results
obtained are reproduced in Table 4.
[0215] With reference to Table 4, in the absence of blocker PNA
there is a 2 fold difference (discrimination factor) between the
fluorescent signal generated when the PNA oligomer hybridizes to
the perfectly complementary wt k-ras DNA as compared with signal
generated upon hybridization to non-complementary mutant k-ras DNA
(single point mutation). Addition of "blocking PNA" increases the
discrimination of the assay by approximately 10 fold (Compare data
in Table 4, the discrimination factor (DF) is increased to 19.6
from 2). This increase in discrimination occurred despite an
approximately 50% reduction in the signal generated by
hybridization to the complementary wt k-ras DNA. However, as
demonstrated by analysis of the data in Table 4, hybridization of
the PNA probe to the non-complementary mu k-ras DNA was effectively
eliminated in the presence of the "blocking PNA". Consequently, the
data again strongly illustrates that the generation of fluorescent
signal was directly related to the sequence specific hybridization
of the nucleic acid to the PNA oligomer. The data further
demonstrates the utility of using blocking probes to enhance
sequence discrimination in nucleic acid hybridization assays when
utilizing PNA Molecular Beacons.
6TABLE 4 Row wt k-ras DNA/ mu k-ras DNA/ No. Conditions PNA .001
PNA .001 DF 1 0 pmol Blocking PNA 541 271 2 2 500 pmol Blocking PNA
255 13 19.6
[0216] Conclusions From the Initial Experimental Data:
[0217] PNA Molecular Beacons can be prepared. They have low
intrinsic fluorescent intensity until hybridized to a complementary
or substantially complementary target nucleic acid. Non-specific
interactions can be eliminated, if desired, using a "blocking PNA".
Thus, we have demonstrated that it is possible to prepare
functional PNA Molecular Beacons which exhibit a good specificity
which can be further enhance by the application of "blocker
probes". Consequently, the PNA oligomer constructs investigated in
these initial experiments demonstrate the effective use of PNA
Molecular Beacons in probe-based hybridization assays.
Example 19
Structural Analysis of PNA Hairpins and Multimers
[0218] The reference entitled "Hairpin-Forming Peptide Nucleic Acid
Oligomers", Armitage et al., Biochemistry, 37: 9417-9425 (1998) is
admitted as prior art to this Example 19 only. It has been recently
reported in the scientific literature that PNAs form hairpin
structures (See: Armitage et al.). Using the hairpin forming PNA
(referred to as "PNA1" in the reference (See: Scheme 3: col. 1, p.
9419), hereinafter referred to as "PNAD") described in the
literature as a model, numerous PNA and analogous DNA oligomers
were prepared and their properties examined in order to obtain a
basis for understanding the physical behavior of PNA hairpins and
multimers. We anticipated this would allow us to better interpret
the results reported in our priority application (U.S. Ser. No.
08/958532). The data presented in this Example 19 demonstrates that
PNA hairpins will form if designed to have long stems (e.g. 9
subunits). However PNA hairpins having shorter stems (e.g. 6
subunits) do not form hairpins as readily as their DNA
counterparts. Furthermore, applicants have observed that the
formation and stability of a PNA hairpin is not substantially
affected by the presence or absence of magnesium or the ionic
strength of the buffer as are DNA hairpins (See Tyagi et al.,
Nature Biotechnology, 14: 303-308 (1996) at p. 305, col. 1, Ins
1-16). Nevertheless, PNA hairpins having stem duplexes of 9
subunits in length exhibit poor signal to noise ratios (less than 4
to 1) upon melting and, contrary to the findings of Armitage et
al., do not appear to substantially hybridize to complementary
nucleic acid. Consequently, PNA hairpins which have long stems
(e.g. 7 or greater subunits), do not appear to be ideally suited
for the analysis of nucleic acids.
[0219] Materials and Methods:
[0220] Probes
[0221] PNAs were prepared and purified as described herein. Labeled
and unlabeled DNA oligonucleotides were synthesized using
commercially available reagents and instrumentation. Dabcylated
DNAs were prepared using the dabcyl synthesis support available
from Glen Research (P/N 20-5911) and other commercially available
DNA reagents and instrumentation. The Fluoredite phosphoramidite
(PerSeptive Biosystems, Inc., P/N GEN080110) was used to label DNAs
with 5(6)carboxyfluorescein. All DNAs were purified by conventional
methods. The DNA and PNA probe compositions are presented in Table
5. Tm data for DNA probes is summarized in Table 6 and the Tm data
for PNA probes is summarized in Table 7. Tm data for both the
melting "M" and the reannealing "R" is presented in Table 6 and
7.
[0222] Preparation of Dilution Series of PNA and DNA Probes for Tm
Analysis
[0223] Purified PNA probes were dissolved in 1:1 DMF/H.sub.2O at
0.05 OD (260 nm) per 20 .mu.L to prepare the PNA Probe Stock.
Purified DNA probes were dissolved in 4:1H.sub.2O/acetonitrile at
0.05 OD (260 nm) per 20 .mu.L to prepare the DNA Probe Stock. Based
on calculated extinction coefficients, the appropriate amount of
PNA Probe Stock or DNA Probe Stock was added to 5 mL of Tm Buffer
(10 mM sodium phosphate, pH 7.0) to prepare a solution of
approximately 7.5 .mu.M of the one or two oligomers needed to
perform the Tm analysis of the unimolecular or bimolecular system.
From this solution was taken 2.5 mL which was added to 2.5 mL of Tm
buffer to thereby prepare the second concentration of a dilution
series of Tm Samples. The remaining 2.5 mL of the first sample was
used for Tm analysis. Serial dilution the samples in Tm Buffer was
continued in this fashion until 2.5 mL of Tm Samples at
concentrations of approximately 7.5 .mu.M, 3.75 pM, 1.87 .mu.M,
0.94 pM and 0.468 .mu.M (5 mL) were prepared. A Tm analysis of
these solutions was then performed as described below.
[0224] Tm Analysis
[0225] 1. Tm Buffer:
[0226] The five Tm Samples of a dilution series of a particular
unimolecular or bimolecular system to be analyzed were
simultaneously examined using a Cary 100 Bio UV-Visible
Spectrophotometer (Varian Instruments) equipped with a 6.times.6
thermostatable multicell block running Win UV Bio application
software package. To a 10.times.10 UV cell (Starna Cells, P/N
21-Q-10) was added a 7.2 mm stir bar and the 2.5 mL of each sample
of the dilution series. The stirring accessory was used during all
analysis. All samples were thermally denatured and reannealed prior
to data collection by having the instrument rapidly ramp the
temperature to a point above the melting temperature and then
holding that temperature for 5-10 minutes before returning to the
starting temperature. Data for both dissociation and reannealing
was collected and analyzed. The temperature range over which data
was collected was varied in response to the expected Tm which was
roughly determined during the premelt and prereannealing step.
Regardless of the temperature range, the temperature ramp rate for
both dissociation and reannealing was always 0.5.degree. C./min.
The absorbance (260 nm, averaged over a 3 second collection) was
plotted vs. the temperature of the multicell block.
[0227] 2. Tm Buffer and 1 mM MgCl.sub.2:
[0228] After the Tm analysis was performed in Tm Buffer, to each
cell was added 0.5 .mu.L of 5M MgCl.sub.2 to thereby prepare a
sample containing 1 mM MgCl.sub.2. The dilution effect was
considered to be negligible. The Tm analysis was then performed
again to determine whether the presence of MgCl.sub.2 had any
effect on the Tm of the unimolecular or bimolecular system.
[0229] 3. Tm Buffer. 1 mM MgCl.sub.2 and 100 mM NaCl:
[0230] After the Tm analysis was performed in Tm Buffer and 1 mM
MgCl.sub.2, to each cell was added 42 .mu.L of saturated NaCl
(approximately 6.11 M/L). The dilution effect was again considered
to be negligible. The Tm analysis was then performed again to
determine whether the presence of approximately 100 mM NaCl had any
effect on the Tm of the unimolecular or bimolecular system.
[0231] Results and Discussion:
[0232] With reference to Table 5, the sequences of complementary
labeled PNA probes P1 and P2 are illustrated. These probes
hybridize to form a 9 subunit duplex identical to the stem duplex
formed in PNA hairpins, P3 and P4. Therefore, P1 and P2 form a
bimolecular system for comparison with the unimolecular PNAs, P3
and P4. Probes P3 and P4 differ only in that the subunits which
form the loop of P4 have been replaced with flexible linkages in
P3. Unlabeled versions of all four PNA probes have likewise been
prepared so as to understand the effects of labels on Tm. In Table
5, the unlabeled probes are designated with an "N" for no
label.
[0233] Also prepared for comparison is the PNA probe "PNAD" which
Armitage et al. teach will form a hairpin. Applicants have also
prepared a version of the PNAD probe which possess arm segments of
6 subunits (PNAD 6S) as compared with the 9 subunits self
complementary arm segments of the PNAD probe. The PNA probe P5 is
complementary to the DNA probe D5B. This bimolecular complex was
prepared to determine its Tm since Armitage et al. teach that this
short 12-mer nucleic acid sequence will hybridize to the PNAD probe
thereby opening the 9 subunit stem.
7TABLE 5 Sequence Probe Sequence ID Seq Id. No. Peptide Nucleic
Probes Flu-O-ATA-TAT-TGG-EE-NH.sub.2 P1
Ac-O-ATA-TAT-TGG-EE-NH.sub.2 P1N
Ac-EE-CCA-ATA-TAT-K(dabcyl)-NH.sub.2 P2 Ac-EE-CCA-ATA-TAT-NH.sub.2
P2N Flu-OEE-ATA-TAT-TGG-OO-CCA-ATA-TAT-- EE-K(dabcyl)-NH.sub.2 P3
H.sub.2N-OEE-ATA-TAT-TGG-OO-CCA-ATA-TAT-EE- K-NH.sub.2 P3N
Flu-OEE-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-EE-K(dabcyl)-- NH.sub.2 P4
H.sub.2N-OEE-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-EEK-NH.sub.- 2 P4N
H.sub.2N-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-KK-NH.sub.2 PNAD
H.sub.2N-TAT-TGG-CTG-ATC-CAA-TA-KK-NH.sub.2 PNAD 6S
H.sub.2N-TTG-GCT-GAT-CCA-NH.sub.2 P5 Synthetic
Oligodeoxynucleotides Flu-ATA-TAT-TGG-OH D1 Seq. Id. No.4
HO-ATA-TAT-TGG-OH D1N Seq. Id. No.5 HO-CCA-ATA-TAT-(dabcyl) D2 Seq.
Id. No.6 HO-CCA-ATA-TAT-OH D2N Seq. Id. No. 7
Flu-ATA-TAT-TGG-(spacer)-CCA-ATA-TAT-dabCyl D3 Seq. Id. No. 8
HO-ATA-TAT-TGG-(spacer)-CCA-ATA-TAT-OH D3N Seq. Id. No. 9
Flu-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-dabCyl D4 Seq. Id. No.10
HO-ATA-TAT-TGG-CTG-ATC-CAA-TAT-AT-OH D4N Seq. Id. No.11
Bio-TGG-ATC-AGC-CAA-OH D5B Seq. Id. No.12 HO-ATA-TAT-TGG-ATC-AGC-C-
AA-TAT-AT-OH D6 Seq. Id. No.13
[0234] PNA sequences are written from the amine to the carboxyl
terminus. DNA sequences are written from the 5' to 3'.
Abbreviations are: Flu=5-(6)-carboxyfluorescein,
dabcyl=4-((4-(dimethylamino)phenyl)azo)benz- oic acid,
O=8-amino-3,6-dioxaoctanoic acid; K=the amino acid L-Lysine and "E"
is the Solubility Enhancer "compound "4" which has been described
in: Gildea et al., Tett. Lett. 39 (1998) 7255-7258. The "spacer"
used for the DNAs was commercially available C3 spacer
phosphoramidite Glen Research (P/N 10-1913).
[0235] With reference to Table 5, the sequences for the synthetic
oligodeoxynucleotides prepared for examination are also
illustrated. DNAs which were conceptually the most equivalent
labeled and unlabeled versions of P1, P2, P3, P4, P1N, P2N, P3N and
P4N were prepared for comparison. As discussed, D5B is a complement
to P5 and D6 is the complement to D4, P4 and PNAD.
8 TABLE 6 UV Tm Analysis [1] [2] [3] [4] [5] Probes (Conditions) M
R M R M R M R M R D1N/D2N (Buf) <10 <10 <10 <10 <10
<10 <10 <10 <10 <10 D1N/D2N (Buf, Mg) 17.1 16.8 14.6
14.8 14.0 13.4 11.4 11.7 <10 <10 D1N/D2N (Buf, Mg & Na)
22.3 21.7 20.7 18.9 19.0 18.2 15.0 15.0 14.2 13.4 D1/D2 (Buf) 15.0
14.8 13.5 13.3 12.0 12.9 11.5 9.9 <10 <10 D1/D2 (Buf, Mg)
24.1 23.8 22.0 21.8 21.0 20.9 19.5 19.9 16.5 17.9 D1/D2 (Buf, Mg
& Na) 29.0 28.4 27.5 26.4 26.0 25.5 23.4 23.0 21.4 20.0 D3N
(Buf) 40.0 40.5 40.5 40.5 40.5 40.5 40.5 41.2 40.5 41.2 D3N (Buf,
Mg) 44.6 44.7 44.6 45.3 44.6 44.8 45.5 45.4 45.5 44.9 D3N (Buf, Mg
& Na) 48.1 48.3 48.6 48.8 49.0 49.4 48.5 49.5 48.0 48.6 D3
(Buf) 43.7 43.6 44.3 44.1 44.3 44.6 43.9 44.0 43.9 44.0 D3 (Buf,
Mg) 50.6 49.9 50.6 49.9 51.1 50.5 51.0 50.5 51.5 51.1 D3 (Buf, Mg
& Na) 54.1 53.8 55.6 53.8 54.6 54.0 54.5 54.5 54.5 54.1 D4N
(Buf) 41.6 41.7 42.1 42.2 42.5 42.3 42.5 42.8 42.0 43.4 D4N (Buf,
Mg) 51.6 51.2 52.1 52.3 52.5 52.4 52.5 53.5 52.5 53.0 D4N (Buf, Mg
& Na) 54.1 54.4 55.1 54.9 55.1 55.0 55.0 55.0 55.0 55.1 D4
(Buf) 45.2 43.4 45.6 44.9 45.6 45.0 45.5 44.5 45.5 44.1 D4 (Buf,
Mg) 54.6 53.9 55.5 55.0 55.5 55.0 54.5 54.1 55.0 54.6 D4 (Buf, Mg
& Na) 57.1 56.5 57.5 57.5 58.0 57.5 58.5 57.6 58.0 57.6
[0236] With reference to Table 6, the Tm for the labeled and
unlabeled bimolecular systems D1/D2 and D1N/D2N, respectively, are
concentration dependent as can be seen by the 8-10.degree. C.
difference between the most and least concentrated samples where Tm
data is available. The low and unmeasurable Tm values under
conditions of low ionic strength are expected for such a short
nucleic acid duplex. However, there is a remarkable stabilizing
effect of approximately 7.degree. C. due to the presence of the
dabcyl/fluorescein labels as can be seen by comparison of the Tm
values at all concentrations and under all conditions examined.
This was a very surprising result.
[0237] The Tm of labeled (D3 and D4) and unlabeled (D3N and D4N)
unimolecular probes exhibited a concentration independent Tm.
Consequently, the data indicates that these DNAs exist as hairpins
in solution. As taught by Tyagi et al., the hairpin stem duplex of
these DNA probes is substantially stabilized by both the addition
of magnesium and an increase in ionic strength (See: Tyagi et al.
Nature Biotechnology, 14: 303-308 (1996) at p. 305, lns. 1-16).
Surprisingly the stem duplex D4 (which contained a polynucleotide
loop) was more stable under all conditions examined than was the
stem duplex of D3 which contained a flexible linkage. Also
noteworthy is the stabilizing effect attributable to the
fluorescein/dabcyl pair. For D4 the stabilizing effect is
approximately 3-4.degree. C. whereas it is approximately
5-6.degree. C. for D3. Thus, the data indicates that as the Tm of
the stem duplex increases there is less of a stabilizing effect
attributable to the dabcyl/fluorescein interactions. Nevertheless,
this observation is very surprising and suggests that the
interaction between the dyes is very strong.
9 TABLE 7 UV Tm Analysis [1] [2] [3] [4] [5] Probes (Conditions) M
R M R M R M R M R P1N/P2N (Buf) 57.1 56.5 55.0 54.6 53.0 52.6 51.5
50.6 48.5 48.7 P1N/P2N (Buf, Mg) 57.1 56.4 55.1 54.5 53.0 53.0 51.0
50.5 49.0 48.6 P1N/P2N (Buf, Mg & Na) 57.6 57.0 55.4 55.1 53.5
53.1 52.0 51.1 49.5 49.2 P1/P2 (Buf) 61.1 60.4 59.1 58.5 57.0 57.0
55.5 54.5 51.5 52.6 P1/P2 (Buf, Mg) 61.1 60.4 59.1 59.0 57.5 57.0
55.5 55.0 52.5 53.1 P1/P2 (Buf, Mg & Na) 61.5 60.9 59.6 59.5
58.0 57.0 56.0 55.5 54.0 53.6 P3N (Buf) 82.6 82.3 82.5 82.4 83.0
82.4 83.0 82.5 83.0 82.6 P3N (Buf, Mg) 83.1 82.4 83.1 81.9 83.0
82.5 83.0 83.1 83.0 82.6 P3N (Buf, Mg & Na) 83.1 82.9 83.1 81.4
83.5 82.9 83.5 83.0 83.5 83.1 P3 (Buf) 82.1 81.9 82.6 82.0 82.6
82.0 83.0 82.6 82.0 81.6 P3 (Buf, Mg) 82.1 81.8 82.1 81.8 82.5 82.4
82.5 82.0 83.0 83.5 P3 (Buf, Mg & Na) 83.1 82.9 83.5 83.0 84.0
83.0 83.5 83.0 83.5 nd P4N (Buf) 81.6 81.4 82.1 81.5 82.1 81.5 82.5
81.1 82.0 80.1 P4N (Buf, Mg) 81.6 81.3 81.6 81.3 82.1 81.4 82.0 nd
82.0 81.1 P4N (Buf, Mg & Na) 82.1 82.3 82.1 82.3 82.5 nd 82.0
82.0 84.0 81.6 P4 (Buf) 81.6 80.9 81.6 81.4 82.1 81.5 82.0 81.5
81.5 81.1 P4 (Buf, Mg) 81.6 81.0 81.6 81.5 82.0 81.5 82.0 81.1 82.0
81.6 P4 (Buf, Mg & Na) 82.6 81.8 82.6 82.4 82.6 82.5 82.5 82.5
83.0 82.1 PNAD (Buf) 81.1 80.4 81.1 81.0 82.0 81.0 81.5 81.5 81.5
81.1 PNAD (Buf, Mg) 80.6 80.3 81.1 80.4 81.1 80.4 80.5 81.0 80.5
80.5 PNAD (Buf, Mg & Na) 80.6 80.4 81.5 nd 81.0 81.4 81.5 80.5
82.1 80.6 PNAD 6S (Buf) 75.0 74.6 75.0 74.1 74.5 73.5 73.5 65.5
67.5 57.5
[0238] With reference to Table 7, Tm data for the PNA constructs is
presented. The Tm values for the PNAs are substantially higher than
for the comparable DNAs. Both the labeled (P1/P2) and unlabeled
(P1N/P2N) bimolecular systems exhibited Tms which were
concentration dependent as is evident by the 8-10.degree. C.
difference in Tm between the most and least concentrated samples.
Again there was a increase of approximately 3-4.degree. C. in Tm
which was attributable to the presence of the fluorescein/dabcyl
moieties. Though clearly dependent upon concentration, the
stability of the duplexes were not substantially affected by the
presence or absence of magnesium or the ionic strength of the
buffer since there was no substantial difference in Tm under any of
the three conditions examined. Most importantly no substantial
hysteresis was observed in the analysis of labeled or unlabeled PNA
bimolecular systems even at the lowest concentration examined. The
lack of hysteresis indicates that the duplex reforms readily.
[0239] For comparison fluorescence analysis of the least
concentrated sample of the P1/P2 bimolecular system was performed.
The least concentrated sample (sample at [5] which had 1 mM
MgCl.sub.2 and 100 mM NaCl) was analyzed for fluorescence
essentially as described in Example 20, below, except that
excitation was at 415 nm and emission was recorded at 520 nm.
Normalized data for fluorescence vs. temperature and absorbance vs.
temperature are overlaid in FIG. 4A. Though the shape of the curves
is similar the data is not superimposible. A similar result was
observed when the absorbance vs. temperature and fluorescence vs.
temperature data for the D1/D2 system was overlaid (data not
shown). The structural basis for this lack of superimposibility is
not known but appears to be consistent for the bimolecular
systems.
[0240] Both the labeled and unlabeled versions of P3 and P4
exhibited a concentration independent Tm. Consequently, the data
indicates that these PNAs form hairpins in solution. Likewise the
probe PNAD also was confirmed to exhibit a concentration
independent Tm of approximately 81-82.degree. C., as had been
reported by Amitage et al. The data clearly demonstrates that the
stem duplex of a PNA hairpin is not substantially affected by the
presence or absence of magnesium or the increase in ionic strength
since the Tm for the probes are the same without regard to the
buffer composition in which the Tm analysis was performed.
Curiously there was no substantial difference in the Tm of labeled
as compared with unlabeled probes. However, it is believed that the
Tm of these duplexes is so high that the fluorescein/dabcyl
interactions cannot be maintained.
[0241] As with the P1/P2 and D1/D2 bimolecular systems,
fluorescence vs. temperature analysis of the least concentrated
samples (each probe at [5] which had 1 mM MgCl.sub.2 and 100 mM
NaCl) of both probes P3 and P4 were performed. With reference to
FIGS. 4B and 4C, normalized fluorescence vs. temperature and
absorbance vs. temperature data are overlaid for P3 and P4,
respectively. Unlike the bimolecular system, the fluorescence vs.
temperature and absorbance vs. temperature data for both P3 and P4
are superimposible. Data was also collected for the D3 and D4
unimolecular probes. The data for these unimolecular probes was
also found to be highly superimposible (data not shown) thereby
indicating they result from the same physical transition of the
probe. Taken as a whole, the excellent correlation between the
fluorescence vs. temperature and absorbance vs. temperature data in
both the DNA and PNA unimolecular systems strongly indicates that
increases in absorbance and fluorescence occur as result of a helix
to coil transition.
[0242] Using the data obtained from melting and reannealing of
D1/D2, D3, D4, P1/P2, P3 and P4 as described above, the difference
between the lowest (helix) and highest (coil) fluorescence
intensities recorded were calculated to determine the signal to
noise value for each probe. This was intended to give an estimate
of the potential increase in signal which could be expected in a
hybridization assay wherein the stem of the probe was opened. In
FIG. 5, the fluorescence signal to noise ratios for melting and
reannealing the PNA and DNA bimolecular and unimolecular systems is
presented. Most striking is the significantly lower signal to noise
ratio for all the PNA systems as compared with the DNA systems. The
low signal to noise ratio is consistent with the data presented by
Armitage et al. though it is not clear that the labeled probes of
Armitage et al. form hairpins. Nevertheless, the low signal to
noise ratios for the PNA probes comprising long self-complementary
arm segments suggests that these constructs are not optimal for
analysis of nucleic acids.
[0243] Though the Tm of labeled and unlabeled hairpins having an
identical 9 bp stem duplex where all very similar (approximately
81-83.degree. C.), normalized data presented in FIG. 6 demonstrates
that several factors can influence thermodynamic parameters of the
stem duplex. In FIG. 6, normalized absorbance vs. temperature data
for melting of probes P3N, P4N and PNAD (each probe at [1]) is
graphically illustrated. As these probes were all unlabeled and
comprised the same nucleobase sequence there were directly
comparable. Probe P3N which comprises a flexible linkage which
links the two arms which form the stem duplex exhibited the most
cooperative sigmoidal transition. Surprisingly, the solubility
enhanced probe, P4, exhibited only a slightly less cooperative a
transition as compared with probe P3. The probe PNAD exhibited the
least cooperative sigmoidal transition.
[0244] The shape of the sigmoidal transition evident in absorbance
vs. temperature plots is a function of the properties of the
duplex. Sharp cooperative transitions are expected for the more
thermodynamically stable duplexes whereas sloping sigmoidal
transitions are expected where the duplex is less thermodynamically
stable. The flexible linkage in P3 was expected to stabilize the
duplex so the sharp transition observed was expected. The
substantial difference between probe P4 and PNAD however was
surprising and can only be attributed to the presence of the
solubility enhancers.
[0245] The data presented in FIG. 6 lead us to believe that
although probe PNAD was a hairpin, it appeared to be borderline in
stability. Therefore we theorized that a probe with shorter arm
segments (e.g. 6 subunits in length) might not exist predominately
as a hairpin since PNAs are known to be organized in solution (See:
Dueholm et al., New J. Chem., 21: 19-31 (1997) at p. 27, col. 2,
lns. 6-30). With reference to Table 7, the data presented for probe
PNAD 6S, which is designed with a six subunit self-complementary
arm segment, as compared with the 9 subunit arm segments of the
PNAD probe, confirms that the probe does not exist primarily as a
hairpin since the Tm is concentration dependent. Moreover, there
are two inflection points in the reannealing curve (data not shown)
at low concentrations (samples at [4] and [5}) which is indicative
of the existence of both hairpin and multimer formation.
Consequently, the data indicates that PNAs having arm segments of 6
or less subunits, and no flexible linkage groups, do not exist
primarily as hairpins.
[0246] The bimolecular duplex, P5/D5B was also analyzed to
determine its Tm. The data obtained by applicants indicated that
the most concentrated sample (approximately 7.5 .mu.M) had a Tm of
71.degree. C. At half concentration the Tm was approximately
70.degree. C. and at one quarter concentration the Tm was
approximately 68.5.degree. C.
[0247] The DNA probe D5B was complementary to only a portion of PNA
probes, P3, P4, PNAD. The DNA probe, D6, was perfectly
complementary to P4 and PNAD and a portion of P3. Hybridization
assays were performed to determine whether probes D5B or D6 would
hybridize with probes P3 or P4, thereby opening the hairpin stem
duplex and generating fluorescent signal. Hybridizations were
performed essentially as described in Example 21, below except that
the DNA target was in excess. The data obtained indicated that very
little hybridization occurred after 30 minutes. As these are the
most favored duplexes given the perfect complementary of the probes
and targets, the lack of detectable signal in the hybridization
reaction indicates that little or no hybridization occurs.
Consequently, the data suggests that probes having long stems (e.g.
7-10) and no flexible linkages are not optimal for analyzing
samples for a nucleic acid target since they do not produce
detectable signal.
[0248] These hybridization results should be expected since the Tm
of the PNA/DNA duplex should be lower than the Tm of the hairpin
stem duplex. For example, the Tm of the perfect complement P5/D5B
is approximately 71.degree. C. at concentrations much higher than
the effective concentration of reactants in the hybridization
reaction whereas the concentration independent Tm of the hairpin
stem duplex is 81-82.degree. C. Thus, it is not reasonable to
expect that the short DNA probe, D5B, will substantially hybridize
to P4 and open the more stable hairpin stem duplex.
[0249] In summary, the data presented in this Example 19
demonstrates that PNAs with long self-complementary arm segments
(e.g. 9 subunits) and no flexible linkages form stable hairpins
while those having shorter arm segments (e.g. 6 subunits) and not
flexible linkages are likely to exist in both the hairpin and
multimer state. When hairpins are formed, the Tm of the stem duplex
is substantially independent of the presence or absence of
magnesium and the ionic strength of the buffer. Unfortunately, the
data compiled by applicants indicates that labeled probes most
likely to form hairpins, because they possess longer
self-complementary arm segments (but do not comprise flexible
linkages), exhibit very poor signal to noise ratios in both
hybridization and thermal melting experiments. This data suggests
that these probes are not well suited for use in the detection of
nucleic acid targets. The most surprising result was the
substantial stabilizing effect attributable to the
dabcyl/fluorescein interactions. Such strong interactions may
explain why quenching occurs regardless of lack of substantial
spectral overlap between dabcyl and fluorescein (i.e. by
non-FRET).
[0250] Detailed Analysis of PNA Oligomers Prepared for Study
[0251] Experiments 20-22 were performed to generate comparative
data on the PNA oligomers in Table 1 so that preferred
configurations of PNA Molecular Beacons could be determined.
Generally the data indicates that the insertion of flexible
linkages within the probes improves signal to noise ratios
particularly when the flexible linkage is inserted at the
N-terminus of the probe to thereby link an arm segment to the
probing nucleobase sequence. The data also indicates that probes
with shorter arm segments also generally exhibit a more favorable
signal to noise ratio. Several of the probes exhibited signal to
noise ratios which were comparable with nucleic acid constructs
which are self-indicating (e.g. a nucleic acid Molecular Beacon).
Therefore, the PNA Molecular Beacons of this invention are useful
for detecting nucleic acid targets in samples of interest. However,
the data is inconclusive with regard to whether or not any of the
PNA probes listed in Table 1 exist primarily as hairpins.
Furthermore, the data indicates that, under the same experimental
conditions, the properties of the probes listed in Table 1 vary
substantially from probe to probe under the conditions examined.
Several of the results are not well understood. Thus, it has not
been possible to characterize the PNA probes listed in Table 1.
Example 20
Analysis of Fluorescent Thermal Profiles:
[0252] General Experimental Procedure:
[0253] Fluorescent measurements were taken using a RF-5000
spectrofluorophotometer (Shimadzu) fitted with a water jacketed
cell holder (P/N 206-15439, Shimadzu) using a 1.6 mL, 10 mm path
length cuvet (Starna Cells, Inc.). Cuvet temperature was modulated
using a circulating water bath (Neslab). The temperature of the
cuvet contents was monitored directly using a thermocouple probe
(Barnant; model No. 600-0000) which was inserted below liquid level
by passing the probe tip through the cap on the cuvet (custom
manufacture).
[0254] Stock solution of HPLC purified PNA oligomer was prepared by
dissolving the PNA in 50% aqueous N,N'-dimethylformamide (DMF).
From each PNA stock was prepared a solution of PNA oligomer, each
at a concentration of 10 pmol in 1.6 mL of Hyb. Buffer (50 mM Tris.
HCl pH 8.3 and 100 mM NaCl) by serial dilution of purified PNA
stock with Hyb. Buffer.
[0255] Samples were exited at 493 nm and the fluorescence measured
at 521 nm. Data points were collected at numerous temperatures as
the cuvet was heated and then again measured as the cuvet was
allowed to cool. Generally, the bath temperature was sequentially
increased by 5.degree. C. and then allowed to equilibrate before
each data point was recorded. Similarly, to generate the cooling
profile, the bath temperature was sequentially decreased by
5.degree. C. and then allowed to equilibrate before each data point
was recorded.
[0256] Data Discussion:
[0257] Nucleic acid Molecular Beacons which form a hairpin
structure are expected to exhibit an increase in fluorescent
intensity when heated which is consistent with the melting of the
hairpin stem and the physical transition of the probe stem from a
helix to a random coil. Consistent with any nucleic acid melting
event, the process is expected to be reversible thereby resulting
in a decrease in fluorescence upon cooling of the sample caused by
the resulting reformation of the helical structure. The expected
melting phenomenon is documented for nucleic acid Molecular Beacons
described by Tyagi et al. (See: Tyagi et al. Nature Biotechnology,
14: 303-308 (1996) at FIG. 3).
[0258] The results of the fluorescent thermal melting analysis of
the PNA Molecular Beacons are summarized in the data presented in
Table 8 and presented graphically in FIGS. 7A, 7B1, 7B2, 7B3 and
7C. With reference to Table 8, there are three different general
Thermal Profiles observed for the different constructs and under
the conditions examined. These are represented in Table 8 as Types
A, B and C.
[0259] Fluorescent Thermal Profile Type A is characterized by a an
increase in fluorescence intensity upon heating (melting) and a
correlating decrease in fluorescence intensity upon cooling
(reannealing). These results are similar to those published for
nucleic acid Molecular Beacons which form a loop and hairpin stem
structure. Thus, a Type A Fluorescent Thermal Profile is consistent
with the formation of a stable hairpin stem and loop structure.
This phenomenon is, therefore, believed to be caused by the melting
and reannealing of a stem and loop structure in the PNA Molecular
Beacon. However, applicants only claim that a Type A Fluorescent
Thermal Profile is indicative of fairly reversible fluorescence
quenching, as other structures may be responsible for or contribute
to the observed phenomenon.
[0260] Representatives of Type A Fluorescent Thermal Profiles are
illustrated in FIG. 7A. The data presented in the Figure is for the
PNA oligomers 0.001, 0.007 and 0.002. Data for both the melting
(open character) and the reannealing (solid character) is
presented. The sigmoidal transitions are consistent with a melting
a reannealing of a duplex.
[0261] Fluorescent Thermal Profile Type B is characterized by an
increase in fluorescence intensity upon heating (melting), but, no
substantial correlating decrease in fluorescence intensity upon
cooling of the sample. Thus, under the conditions examined, the
interactions which initially cause the quenching of fluorescence do
not appear to be readily reversible. Consequently, the data
suggests that a PNA oligomer exhibiting a Type B Fluorescent
Thermal Profile, does not exhibit all the features of a True
Molecular Beacon. Nonetheless, as seen by the hybridization assay
data, a Type B Fluorescent Thermal Profile does not prohibit the
PNA oligomer from functioning as a PNA Beacon.
[0262] Representatives of Type B Fluorescent Thermal Profiles are
illustrated in FIGS. 8B1, 8B2 and 8B3. The data is presented in
three sets so that each trace may be more clearly viewed. The data
presented in the Figures are for the PNA oligomers 0.010, 0.008,
0.009 (FIG. 7B1), 0.018, 0.011A, 0.017, (FIGS. 7B2), and 0.003 and
0.004, (FIG. 7B3). Data for both the melting (open character) and
the reannealing (solid character) is presented.
[0263] Fluorescent Thermal Profile Type C is characterized by a
high initial fluorescent intensity which initially decreases with
heating and again decreases even further upon cooling of the
sample. The high initial fluorescent intensity suggests that this
construct does not exhibit the initial fluorescence quenching
observed with most of the other PNA constructs examined. The
constant decrease in fluorescent intensity upon cooling is not well
understood. Nevertheless, as seen by the hybridization assay data,
a Type C, Fluorescent Thermal Profile does not prohibit the PNA
oligomer from functioning as a PNA Beacon.
[0264] Representatives of Type C Fluorescent Thermal Profiles are
illustrated in FIG. 7C. The data presented in the FIG. 7C is for
the PNA oligomers 0.005 and 0.006. Data for both the melting (open
character) and the reannealing (solid character) is presented.
10TABLE 8 Summary of Data Compiled In Experiments 20-22 Fluorescent
Thermal Hybridization Profile Profile Thermal Profile Probe No.
CODE Observed Observed Observed N-terminal Arm Forming Segments
.001 5205 A A Sig, 6% .007 5105 A A Sig, 7% .010 5005 B A Sig, 19%
.002 3203 A A Sig, 8% .008 3103 B A Sig, 8% .009 4004 B A Sig, 8%
C-terminal Arm Forming Segments .018 7027 B A, B Sig, 6% .011A 5025
B A N. Sig, 5% .006 3023 C C N. Sig, 8% Probing Sequence External
To Arm Segments .017 5115 B B N. Sig, 14% .005 3113 C C N. Sig, 10%
Control Probes: No Arm Forming Segments .003 0000 B B No Data .004
0110 B B N. Sig, 5% For a definition of CODE, see Table 1
Example 21
Analysis of Hybridization Assay Data
[0265] General Experimental Procedures:
[0266] All hybridization assay data was collected using a Wallac
1420 VICTOR equipped with a F485 CW-lamp filter and a F535 Emission
filter. The NUNC MaxiSorp, breakapart microtitre plate was used as
the reaction vessel. Each microtitre plate was prewashed with Hyb.
Buffer at room temperature for 15 minutes before the reaction
components were added. Total reaction volume was 100 .mu.L in Hyb.
Buffer.
[0267] Stock solution of purified PNA probe was prepared by
dissolving the PNA in 50% aqueous N,N'-dimethylformamide (DMF).
From this PNA Stock was prepared a solution of each PNA at a
concentration of 25 pmole/1 .mu.L by serial dilution of the PNA
Stock with 50% aqueous DMF.
[0268] Stock solution of purified wt k-ras DNA was prepared by
dissolving the purified DNA in TE (10 mM Tris. HCl pH 8.0; 1.0 mM
EDTA, Sigma Chemical). From this DNA Stock was prepared a solution
of wt k-ras DNA at a concentration of 100 pmol/99 .mu.L by serial
dilution of the DNA Stock with Hyb. Buffer.
[0269] Each reaction sample used for analysis was prepared by
combining 1 .mu.L of the appropriate PNA oligomer (25 pmole/.mu.L)
with either of 99 .mu.L of wt k-ras DNA stock or 99 .mu.L of Hyb.
Buffer (control) as needed to prepare 100 .mu.L of sample.
[0270] Samples were mixed and then fluorescence intensity monitored
with time using the Wallac VICTOR instrument. Samples were run in
triplicate to insure reproducible results. Data was acquired for
20-25 minutes after the reactants were mixed and then the wells
were sealed and the plate heated to 42-50.degree. C. in an
incubator for 30-40 minutes. After cooling to ambient temperature,
the wells were unsealed and then another 10 data points were
collected over approximately five minutes.
[0271] Data Discussion:
[0272] Nucleic acid Molecular Beacons which form a hairpin stem and
loop structure are expected to exhibit an increase in fluorescent
intensity upon hybridization of the probing sequence to
complementary nucleic acid. The expected increase in fluorescent
intensity is documented for DNA Molecular Beacons described by
Tyagi et al. (See: Tyagi et al. Nature Biotechnology, 14: 303-308
(1996)).
[0273] The results of the hybridization analysis of the PNA
oligomers are summarized in Table 8 and presented graphically in
FIGS. 8A1, 8A2, 8A3, 8B and 8C. With reference to Table 8, there
are three different general Hybridization Profiles observed for the
different constructs examined. These are represented in Table 8 as
Types A, B and C. In FIG. 10, the signal to noise ratio (before and
after heating) for all probes examined are graphically
illustrated.
[0274] Hybridization Profile Type A is characterized by the
increase in fluorescence intensity in samples containing
complementary target DNA as compared with samples containing only
the PNA oligomer. After heating, the fluorescent intensity of
samples containing target sequence increases but the background
fluorescence of the control sample(s) does not significantly
change. Because the PNAs possess a very low inherent fluorescence,
the probes which exhibit a Type A, Hybridization Profile generally
have the highest signal to noise ratios. Representatives of Type A
Hybridization Profiles are illustrated in FIGS. 8A1, 8A2 and 8A3.
The data is presented in two separate graphical illustrations to
clarify the presentation. The data presented in the Figures is for
the PNA oligomers 0.001, 0.007, 0.010 (FIG. 8A1), and 0.002, 0.008,
0.009 (FIG. 8A2), and 0.011A, 0.017 and 0.018 (FIG. 8A3).
[0275] Hybridization Profile Type B is characterized by the very
rapid increase in fluorescence intensity in samples containing
complementary target DNA as compared with samples containing only
the PNA oligomer. The fluorescence intensity quickly reaches a
plateau which does not significantly change (if at all) after
heating. The background fluorescence of the control sample(s) does
not change significantly even after heating. This suggest that the
hybridization event rapidly, and with little resistance, reaches a
binding equilibrium without any-requirement for heating.
Representatives of Type B Hybridization Profiles are illustrated in
FIG. 8B. The data presented in FIG. 8B is for the PNA oligomers
0.018, 0.003 and 0.004 though PNA oligomer 0.018 does not exhibit
all the characteristics of a Type B Hybridization Profile.
Specifically, the signal for probe 0.018 does not appear to
increase after heating (Type B) but the hybridization kinetics
appear to be more like a Type A Hybridization Profile.
[0276] Control probes 0.003 and 0.004 (herein referred to as PNA
Molecular Beacons) exhibit a Type B Hybridization Profile. Thus,
the rapid hybridization kinetics of the Type B Hybridization
Profile is probably the result of having no stable hairpin stem, or
any other strong force, which can stabilize the non fluorescent
polymer form. Nonetheless, the dynamic range (signal to noise
ratio) observed in the hybridization assay of these probes is
typically quite high and suggests that forces other than the
hydrogen bonding of complementary nucleobases of arm segments can
stabilize the interactions between the donor and acceptor moieties.
The data presented in Example 19 suggests that label/label
interactions can be quite strong and may be an important factor in
this surprising result.
[0277] Though the background (noise) is higher for the 0.003 and
0.004 probes, as compared with the 0.001, 0.002, 0.007, 0.009 and
0.010 probes, the fluorescence intensity after hybridization is
higher than that observed in any probes yet examined. As a result
of the higher background, PNA oligomers 0.003 and 0.004 have a very
favorable signal to noise ratio. This S/N ratio is nearly as
favorable as any (and better than some) of the other PNA oligomers
examined whether or not they possess arm segments. The data
demonstrates that it is not necessary to have arm forming segments
to create a probe which exhibits an initial low fluorescence
intensity and a corresponding increase in fluorescence signal upon
the binding of the probe to a target sequence.
[0278] Hybridization Profile C is characterized by a moderate
increase in fluorescence intensity in samples containing target DNA
as compared with samples containing only the PNA oligomer. The
fluorescence intensity quickly reaches a plateau which does not
significantly change (if at all) after heating. The background
fluorescence of the control sample(s) is relatively high but does
not change significantly even after heating. Hybridization Profiles
B and C differ primarily because the background fluorescence in the
control samples, containing no target nucleic acid, are
dramatically higher in Hybridization Profile Type C. The
hybridization data obtained for samples containing complementary
nucleic acid, suggests that the hybridization event rapidly, and
with little resistance, reaches equilibrium. However, the very high
background signal suggests that the forces which should hold the
donor and acceptor moieties in close proximity are not strong
enough in these constructs to effectively quench the fluorescent
signal. As a consequence of the moderate increase in fluorescence
upon binding to the target sequence and the higher than usual
intrinsic fluorescence a PNA Molecular Beacon, which exhibits a
Type C Hybridization Profile, has a very low signal to noise ratio.
Representatives of Type C Hybridization Profiles are illustrated in
FIG. 8C. The data presented in FIG. 8C is for the PNA oligomers
0.006 and 0.005, respectively.
Example 22
Ultraviolet Thermal Profile Analysis
[0279] The data collected for this Example was intended to
determine whether the fluorescence vs. temperature analysis
presented in Example 20 correlated with ultraviolet (UV) absorbance
(260 nm) vs. temperature plots. Additionally, concentration
dependency of the traces was also examined in order to determine
whether the PNA Molecular Beacons listed in Table 1 (except probe
0.003) existed as a hairpin or as a multimer.
[0280] Materials and Methods:
[0281] The purified probes were dissolved in the Hyb. Buffer to a
concentration which was intended to be approximately 5-7.5 .mu.M.
However it was determined that the PNA Molecular Beacons were too
insoluble such that a large proportion of the PNA probe existed in
a suspension. The solutions were centrifuged to remove suspended
matter and therefore the most concentrated samples examined were
estimated to have a concentration of approximately 2.5 .mu.M or
less. The most concentrated stocks were then serially diluted with
Hyb. Buffer two times so that for each sample, a total of three
concentrations could be examined. All samples were analyzed using a
Cary 100 Bio UV-Visible Spectrophotometer (Varian Instruments)
equipped with a 6.times.6 thermostatable multicell block running
Win UV Bio application software package. To a 10.times.10 UV cell
(Starna Cells, P/N 21-Q-10) was added a 7.2 mm stir bar and the 2.5
mL of each sample of the dilution series. The stirring accessory
was used during all analysis. All samples were thermally denatured
and reannealed prior to data collection by having the instrument
rapidly ramp to at least 90C and then holding for 5 minutes before
returning to the starting temperature of 20.degree. C. After the
premelt, it was preferable to allow the samples to remain at the
starting temperature for at least 30 minutes to reach equilibrium
before beginning data collection. Data for both dissociation and
reannealing was collected and analyzed. The temperature range over
which data was collected was 20-90.degree. C. The temperature ramp
rate for both dissociation and reannealing was 0.5.degree. C./min.
The absorbance (260 nm, averaged over a 2-3 second collection) was
plotted vs. the temperature of the multicell block.
[0282] Results:
[0283] Factors to be considered in analyzing the absorbance vs.
temperature plots include whether the transition was sigmoidal,
whether and to what extent there was any hysteresis and the percent
hyperchromicity (for the purposes of this discussion the percent
hyperchromicity will be defined as the approximate percent
difference between the absorbance at 20.degree. C. and the
absorbance at 90.degree. C.). Summary of the data obtained by
analysis of the absorbance vs. temperature plots is presented in
Table 8.
[0284] Probes 0.001, 0.002, 0.007, 0.008, 0.009, 0.010 and 0.018
exhibited a sigmoidal transition as indicated by "Sig" in Table 8.
The sigmoidal transition is characteristic of the melting and
reannealing of a duplex or hairpin. Probes. 0.004, 0.005, 0.006,
011A and 0.017 all exhibited a non-sigmoidal transition as
indicated by "N. Sig" in Table 8. Curiously, the shape of the
non-sigmoidal transition was essentially the same in all cases
except for 0.011A. For these probes the increase in absorbance as a
function of temperature appeared to be almost linear. The
non-sigmoidal shape of these curves suggest that the transition is
not the result of the melting and reannealing of a duplex
structure. Thus, these probe are not likely to exist as hairpins or
multimers.
[0285] Not a single probe examined was without a noticeable
hysteresis. Though the extent of hysteresis varied widely, the
presence of a conspicuous hysteresis indicates that the probes are
resistant to returning to their original confirmation as a hairpin,
mulitmer or other confirmation. Though this result was generally
observed in the fluorescence vs. temperature plots, the absorbance
traces were far more reversible upon cooling as compared with the
data observed in the fluorescence vs. temperature plots. Therefore,
the substantial differences between the fluorescence vs.
temperature and absorbance vs. temperature plots are not
understood. Moreover, it is unclear why only probes 0.001, 0.002
and 0.007 exhibited a reversible decrease in fluorescence upon
cooling whereas other probes did not. However, the data suggests
that the longer flexible linkages and longer arm segments promote
favorable properties since probes 0.001 and 0.002 both possess 2
flexible linkages and probe 0.007, though is possesses only one
flexible linkage, it comprises a 5 subunit arm segments.
[0286] For comparison, normalized absorbance vs. temperature and
fluorescence vs. temperature data for probe 0.001 was overlaid
since the plots looked relatively similar. The overlaid data is
presented in FIG. 9. With reference to FIG. 9, the absorbance vs.
temperature and fluorescence vs. temperature data is fairly
superimposible. The absorbance vs. temperature and fluorescence vs.
temperature plots for probe 0.002 and 0.007 were likewise very
similar thought the data has not yet been overlaid. The good
correlation between the absorbance vs. temperature and fluorescence
vs. temperature plots suggests that the same transition is being
measured in both analyses and it is likely to be a helix to coil
transition.
[0287] Except for probes 0.010, 0.005 and 0.017, the percent
hyperchromicity is less than 10 percent. Generally, the
hyperchromic effect for a duplex to random coil transition is
greater than 10 percent. The hyperchromicity for the DNA and PNA
probes examined in Example 19 were all better than 15 percent.
Thus, the lower than expected hyperchromic effect for substantially
all probes, except probe, 010 which exhibits a 19 percent
hyperchromic effect, is not well understood. Nevertheless, the
values are not consistent with at melting of a duplex of hairpin
even for probes which exhibited a sigmoidal transition.
[0288] Finally, the data obtained by applicants was inconclusive
with regard to whether the PNA Molecular Beacons listed in Table 1
exist as a hairpin or multimer because the scatter in the data at
the lower concentrations made it impossible to obtain reliable
derivative information from which the Tm values are determined.
Though the data generated in Example 19 would suggest that probes
comprising arm segments of six or less subunits are not likely to
form hairpins, the effect of flexible linkers was not fully
evaluated in that Example. Thus, it remains unknown whether any of
the probes listed in Table 1 exist primarily as hairpins.
[0289] In summary, the data presented in this Example 22 is
inconclusive as to whether the PNA Molecular Beacons listed in the
Table exist primarily as hairpins or mulitmers. It does however
show there is good agreement between the absorbance vs. temperature
plots and the fluorescence vs. temperature plots for probes 0.001,
0.002 and 0.007. The lack of correlation between absorbance vs.
temperature plots and the fluorescence vs. temperature plots for
other probes supports the theory that the probes may adopt
structures other than hairpins or multimers. This theory is
supported by the non-sigmoidal transitions, the substantial
hysteresis and the very low percent hyperchromicity for most of the
probes.
[0290] General Discussion of the Data Presented in Examples
19-22
[0291] Though all the probes examined exhibited a detectable
increase in fluorescent signal in the presence of a target
sequence, the probes which exhibit properties which are most like
nucleic acid Molecular Beacons are probes 0.001,0.002 and 0.007.
These probes process very favorable signal to noise ratios, exhibit
sigmoidal transitions during melting and also readily reannealed
upon cooling whether the analysis was by fluorescence or
absorbance. This data indicates that probes of this configuration
form duplexes which dissociate upon hybridization or thermal
melting to produce an increase in detectable signal though it is
not known whether or not these probes exist primarily as hairpins.
Whether hairpins or not, the favorable characteristics of these
probes correlate with the presence of flexible linkages and arm
segments in the range of 3 to 5 subunits in length. Furthermore,
the data in Example 19 in combination with the data for probe
0.018, in Examples 20-22, demonstrate that long arm segments of 7
to 9 subunits substantially reduce signal thereby resulting in very
poor signal to noise ratios. Consequently, long arm segments are a
disfavored configuration for a PNA Molecular Beacon.
[0292] Though probes 0.001, 0.002 and 0.007 exhibited the most
favorable properties, all the probes listed in Table 1, except for
control probes 0.003 and 0.004, are PNA Molecular Beacons because
they comprise arm segments and appropriate labeling and also
exhibit a detectable change in a property of a label which
correlates with the binding of the probe to a target sequence. The
nature of the forces which result in fluorescence quenching of the
other PNA probes is not well understood, though it is likely that
nucleobase-nucleobase, electrostatic and hydrophobic-hydrophobic
interactions contribute to fix the probes in a favorable secondary
structure until this is altered by hybridization.
[0293] Surprisingly, the control probes 0.003 and 0.004, which have
no arm forming segments, exhibit a correlation between increased
fluorescence intensity and binding of the probe to target sequence.
Remarkably these probe exhibit a very good signal to noise ratio in
hybridization assays. Thus, it has been demonstrated that PNA
oligomers need not comprise regions of complementary nucleobases
which are, by design, intended to form a hairpin to thereby exhibit
many of the favorable characteristics of a nucleic acid Molecular
Beacon. Since PNA oligomers 0.003 and 0.004 cannot form duplexes,
this result demonstrates that other types of secondary structures
can result in fluorescence quenching until the probe hybridizes to
a target sequence.
[0294] Equivalents
[0295] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those of ordinary skill in the art that various
changes in form and details may be made therein without departing
from the spirit and scope of the invention as defined by the
appended claims. Those skilled in the art will be able to
ascertain, using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed in the
scope of the claims.
Sequence CWU 1
1
13 1 24 DNA Artificial Sequence misc_feature (1) 5' Biotin 1
gtggtagttg gagctggtgg cgta 24 2 24 DNA Artificial Sequence
misc_feature (1) 5' Biotin 2 gtggtagttg gagcttgtgg cgta 24 3 18 DNA
Artificial Sequence misc_feature (1) 5' Biotin 3 actcctacgg
gaggcagc 18 4 9 DNA Artificial Sequence misc_feature (1)
5'-Fluorescein 4 atatattgg 9 5 9 DNA Artificial Sequence
Description of Artificial SequenceSYNTHETIC PROBE OR TARGET 5
atatattgg 9 6 9 DNA Artificial Sequence misc_feature (9) 3' Dabcyl
6 ccaatatat 9 7 9 DNA Artificial Sequence Description of Artificial
SequenceSYNTHETIC PROBE OR TARGET 7 ccaatatat 9 8 18 DNA Artificial
Sequence misc_feature (1) 5' Fluorescein 8 atatattggc caatatat 18 9
18 DNA Artificial Sequence misc_feature (9) spacer 9 atatattggc
caatatat 18 10 23 DNA Artificial Sequence misc_feature (1) 5'
Fluorescein 10 atatattggc tgatccaata tat 23 11 23 DNA Artificial
Sequence Description of Artificial SequenceSYNTHETIC PROBE OR
TARGET 11 atatattggc tgatccaata tat 23 12 12 DNA Artificial
Sequence misc_feature (1) 5' Biotin 12 tggatcagcc aa 12 13 23 DNA
Artificial Sequence Description of Artificial SequenceSYNTHETIC
PROBE OR TARGET 13 atatattgga tcagccaata tat 23
* * * * *