U.S. patent application number 10/909914 was filed with the patent office on 2005-03-03 for higher order structure and binding of peptide nucleic acids.
This patent application is currently assigned to Isis Pharmaceuticals, Inc.. Invention is credited to Berg, Rolf H., Buchardt, Ole, Ecker, David J., Egholm, Michael, Mollegaard, Niels E., Nielsen, Peter E..
Application Number | 20050048552 10/909914 |
Document ID | / |
Family ID | 32775387 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050048552 |
Kind Code |
A1 |
Ecker, David J. ; et
al. |
March 3, 2005 |
Higher order structure and binding of peptide nucleic acids
Abstract
Peptide nucleic acids and analogues of peptide nucleic acids are
used to form duplex, triplex, and other structures with nucleic
acids and to modify nucleic acids. The peptide nucleic acids and
analogues thereof also are used to modulate protein activity
through, for example, transcription arrest, transcription
initiation, and site specific cleavage of nucleic acids.
Inventors: |
Ecker, David J.; (Leucadia,
CA) ; Buchardt, Ole; (Vaerlose, DK) ; Egholm,
Michael; (Fredriksberg, DK) ; Nielsen, Peter E.;
(Koddedal, DK) ; Berg, Rolf H.; (Rungsted Kyst,
DK) ; Mollegaard, Niels E.; (Virum, DK) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE - 46TH FLOOR
PHILADELPHIA
PA
19103
US
|
Assignee: |
Isis Pharmaceuticals, Inc.
|
Family ID: |
32775387 |
Appl. No.: |
10/909914 |
Filed: |
August 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10909914 |
Aug 2, 2004 |
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09442054 |
Nov 16, 1999 |
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6770738 |
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09442054 |
Nov 16, 1999 |
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08471907 |
Jun 7, 1995 |
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5986053 |
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08471907 |
Jun 7, 1995 |
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08088658 |
Jul 2, 1993 |
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5641625 |
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08088658 |
Jul 2, 1993 |
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08054363 |
Apr 26, 1993 |
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5539082 |
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08054363 |
Apr 26, 1993 |
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PCT/EP92/01219 |
May 19, 1992 |
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Current U.S.
Class: |
435/6.14 ;
536/23.1 |
Current CPC
Class: |
B82Y 5/00 20130101; C12Q
1/6813 20130101; C12N 2310/15 20130101; A61K 38/00 20130101; C12N
2310/3181 20130101; C12Q 1/6813 20130101; C12Q 1/6813 20130101;
C07H 21/00 20130101; C12N 15/113 20130101; C12Q 2537/119 20130101;
C07K 14/003 20130101; C12Q 2525/107 20130101 |
Class at
Publication: |
435/006 ;
536/023.1 |
International
Class: |
C12Q 001/68; C07H
021/02 |
Claims
We claim:
1. A complex comprising a first nucleic acid strand and second and
third strands; said second and third strands independently
containing a sequence of ligands covalently bound by linking
moieties; at least one of said second strand linking moieties
comprising an amide, a thioamide, a sulfinamide or a sulfonamide
linkage and a plurality of said ligands on said second strand
interacting with said first strand; and at least one of said third
strand linking moieties comprising an amide, a thioamide, a
sulfinamide or a sulfonamide linkage and a plurality of said
ligands on said third strand interacting with said first strand or
with said ligands on said second strand.
2. The complex of claim 1 wherein each of said second strand and
said third strand, independently, include a plurality of monomeric
units connected via amide, thioamide, sulfinamide or sulfonamide
linkages.
3. The complex of claim 1 wherein said first strand is a DNA
strand.
4. The complex of claim 1 wherein said first stand is a RNA
strand.
5. The complex of claim 2 wherein said monomeric units are
connected via amide linkages.
6. The complex of claim 2 wherein said monomeric units each include
a first nitrogen that connects said ligands to said monomeric units
and a second nitrogen atom that forms a portion of said amide,
thioamide, sulfinamide or sulfonamide linkage.
7. The complex of claim 2 wherein a majority of said ligands of at
least one of said second strand or third strand are pyrimidine
bases.
8. The complex of claim 7 wherein said pyrimidine bases are
selected from thymine and cytosine.
9. The complex of claim 2 wherein said nucleic acid strand is a
purine rich strand.
10. The complex of claim 2 wherein said monomeric units comprise
aminoethylglycine units.
11. The complex of claim 1 wherein said second and said third
strands, independently, comprise a compound of the formula:
5wherein: n is at least 2, each of L.sup.1-L.sup.n is independently
selected from the group consisting of hydrogen, hydroxy,
(C.sub.1-C.sub.4)alkanoyl, naturally occurring nucleobases,
non-naturally occurring nucleobases, aromatic moieties, DNA
intercalators, nucleobase-binding groups, heterocyclic moieties,
and reporter ligands; each of C.sup.1-C.sup.n is
(CR.sup.6R.sup.7).sub.7 where R.sup.6 is hydrogen and R.sup.7 is
selected from the group consisting of the side chains of naturally
occurring alpha amino acids, or R and R.sup.7 are independently
selected from the group consisting of hydrogen,
(C.sub.2-C.sub.5)alkyl, aryl, aralkyl, heteroaryl, hydroxy,
(C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.6)alkylthio,
NR.sup.3R.sup.4 and SR.sup.5, where R.sup.3 and R.sup.4 are as
defined above, and R.sup.5 is hydrogen, (C.sub.1-C.sub.6)alkyl,
hydroxy-, alkoxy-, or alkylthio-substituted (C.sub.1-C.sub.5)alkyl,
or R.sup.5 and R.sup.7 taken together complete an alicyclic or
heterocyclic system; each of D.sup.1-D.sup.n is
(CR.sup.6R.sup.7).sub.z where R.sup.5 and R.sup.7 are as defined
above; each of y and z is zero or an integer from 1 to 10, the sum
y+z being greater than 2 but not more than 10; each of
G.sup.1-G.sup.n-1 is --NR.sup.3CO--, --NR.sup.3CS--, --NR.sup.3SO--
or --NR.sup.3SO.sub.2--, in either orientation, where R.sup.3 is as
defined above; each of A.sup.1-A.sup.n and B.sup.1-B.sup.n are
selected such that: (a) A is a group of formula (IIa), (IIb), (IIc)
or (IId), and B is N or R.sup.3N.sup.+; or (b) A is a group of
formula (IId) and B is CH; 6 where: X is O, S, Se, NR.sup.3,
CH.sub.2 or C(CH.sub.3).sub.2; Y is a single bond, O, S or
NR.sup.4; each of p and q is zero or an integer from 1 to 5, the
sum p+q being not more than 10; each of r and s is zero or an
integer from 1 to 5, the sum r+s being not more than 10; each
R.sup.1 and R.sup.2 is independently selected from the group
consisting of hydrogen, (C.sub.1-C.sub.4)alkyl which may be
hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy,
alkylthio, amino and halogen; and each R.sup.3 and R.sup.4 is
independently selected from the group consisting of hydrogen,
(C.sub.1-C.sub.4)alkyl, hydroxy- or alkoxy- or
alkylthio-substituted (C.sub.1-C.sub.4)alkyl, hydroxy, alkoxy,
alkylthio and amino; Q is --CO.sub.2H, --CONR'R", --SO.sub.3H or
--SO.sub.2NR'R" or an activated derivative of --CO.sub.2H or
--SO.sub.3H; and I is --NHR'"R"" or --NR'"C(O)R"", where R', R",
R'" and R"" are independently selected from the group consisting of
hydrogen, alkyl, amino protecting groups, reporter ligands,
intercalators, chelators, peptides, proteins, carbohydrates,
lipids, steroids, nucleosides, nucleotides, nucleotide
diphosphates, nucleotide triphosphates, oligonucleotides,
oligonucleosides and soluble and non-soluble polymers.
12. The complex of claim 1 wherein said second strand and said
third strand, independently, comprise a compound of the formula
III, IV or V: 7wherein: each L is independently selected from the
group consisting of hydrogen, phenyl, heterocyclic moieties,
naturally occurring nucleobases, and non-naturally occurring
nucleobases; each R.sup.7' is independently selected from the group
consisting of hydrogen and the side chains of naturally occurring
alpha amino acids; n is an integer greater than 1, each k, l, and m
is, independently, zero or an integer from 1 to 5; each p is zero
or 1; R.sup.h is OH, NH.sub.2 or --NHLysNH.sub.2; and R.sup.1 is H
or COCH.sub.3.
13. The complex of claim 1 wherein said interaction between said
first and second strands occurs between heteocyclic bases of said
first strand and ligands on said second strand.
14. The complex of claim 1 wherein said interaction between said
first and third strands occurs between heteocyclic bases of said
first strand and ligands on said third strand.
15. The complex of claim 1 wherein said interaction includes
formation of non-covalent bonds.
16. The complex of claim 1 wherein said interaction of ligands is
via hydrogen bonding.
17. The complex of claim 1 wherein said interaction between said
first and second strands occurs between heteocyclic bases of said
first strand and ligands on said second strand; said interaction
between said first and third strands occurs between heteocyclic
bases of said first strand and ligands on said third strand; and
said interaction of ligands of said second strand and heterocyclic
bases of said first strand is via Watson/Crick hydrogen bonding,
and said interaction of ligands of said second strand with
heterocyclic bases of said first strand is via one of Watson/Crick
hydrogen bonding or Hoogsteen hydrogen bonding.
18. A complex comprising a first nucleic acid strand, a second
nucleic acid strand, and a third strand; said third strand
containing a sequence of ligands covalently bound by linking
moieties and wherein a plurality of said ligands interact with at
least one of said first strand and said second strand; and at least
one of said third strand linking moieties comprises an amide, a
thioamide, a sulfinamide or asulfonamide linkage.
19. The complex of claim 18, wherein said third strand includes a
plurality of monomeric units that are connected via amide,
thioamide, sulfinamide or sulfonamide backbone linkages.
20. The complex of claim 18 wherein said interaction includes
formation of non-covalent bonds.
21. The complex of claim 20 wherein said interaction includes
hydrogen bonding.
22. The complex of claim 18 wherein said interaction occurs between
heteocyclic bases on said first strand or said second strand and
ligands on said third strand.
23. The complex of claim 18 wherein said first strand and said
second strand are DNA strands.
24. The complex of claim 23 wherein said DNA strands are
double-stranded DNA.
25. The complex of claim 23 wherein said DNA strands are
complementary DNA strands.
26. The complex of claim 19 wherein said monomeric units are
connected via amide linkages.
27. The complex of claim 19 wherein said monomeric units each
include a first nitrogen that connects said ligands to said
monomeric units and a second nitrogen atom that forms a portion of
said amide, thioamide, sulfinamide or sulfonamide linkage.
28. The complex of claim 18 wherein a majority of said ligands are
pyrimidine bases.
29. The complex of claim 28 wherein said pyrimidine bases are
selected from thymine and cytosine.
30. The complex of claim 18 wherein one of said nucleic acid
strands is a purine rich strand.
31. The complex of claim 19 wherein said monomeric units comprise
aminoethylglycine units.
32. The complex of claim 18 wherein said third strand comprises a
compound of the formula: 8wherein: n is at least 2, each of
L.sup.1-L.sup.n is independently selected from the group consisting
of hydrogen, hydroxy, (C.sub.1-C.sub.4)alkanoyl, naturally
occurring nucleobases, non-naturally occurring nucleobases,
aromatic moieties, DNA intercalators, nucleobase-binding groups,
heterocyclic moieties, and reporter ligands; each of
C.sup.1-C.sup.n is (CR.sup.6R.sup.7).sub.y where R.sup.6 is
hydrogen and R.sup.7 is selected from the group consisting of the
side chains of naturally occurring alpha amino acids, or R.sup.6
and R.sup.7 are independently selected from the group consisting of
hydrogen, (C.sub.2-C.sub.6)alkyl, aryl, aralkyl, heteroaryl,
hydroxy, (C.sub.1-C.sub.6)alkoxy, (C.sub.1-C.sub.6)alkylthio,
NR.sup.3R and SR.sup.5, where R.sup.3 and R.sup.4 are as defined
above, and R.sup.5 is hydrogen, (C.sub.1-C.sub.6)alkyl, hydroxy-,
alkoxy-, or alkylthio-substituted (C.sub.1-C.sub.6)alkyl, or
R.sup.6 and R.sup.7 taken together complete an alicyclic or
heterocyclic system; each of D.sup.1-D.sup.5 is
(CR.sup.6R.sup.7).sub.z where R.sup.6 and R.sup.7 are as defined
above; each of y and z is zero or an integer from 1 to 10, the sum
y+z being greater than 2 but not more than 10; each of
G.sup.1-G.sup.n-1 is --NR.sup.3CO--, --NR.sup.3CS--, --NR.sup.3SO--
or --NR.sup.3SO.sub.2--, in either orientation, where R.sup.3 is as
defined above; each of A.sup.1-A.sup.n and B.sup.1-B.sup.n are
selected such that: (a) A is a group of formula (IIa), (IIb), (IIc)
or (IId), and B is N or R.sup.3N.sup.+; or (b) A is a group of
formula (IId) and B is CH; 9 where: X is O, S, Se, NR.sup.3,
CH.sub.2 or C(CH.sub.3).sub.2; Y is a single bond, O, S or
NR.sup.4; each of p and q is zero or an integer from 1 to 5, the
sum p+q being not more than 10; each of r and s is zero or an
integer from 1 to 5, the sum r+s being not more than 10; each
R.sup.1 and R.sup.2 is independently selected from the group
consisting of hydrogen, (C.sub.1-C.sub.4)alkyl which may be
hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy,
alkylthio, amino and halogen; and each R.sup.3 and R.sup.4 is
independently selected from the group consisting of hydrogen,
(C.sub.1-C.sub.4)alkyl, hydroxy- or alkoxy- or
alkylthio-substituted (C.sub.1-C.sub.4)alkyl, hydroxy, alkoxy,
alkylthio and amino; Q is --CO.sub.2H, --CONR'R", --SO.sub.2H or
--SO.sub.2NR'R" or an activated derivative of --CO.sub.2H or
--SO.sub.2H; and I is --NHR'"R"" or --NR'"C(O)R"", where R', R",
R'" and R"" are independently selected from the group consisting of
hydrogen, alkyl, amino protecting groups, reporter ligands,
intercalators, chelators, peptides, proteins, carbohydrates,
lipids, steroids, nucleosides, nucleotides, nucleotide
diphosphates, nucleotide triphosphates, oligonucleotides,
oligonucleosides and soluble and non-soluble polymers.
33. The complex of claim 18 wherein said third strand comprises a
compound of the formula III, IV or V: 10wherein: each L is
independently selected from the group consisting of hydrogen,
phenyl, heterocyclic moieties, naturally occurring nucleobases, and
non-naturally occurring nucleobases; each R.sup.7' is independently
selected from the group consisting of hydrogen and the side chains
of naturally occurring alpha amino acids; n is an integer greater
than 1, each k, l, and m is, independently, zero or an integer from
1 to 5; 8 each p is zero or 1; R.sup.h is OH, NH.sub.2 or
--NHLysNH.sub.2; and R.sup.i is H or COCH.sub.3.
34. A process for modifying double-stranded DNA, comprising the
steps of: contacting said double-stranded DNA with a compound that
comprises a sequence of ligands covalently bound by amide,
thioamide, sulfinamide, or sulfonamide linking moieties, wherein a
plurality of said ligands interact with said double-stranded DNA
and thereby displace one of said DNA strands; and modifying said
displaced strand.
35. The process of claim 34 wherein modifying said displaced strand
comprises cleaving said strand.
36. The process of claim 35 wherein said cleavage is effected by an
enzyme.
37. The process of claim 36 wherein said double-stranded DNA is
contacted with said compound intercellular and said enzymatic
cleavage is effect by enzymes naturally present intercellular.
38. The process of claim 37 wherein said enzyme is a nuclease.
39. The process of claim 38 wherein said nuclease is nuclease
S.sub.1.
40. The process of claim 34 wherein said compound further includes
a moiety that modifies said displaced strand.
41. The process of claim 40 wherein moiety cleaves said displaced
strand.
42. The process of claim 34 wherein a plurality of said ligands
hydrogen bond with said DNA.
43. The process of claim 34 wherein said compound forms
Watson-Crick hydrogen bonds with one strand of said double-stranded
DNA.
44. The process of claim 31 wherein a first molecule of said
compound forms Watson-Crick hydrogen bonds with a first strand of
said double-stranded DNA and a second molecule of said compound
forms Hoogsteen hydrogen bonds with said first strand.
45. The process of claim 34 wherein modifying said displaced strand
comprises covalently bonding a bonding moiety to said displaced
strand.
46. The process of claim 34 wherein modifying said displaced strand
comprises activating cellular repair mechanisms to effects
digestion of said displaced strand.
47. The process of claim 34 wherein said displaced strand is
modified by covalently bonding a bonding moiety to said displaced
stand, thereby initiating a cellular repair mechanism that effects
digestion of said displaced strand.
48. A process of inhibiting the expression of a gene comprising
contacting said gene with a compound that comprises a sequence of
ligands covalently bound by amide, thioamide, sulfinamide, or
sulfonamide linking moieties, and wherein a plurality of said
ligands interact with said gene.
49. The process of claim 48 wherein a plurality of said ligands
hydrogen bond with DNA of said gene.
50. The process of claim 48 wherein inhibition of gene expression
comprises transcription interference.
51. The process of claim 50 wherein said transcription
interferences comprises RNA polymerase arrest.
52. The process of claim 51 wherein said transcription interference
comprises transcription arrest outside of a promoter region of said
gene.
53. The process of claim 49 wherein a plurality of said ligands
hydrogen bond to a template strand of said gene.
54. The process of claim 48 wherein said compound includes at least
six monomeric units connected via a polyamide backbone and each of
said monomeric units includes one of said ligands.
55. A process for arresting transcription of a gene comprising
contacting said gene with a compound that comprises a sequence of
ligands covalently bound by amide, thioamide, sulfinamide, or
sulfonamide linking moieties, thereby binding a plurality of said
ligands to a template DNA strand of said gene.
56. A process for modulating the activity of a restriction enzyme
at a DNA restriction site, comprising contacting said DNA with a
compound that comprises a sequence of ligands covalently bound by
amide, thioamide, sulfinamide, or sulfonamide linking moieties,
thereby binding a plurality of said ligands to said DNA proximal to
said restriction site.
57. A process for sequencing DNA, comprising: contacting said DNA
with a compound that comprises a sequence of ligands covalently
bound by amide, thioamide, sulfinamide, or sulfonamide linking
moieties, thereby binding a plurality of said ligands to said DNA
proximal to a DNA restriction site; treating said DNA with a
restriction enzyme that recognizes and cleaves said DNA at said
restriction site; and identifying at least one product of said
cleavage.
58. A process for inhibiting transcription of DNA comprising:
selecting DNA for which transcription is to be inhibited; and
adding to a mixture containing said DNA and a transcription factor
for said DNA a compound that comprises a sequence of ligands
covalently bound by aside, thioamide, sulfinamide, or sulfonamide
linking moieties, thereby binding a plurality of said ligands to
said-transcription factor.
59. A method for modulating binding of RNA polymerase to
double-stranded DNA comprising: contacting said DNA with a compound
that comprises a sequence of ligands covalently bound by amide,
thioamide, sulfinamide, or sulfonamide linking moieties, thereby
binding a plurality of said ligands to said DNA; and exposing said
complex to RNA polymerase.
60. A process for initiating transcription of a gene comprising
contacting said gene with a compound that comprises a sequence of
ligands covalently bound by amide, thioamide, sulfinamide, or
sulfonamide linking moieties, wherein a plurality of said ligands
interact with said gene to melt double-stranded DNA of said gene
and thereby expose a template strand of said DNA to recognition by
RNA polymerase.
61. The process of claim 60 wherein said gene is contacted with
said compound under conditions that form a hybrid strand with a
non-coding strand of said DNA.
62. The process of claim 60 wherein said gene is contacted with
said compound under conditions that form both a hybrid strand with
said DNA and a template strand "D" loop.
63. A process for initiating transcription of a gene, comprising
contacting said gene with a compound that comprises a sequence of
ligands covalently bound by amide, thioamide, sulfinamide, or
sulfonamide linking moieties, wherein a plurality of said ligands
interact with said gene to melt double-stranded DNA of said gene
and thereby form a transcription elongation loop.
64. A method for binding RNA polymerase to double-stranded DNA
comprising: contacting said DNA with a compound that comprises a
sequence of ligands covalently bound by amide, thioamide,
sulfinamide, or sulfonamide linking moieties, wherein a plurality
of said ligands interact with said DNA; and exposing said compound
and said DNA to said RNA polymerase.
65. A hybrid complex for modulating transcription comprising:
double-stranded DNA; a compound that comprises a sequence of
ligands covalently bound by amide, thioamide, sulfinamide, or
sulfonamide linking moieties, wherein a plurality of said ligands
interact with said double-stranded DNA; and RNA polymerase in
contact with at least one of said DNA and said compound.
66. A synthetic transcription factor comprising: double-stranded
DNA; and a compound that comprises a sequence of ligands covalently
bound by amide, thioamide, sulfinamide, or sulfonamide linking
moieties, wherein a plurality of said ligands interact with said
double-stranded DNA.
67. A sequence specific gene activator comprising a compound that
comprises a sequence of ligands covalently bound by amide,
thioamide, sulfinamide, or sulfonamide linking moieties, wherein a
plurality of said ligands interact with a specific sequence of DNA
of said gene.
68. A sequence specific gene activator comprising first and second
synthetic strands, wherein each strand comprises a sequence of
about 6-50 ligands covalently bound by amide, thioamide,
sulfinamide, or sulfonamide linking moieties, and each of said
strands has a ligand sequence that recognizes a base sequence
within proximal regions on at least one DNA strand of said gene
such that said strands bind proximal to one another on said DNA
strand.
69. The gene activator of claim 68 wherein the ligand sequence of
said synthetic strands are complementary to a non-template strand
of said DNA.
70. The gene activator of claim 68 wherein the ligand sequence of
said synthetic strands is selected such that binding of said
synthetic strands on said gene results in creation of a loop of DNA
that includes the binding sites of said synthetic strands on said
gene.
71. A sequence specific gene activator comprising first and second
synthetic strands, wherein: each strand comprises a sequence of
about 6-50 ligands covalently bound by amide, thioamide,
sulfinamide, or sulfonamide linking moieties: said first strand has
a ligand sequence that recognizes a base sequence on one of the DNA
strands of said gene; said second strand has a ligand sequence that
recognizes a base sequence on the other of the DNA strands of said
gene; and the recognized base sequence on said one DNA strand and
the recognized base sequence of said other DNA strand are
positioned proximal to one another on said gene.
72. The gene activator of claim 71 wherein ligand sequences of said
synthetic strands is selected such that binding of said synthetic
strands on said gene results in creation of a loop of DNA on said
gene that includes the binding sites of said synthetic strands on
said gene.
73. A chimeric compound comprising a first strand section including
DNA or RNA, and a second strand section including a sequence of
ligands covalently bound by amide, thioamide, sulfinamide, or
sulfonamide linking moieties, said first strand section and said
second strand section being covalently bound.
74. The chimeric structure of claim 73 wherein said first stand
section includes DNA.
75. A double-stranded chimeric complex, wherein: a first strand
comprises a first strand section including DNA or RNA and a second
strand section comprises a sequence of ligands covalently bound by
amide, thioamide, sulfinamide, or sulfonamide linking moieties,
said first and said second strand sections being covalently bound;
a second strand comprises DNA, RNA, or a sequence of ligands
covalently bound by amide, thioamide, sulfinamide, or sulfonamide
linking moieties; and said first strand and said second strand
interact with one another to form a duplex structure.
76. A method for intercellularly modulating the activity of a
transcription factor comprising: forming a chimeric structure by
covalently bonding a first strand section including DNA or RNA and
a second strand section including a sequence of ligands covalently
bound by amide, thioamide, sulfinamide, or sulfonamide linking
moieties, wherein a plurality of said ligands bind with said
transcription factor; and introducing said chimeric structure into
a cell.
77. The method of claim 76 further including selecting said ligands
and said DNA or RNA such that a plurality of said ligands bind with
the sugar-phosphate backbone of said RNA or DNA.
78. A process for intercellularly inhibiting the binding of a
protein comprising: forming a compound that comprises a sequence of
ligands covalently bound by amide, thioamide, sulfinamide, or
sulfonamide linking moieties, wherein a plurality of said bind to
said protein; and introducing said structure into a cell.
Description
RELATED APPLICATION
[0001] This patent application is related to the patent application
Ser. No. ______ entitled Double-Stranded Peptide Nucleic Acids,
filed herewith bearing attorney docket number ISIS-1108. This
patent application also is a continuation-in-part of patent
application Ser. No. 08/054,363, filed Apr. 26, 1993, which is a
continuation-in-part of application PCT EP92/01219, filed May 19,
1992 and published Nov. 26, 1992 as WO 92/20702. The entire
contents of each of the foregoing patent applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention is directed to compounds that form
triple-stranded structures with single-stranded and double-stranded
nucleic acids. It is further directed to the use of such compounds
to cause strand displacement in double-stranded nucleic acids. The
invention further is directed to processes for modifying
double-stranded nucleic acid utilizing such strand displacement.
Such processes for modifying double-stranded nucleic acids include
cleavage of the nucleic acid strand or strands. In particular, such
cleavage includes sequence specific cleavage of double-stranded
nucleic acids using a nuclease which normally is nonsequence
specific. Such processes also include transcription inhibition or
arrest as well as transcription initiation. The processes of the
invention are effected, in particular, with compounds that include
naturally-occurring nucleobases or other nucleobase-binding
moieties covalently bound to a polyamide backbone.
BACKGROUND OF THE INVENTION
[0003] The function of a gene starts by transcription of its
information to a messenger RNA (mRNA) which, by interaction with
the ribosomal complex, directs the synthesis of a protein coded for
by the mRNA sequence. The synthetic process is known as
translation. Translation requires the presence of various
co-factors and building blocks, the amino acids, and their transfer
RNAs (tRNA), all of which are present in normal cells.
[0004] Transcription initiation requires specific recognition of a
promoter DNA sequence by the RNA-synthesizing enzyme, RNA
polymerase. In many cases in prokaryotic cells, and probably in all
cases in eukaryotic cells, this recognition is preceded by
sequence-specific binding of a protein transcription factor to the
promoter. Other proteins which bind to the promoter, but whose
binding prohibits action of RNA polymerase, are known as
repressors. Thus, gene activation typically is regulated positively
by transcription factors and negatively by repressors.
[0005] Most conventional drugs function by interaction with and
modulation of one or more targeted endogenous proteins, e.g.,
enzymes. Typical daily doses of drugs are from 10.sup.-5-10.sup.-1
millimoles per kilogram of body weight or 10.sup.-3-10 millimoles
for a 100 kilogram person. If this modulation instead could be
effected by interaction with and inactivation of mRNA, a dramatic
reduction in the necessary amount of drug necessary likely could be
achieved, along with a corresponding reduction in side effects.
Further reductions could be effected if such interaction could be
rendered site-specific. Given that a functioning gene continually
produces mRNA, it would thus be even more advantageous if gene
transcription could be arrested in its entirety.
[0006] Synthetic reagents that bind sequence selectively to
single-stranded and especially to double-stranded nucleic acids are
of great interest in molecular biology and medicinal/chemistry,
since such reagents may provide the tools for developing
gene-targeted drugs and other sequence-specific gene modulators.
Until now oligonucleotides and their close analogues have presented
the best candidates for such reagents.
[0007] Oligodeoxyribonucleotides as long as 100 base pairs (bp) are
routinely synthesized by solid phase methods using commercially
available, fully automatic synthesis machines.
Oligoribonucleotides, however, are much less stable than
oligodeoxyribonucleotides, a fact which has contributed to the more
prevalent use of oligodeoxyribonucleotides in medical and
biological research directed to, for example, gene therapy and the
regulation of transcription and translation. Synthetic
oligodeoxynucleotides are being investigated for used as antisense
probes to block and eventually breakdown mRNA.
[0008] It also may be possible to modulate the genome of an animal
by, for example, triple helix formation using oligonucleotides or
other DNA recognizing agents. However, there are a number of
drawbacks associated with oligonucleotide triple helix formation.
For example, triple helix formation generally has only been
obtained using homopurine sequences and requires unphysiologically
high ionic strength and low pH. Whether used as antisense reagents
or a triplexing structures, unmodified oligonucleotides are
unpractical because they have short in vivo half-lives. To
circumvent this, oligonucleotide analogues have been used.
[0009] These areas for concern have resulted in an extensive search
for improvements and alternatives. For example, the problems
arising in connection with double-stranded DNA (dsDNA) recognition
through triple helix formation have been diminished by a clever
"switch back" chemical linking whereby a sequence of polypurine on
one strand is recognized, and by "switching back", a homopurine
sequence on the other strand can be recognized. See, e.g., McCurdy,
Moulds, and Froehler, Nucleosides, in press. Also, helix formation
has been obtained by using artificial bases, thereby improving
binding conditions with regard to ionic strength and pH.
[0010] In order to improve half-life as well as membrane
penetration, a large number of variations in polynucleotide
backbones has been undertaken. These variations include the use of
methylphosphonates, monothiophosphates, dithiophosphates,
phosphoramidates, phosphate esters, bridged phosphoramidates,
bridged phosphorothioates, bridged methylenephosphonates, dephospho
internucleotide analogs with siloxane bridges, carbonate bridges,
carboxymethyl ester bridges, acetamide bridges, carbamate bridges,
thioether, sulfoxy, sulfono bridges, various "plastic" DNAs,
.alpha.-anomeric bridges, and borane derivatives. The great
majority of these backbone modifications have decreased the
stability of hybrids formed between a modified oligonucleotide and
its complementary native oligonucleotide, as assayed by measuring
T.sub.m values. Consequently, it is generally believed in the art
that backbone modifications destabilize such hybrids, i.e., result
in lower T.sub.m values, and should be kept to a minimum.
[0011] The discovery of sequence specific endonucleases
(restriction enzymes) was an essential step in the development of
biotechnology, enabling DNA to be cut at precisely specified
locations containing specific base sequences. However, although the
range of restriction enzymes now known is extensive, there is still
a need to obtain greater flexibility in the ability to recognize
particular sequences in double-stranded nucleic acids and to cleave
the nucleic acid specifically at or about the recognized
sequence.
[0012] Most restriction enzymes recognize quartet or sextet DNA
sequences and only a very few require octets for recognition.
However, restriction enzymes have been identified and isolated only
for a small subset of all possible sequences within these
constraints. A need exists, especially in connection with the study
of large genomic DNA molecules, in general, and with the human
genome project, in particular, to recognize and specifically cleave
DNA molecules at more rarely occurring sites, e.g., sites defined
by about fifteen base pairs.
[0013] Efforts have therefore been made to create artificial
"restriction enzymes" or to modify the procedures for using
existing restriction enzymes for this purpose. Methods investigated
include the development of oligonucleotides capable of binding
sequence specifically via triple helix formation to double-stranded
DNA tagged with chemical groups (e.g., photochemical groups) able
to cleave DNA or with non-specific DNA cleaving enzymes and other
such modifications consistent with an "Achilles heel" general
strategy. Such methods are described by: Francois, J. C., et al.
PNAS 86, 9702-9706 (1989); Perrouault, L., et al Nature 344,
358-360 (1990); Strobel, S. A. & Dervan P. B. Science 249,
73-75 (1990); Pei, D., Corey D. R. & Schultz P. G. PNAS 87,
9858 (1990); Beal, P. A. & Dervan P. B., Science 251, 1360
(1991); Hanvey, J. C., Shimizu M. & Wells R. D. NAR 18, 157-161
(1990); Koob, H. & Szybalski W. Science 250, 271 (1990);
Strobel, S. A. & Dervan P. B. Nature #50, 172 (1991); and
Ferrin, L. J. & Camerini-Otero R. D. Science 254, 1494-1497
(1991).
[0014] In Patent Cooperation Treaty Applications No.
PCT/EP92/01220, and PCT/EP92/01219, both filed on 22nd May 1992, we
described certain nucleic acid analogue compounds that have a
strong sequence specific DNA binding ability. Examples of such
compounds were also disclosed by us in Science 1991, 254 1497-1500.
We have shown that a nucleic acid analogue of this type containing
10 bases hybridized to a non-terminal region of a double-stranded
DNA and rendered the strand of DNA which was non-complementary to
the nucleic acid analogue susceptible to degradation by S.sub.1
nuclease. No increased cleavage of the DNA strand complementary to
the nucleic acid analogue was seen, so no cleavage of
double-stranded DNA was obtained.
OBJECTS OF THE INVENTION
[0015] It is an object of the invention to provide compounds that
bind DNA and RNA strands.
[0016] It is a further object of the invention to provide triplex
structures between DNA or RNA strands and these compounds.
[0017] It is yet another object to provide compounds other than RNA
that can bind one strand of a double-stranded polynucleotide,
thereby displacing the other strand.
[0018] It is still another object to provide therapeutic,
diagnostic, and prophylactic methods that employ such
compounds.
BRIEF DESCRIPTION OF THE INVENTION
[0019] In the cell, DNA exists as a double stranded structure.
During certain cellular events, as for instance transcription or
during cell division, portions of the double stranded DNA are
transiently denatured to single strand. Further DNA can be isolated
outside of a cell and can be purposefully denatured to single
stranded DNA. RNA generally exist as a single stranded structure;
however, in a local area of secondary structure a RNA, as for
instance the stem of a stem loop structure, the RNA can exist as a
double stranded structure.
[0020] We have found that certain compounds that have nucleobases
attached to an aminoethylglycine backbone and other like backbones
including polyamides, polythioamides, polysulfinamides and
polysulfonamides, which compounds we call peptide nucleic acids or
PNA, surprisingly bind strongly and sequence selectively to both
RNA and DNA.
[0021] We have surprisingly found that these PNA compounds
recognize and bind sequence-selectively and strand-selectively to
double-stranded DNA (dsDNA). We have found that the binding to
double-stranded DNA is accomplished via strand displacement, in
which the PNA binds via Watson-Crick binding to its complementary
strand and extrudes the other strand in a virtually single-stranded
conformation. We have also surprisingly found that these PNA
compounds recognize and bind sequence-selectively to
single-stranded DNA (ssDNA) and to RNA.
[0022] The recognition of PNA to RNA, ssDNA or dsDNA can take place
in sequences at least 5 bases long. A more preferred recognition
sequence length is 5-60 base pairs long. Sequences between 10 and
20 bases are of particular interest since this is the range within
which unique DNA sequences of prokaryotes and eukaryotes are found.
Sequences of 17-18 bases are of special interest since this is the
length of unique sequences in the human genome.
[0023] We have further surprisingly found that the PNA compounds
are able to form triple helices with dsDNA. We have found that PNA
compounds are able to form triple helices with RNA and ssDNA. The
resulting triplexes, e.g., (PNA).sub.2/DNA or (PNA).sub.2/RNA,
surprisingly have very high thermal stability. It has been found
that the PNA binds with a DNA or RNA in either orientation, i.e.,
the antiparallel orientation where the amino-terminal of the PNA
faces the 3' end of the nucleic acid or the parallel orientation
where the amino-terminal of the PNA faces the 5' end of the nucleic
acid. PNAs are able to form triple helices wherein a first PNA
strand binds with RNA or ssDNA and a second PNA strand binds with
the resulting double helix or with the first PNA strand.
[0024] We further have found that the PNA compounds are able to
form triple helices wherein a first PNA strand binds with the ssDNA
or RNA or to one of the strands of dsDNA and in doing so displaces
the other strand, and a second PNA strand then binds with the
resulting double helix. While we do not wish to be bound by theory,
it is further believed that other triple helices might be formed
wherein a single PNA strand binds to two single stranded nucleic
acids strands. In binding with nucleic acids both Watson-Crick and
Hoogsteen bind is utilized. It is further believed that PNA might
also bind via reverse Hoogsteen binding.
[0025] We have further surprisingly found that the PNA compounds
form double helices with RNA and ssDNA. Such double helices are
hetero duplex structures between the PNA and the respective nucleic
acid. Such double helices are preferably helices formed when the
PNA strand includes a mixture of both pyrimidine and purines
nucleobases.
[0026] For therapeutic use of PNA compounds the targets of the PNA
compounds would generally be double stranded DNA and RNA. For
diagnostic use, investigations methods and reagents where DNA is
isolated outside of a cell, the DNA can be denatured to single
stranded DNA and use of the PNA compound would be targeted to such
single stranded DNA as well as RNA.
[0027] PNA compounds useful to effect binding to RNA, ssDNA and
dsDNA and to form duplex and triplex complexes are in one sense
polymeric strands formed from a polyamide, polythioamide,
polysulfinamide or polysulfonamide backbone with a plurality of
ligands located at spaced locations along the backbone. At least
some of the ligands are capable of hydrogen bonding with other
ligands either on the compounds or nucleic acid ligands.
[0028] More preferred PNA compounds according to the invention have
the formula: 1
[0029] wherein:
[0030] n is at least 2,
[0031] each of L.sup.1-L.sup.n is independently selected from the
group consisting of hydrogen, hydroxy, (C.sub.1-C.sub.4)alkanoyl,
naturally occurring nucleobases, non-naturally occurring
nucleobases, aromatic moieties, DNA intercalators,
nucleobase-binding groups, heterocyclic moieties, and reporter
ligands;
[0032] each of C.sup.1-C.sup.n is (CR.sup.6R.sup.7).sub.y where
R.sup.6 is hydrogen and R.sup.7 is selected from the group
consisting of the side chains of naturally occurring alpha amino
acids, or R.sup.6 and R.sup.7 are independently selected from the
group consisting of hydrogen, (C.sub.2-C.sub.6)alkyl, aryl,
aralkyl, heteroaryl, hydroxy, (C.sub.1-C.sub.6)alkoxy,
(C.sub.2-C.sub.6)alkylthio, NR.sup.3R.sup.4 and SR.sup.5, where
R.sup.3 and R.sup.4 are as defined above, and R.sup.5 is hydrogen,
(C.sub.1-C.sub.6)alkyl, hydroxy-, alkoxy-, or alkylthio-substituted
(C.sub.1-C.sub.6)alkyl, or R.sup.6 and R.sup.7 taken together
complete an alicyclic or heterocyclic system;
[0033] each of D.sup.1-D.sup.n is (CR.sup.6R.sup.7).sub.s where
R.sup.6 and R.sup.7 are as defined above;
[0034] each of y and z is zero or an integer from 1 to 10, the sum
y+z being greater than 2 but not more than 10;
[0035] each of G.sup.1-G.sup.n-1 is --NR.sup.3CO--, --NR.sup.3CS--,
--NR.sup.3SO-- or --NR.sup.3SO.sub.2--, in either orientation,
where R.sup.3 is as defined above;
[0036] each of A.sup.1-A.sup.n and B.sup.1-B.sup.n are selected
such that:
[0037] (a) A is a group of formula (IIa), (IIb), (IIc) or (IId),
and B is N or R.sup.3N.sup.+; or
[0038] (b) A is a group of formula (IId) and B is CH; 2
[0039] where:
[0040] X is O, S, Se, NR.sup.3, CH.sub.2 or C(CH.sub.3).sub.2;
[0041] Y is a single bond, O, S or NR.sup.4;
[0042] each of p and q is zero or an integer from 1 to 5, the sum
p+q being not more than 10;
[0043] each of r and s is zero or an integer from 1 to 5, the sum
r+s being not more than 10;
[0044] each R.sup.1 and R.sup.2 is independently selected from the
group consisting of hydrogen, (C.sub.1-C.sub.4)alkyl which may be
hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy,
alkylthio, amino and halogen; and
[0045] each R.sup.3 and R.sup.4 is independently selected from the
group consisting of hydrogen, (C.sub.1-C.sub.4)alkyl, hydroxy- or
alkoxy- or alkylthio-substituted (C.sub.1-C.sub.4)alkyl, hydroxy,
alkoxy, alkylthio and amino;
[0046] Q is --CO.sub.2H, --CONR'R", --SO.sub.3H or --SO.sub.2NR'R"
or an activated derivative of --CO.sub.2H or --SO.sub.3H; and
[0047] I is --NHR'"R"" or --NR'"C(O)R"", where R', R", R'" and R""
are independently selected from the group consisting of hydrogen,
alkyl, amino protecting groups, reporter ligands, intercalators,
chelators, peptides, proteins, carbohydrates, lipids, steroids,
nucleosides, nucleotides, nucleotide diphosphates, nucleotide
triphosphates, oligonucleotides, oligonucleosides and soluble and
non-soluble polymers.
[0048] In the above structures wherein R', R", R'" and R"" are
oligonucleotides or oligonucleosides, such structures can be
considered chimeric structures between PNA compounds and the
oligonucleotide or oligonucleoside.
[0049] Preferred PNA-containing compounds useful to effect binding
to RNA, ssDNA and dsDNA and to form triplexing structure are
compounds of the formula III, IV or V: 3
[0050] wherein:
[0051] each L is independently selected from the group consisting
of hydrogen, phenyl, heterocyclic moieties, naturally occurring
nucleobases, and non-naturally occurring nucleobases;
[0052] each R.sup.7' is independently selected from the group
consisting of hydrogen and the side chains of naturally occurring
alpha amino acids;
[0053] n is an integer greater than 1,
[0054] each k, l, and m is, independently, zero or an integer from
1 to 5;
[0055] each p is zero or 1;
[0056] R.sup.h is OH, NH.sub.2 or --NHLysNH.sub.2; and
[0057] R.sup.i is H or COCH.sub.3.
[0058] The improved binding of the PNA compounds of the invention
with single-stranded RNA and DNA renders them useful as antisense
agents. In addition, the binding to double-stranded DNA renders
these compounds useful for gene inhibition via various mechanisms.
Further, the binding to double-stranded DNA renders these compounds
useful as gene activators to initiate transcription.
[0059] In one embodiment, the present invention provides methods
for inhibiting the expression of particular genes in the cells of
an organism, comprising administering to said organism a reagent as
defined above which binds specifically to sequences of said
genes.
[0060] In a further embodiment, the invention provides methods for
inhibiting transcription and/or replication of particular genes or
for modifying double-stranded DNA as, for instance, by inducing
degradation of particular regions of double-stranded DNA in cells
of an organism comprising administering to said organism a reagent
as defined above.
[0061] In a still further embodiment, the invention provides
methods for killing cells or virus by contacting said cells or
virus with a reagent as defined above which binds specifically to
sequences of the genome of said cells or virus.
[0062] A novel strategy for sequence-selective cleavage of
double-stranded DNA is described. For cases were two closely
positioned homo-pyrimidine stretches (of 7-10 bases and preferably
on opposite strands) can be identified, this can be done by
synthesizing pairs of PNAs complementary to two parts of this DNA
sequence and separated by several base pairs. These PNA molecules
are then reacted with the dsDNA and the resulting complex is
allowed to react with an endonuclease.
[0063] In practicing certain embodiments of the invention, the PNA
compounds are able to recognize duplex DNA by displacing one
strand, thereby presumably generating a hetero duplex with the
other one. Such recognition can take place with dsDNA sequences
5-60 base pairs long. Sequences between 10 and 20 bases are of
interest since this is the range within which unique DNA sequences
of prokaryotes and eukaryotes are found. Reagents which recognize
17-18 bases are of particular interest since this is the length of
unique sequences in the human genome.
[0064] The PNA compounds are able to form triple helices with
dsDNA, ssDNA or RNA and double helices with RNA or ssDNA. In one
embodiment of the invention, the PNA compounds form triple helices
wherein a first PNA strand binds with a nucleic acid strand forming
a hetero duplex and a second PNA strand then binds with the
resulting hetero duplex. In other embodiments of the invention, a
PNA compound or a PNA chimera compound forms triple helices wherein
a single PNA strand or PNA chimera strand binds with two nucleic
acid strands, with a nucleic acid strand and a PNA chimera strand
or with two chimera PNA strands.
[0065] The invention further provides methods for inhibiting the
action of restriction enzymes at restriction sites in nucleic
acids. Such methods comprise contacting a nucleic acid with a
reagent as defined above under conditions effective to bind such
reagent to the nucleic acid proximal to a restriction site.
[0066] The invention further provides methods of sequencing DNA by
binding the DNA with a reagent as defined above at a site proximal
to a restriction site, cleaving the DNA with a restriction enzyme,
and identifying the cleaved products.
[0067] The invention further provides methods for initiating
transcription in cells or organisms comprising administering to the
organism a reagent as defined above which initiates transcription
of a gene in such cells or organisms.
[0068] The invention further provides methods for modulating
binding of RNA polymerase to dsDNA by binding the dsDNA with a
reagent as defined above that binds with the DNA and then exposing
the complex formed thereby to a RNA polymerase.
[0069] The invention further provides methods for initiating
transcription of a gene by binding the gene with a reagent as
defined above that interacts with the gene to melt the
double-stranded DNA of the gene and to form a transcription
elongation loop.
[0070] The invention further provides methods for binding RNA
polymerase to dsDNA by contacting the dsDNA with a reagent as
defined above that is capable of interacting with the DNA and then
exposing the complex formed thereby to a RNA polymerase. More
particularly, the interaction is via binding to said dsDNA.
[0071] The invention further provides a hybrid complex for
modulating transcription wherein the complex comprises dsDNA and a
reagent as defined above that binds with the dsDNA, and a RNA
transferase.
[0072] The invention further provides a synthetic transcription
factor comprising a dsDNA and a reagent as defined above that is
capable of interaction with the dsDNA. More particularly, the
interaction is via binding to said dsDNA.
[0073] The invention further provides specific gene activators
comprising first and second strands, as defined above, that have
specific sequences that bind to selected DNA regions of the
gene.
[0074] The invention further provides chimeric structures
comprising PNAs and DNA or RNA. Such chimeric structures will be
used in place of or in addition to a normal PNA strand to effect
duplexing, triplexing, nucleic acid binding or protein binding.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1 is an ethidium bromide stained gel showing cleavage
in a sequence specific manner by S.sub.1 nuclease and a nucleic
acid analogue of a double-stranded DNA having a single site for
hybridization to the nucleic acid analogue (Example 1);
[0076] FIG. 2 is an ethidium bromide stained gel showing a similar
cleavage of a double-stranded DNA having two closely spaced sites
for hybridization to the nucleic acid analogue on the same DNA
strand (Example 2);
[0077] FIG. 3 is an ethidium bromide stained gel showing a similar
cleavage of a double-stranded DNA having two closely spaced sites
for hybridization to a nucleic acid analogue on opposite strands
(Example 3).
[0078] FIG. 4 shows a PAGE autoradiograph demonstrating that
PNAs-T.sub.10, -T.sub.9C and -T.sub.8C.sub.2 bind to
double-stranded DNA with high sequence specificy;
[0079] FIG. 5 shows an electrophoretic gel staining demonstrating
that restriction enzyme activity towards DNA is inhibited when PNA
is bound proximal to the restriction enzyme recognition site;
[0080] FIG. 6 shows a graph based on densitometric scanning of PAGE
autoradiographs demonstrating the kinetics of the binding of
PNA-T.sub.10 to a double-stranded target;
[0081] FIG. 7 shows a graph based on densitometric scanning of PAGE
autoradiographs demonstrating the thermal stabilities of PNAs of
varying lengths bound to an A.sub.10/T.sub.10 double-stranded DNA
target;
[0082] FIG. 8 is a schematic model of C.sup.+G-G and T A-T triplets
with Hoogsteen and Watson-Crick hydrogen bonds;
[0083] FIG. 9 is a schematic model of the binding of PNA
(T.sub.2CT.sub.2CT.sub.4) to a double-stranded DNA target;
[0084] FIG. 10 shows a footprinting experiment of the binding of
PNA to a dimeric target;
[0085] FIG. 11 shows a footprinting experiment of PNA binding to a
monomeric target;
[0086] FIG. 12 is a footprinting experiment showing the effect of
pH and salt on the binding of a thymine/cytosine containing PNA to
double stranded DNA;
[0087] FIG. 13 is an electron micrograph of a PNA double strand DNA
complex;
[0088] FIG. 14 is a histogram representation of the results
obtained in the experiments of FIG. 13;
[0089] FIG. 15 is a one dimensional gel electrophoresis experiment
showing the DNA unwinding upon PNA binding;
[0090] FIG. 16 is a two dimensional gel electrophoresis experiment
showing the DNA unwinding upon PNA binding;
[0091] FIG. 17a shows the salt dependent binding of PNA in a gel
electrophoresis experiment;
[0092] FIG. 17b shows the salt resistance of the PNA-DNA complex in
a gel electrophoresis experiment;
[0093] FIG. 18 is an autoradiogram showing sequence selective
transcription inhibition by PNA;
[0094] FIG. 19 is an autoradiogram showing sequence specific
inhibition of Taq DNA polymerase by PNA;
[0095] FIG. 20 is an autoradiogram showing the concentration
dependence of transcription inhibition by PNA T.sub.10;
[0096] FIG. 21 is a FIG. 20 but using PNA
T.sub.4CT.sub.2CT.sub.2;
[0097] FIG. 22 is a quantitative representation of the results of
FIGS. 20 & 21;
[0098] FIG. 23 is an autoradiogram showing the results of
transcription arrest using various PNA and RNA polymerases;
[0099] FIG. 24 is a continuation of FIG. 23;
[0100] FIG. 25 is an autoradiogram showing the specific effect on
transcription of PNA bound to the template versus the non-template
strand;
[0101] FIG. 26 is an autoradiogram showing transcription arrest by
short (T.sub.6&T.sub.8) PNAs;
[0102] FIG. 27 is a schematic drawing of PNA-DNA transcription
initiation complexes;
[0103] FIG. 28 is an autoradiogram showing transcription initiation
by PNA;
[0104] FIG. 29 is an autoradiogram showing the efficient
competition of a "PNA promoter" versus the strong lacUV5
promoter;
[0105] FIG. 30 illustrates schematic structures of PNA/DNA
complexes and sequences of the PNA targets;
[0106] FIG. 31 is a plasmid map;
[0107] FIG. 32 shows a PAGE autoradiography illustrating site
specific termination of in vitro transcription;
[0108] FIG. 33 shows a PAGE autoradiography the effect of target
site orientation on PNA mediated termination of in vitro
transcription;
[0109] FIG. 34 shows a PAGE autoradiography illustrating the effect
of salt concentration on PNA activity;
[0110] FIG. 35 shows a PAGE autoradiography illustrating the effect
of pH on PNA activity;
[0111] FIG. 36 are charts plotting K.sub.0 vs ph and K.sub.0 vs
ionic strength on in vitro binding of PNA to complement;
[0112] FIG. 37 is a chart illustrating melting temperature for
hybrid duplex binding;
[0113] FIG. 38 is a chart illustrating melting temperature for
hybrid duplex binding;
[0114] FIG. 39 shows a PAGE autoradiography illustrating titration
by gel-shift binding of PNA to a end labeled oligonucleotide;
[0115] FIG. 40 is a circular dichroism spectra;
[0116] FIG. 41 is a further circular dichroism spectra;
[0117] FIG. 42 is a chemical schematic; and
[0118] FIG. 43 is a further chemical schematic.
DETAILED DESCRIPTION OF THE INVENTION
[0119] For the purposes of this invention the term "purine rich"
shall mean a preponderance (i.e., greater than 50%) of purine
bases, preferably greater than about 65% and most preferrably
greater than about 90%. Further, the term "modify" shall mean to
change properties by addition to, substraction from, cleavage of or
otherwise, such that which results is intrinsically different from
that which is modified. The term "modulate" shall mean to change
(i.e., increase or decrease) the magnitude of a property.
[0120] In accordance with the present inventions, the ligand L is
primarily a naturally occurring nucleobase attached at the position
found in nature, i.e., position 9 for purines including adenine,
guanine or inosine, and position 1 for pyrimidines including
thymine, uridine, or cytosine. Alternatively, L may be a
non-naturally occurring nucleobase (nucleobase analog), another
base-binding moiety, an aromatic moiety, (C.sub.1-C.sub.4)alkanoyl,
hydroxy or even hydrogen. It will be understood that the term
nucleobase includes nucleobases bearing removable protecting
groups. Some typical nucleobase ligands and illustrative synthetic
ligands are shown in FIG. 2 of WO 92/20702. Two particular
nucleobase ligands, 5-propynylthymine and 3-deazauracil, have been
shown to increase binding affinity of an oligonucleotide to a
target nucleotide (see Froehler, B. C. et. al., Tetrahedron
Letters, 1992 33:5307-5310 and U.S. Pat. No. 5,134,066). Other like
analogues are shown by Sagi, J. et. al., Tetrahedron Letters, 1993
34:2191-2194. Incorporation of these nucleobase can be effected to
increase the binding affinity of the PNA product with a nucleic
acid target. Further useful non-naturally occurring nucleobases
include 6-thioguanine, i.e., purine-6(1H)-thione, and
pyrazolo[4,3-d]pyrimidines especially useful as triplexing bases
(see PCT application PCT/US92/04795).
[0121] Furthermore, L can be a DNA intercalator, a reporter ligand
such as, for example, a fluorophor, a radio label, a spin label,
hapten, or a protein-recognizing ligand such as biotin. In monomer
synthons, L may be blocked with protecting groups, as illustrated
in FIG. 4 of WO 92/20702.
[0122] Linker A can be a wide variety of groups such as
--CR.sup.1R.sup.2CO--, --CR.sup.1R.sup.2CS--,
--CR.sup.1R.sup.2CSe--, --CR.sup.1R.sup.2CNHR.sup.3--,
--CR.sup.1R.sup.2C.dbd.CH.sub.2-- and
--CR.sup.1R.sup.2C.dbd.C(CH.sub.3).sub.2--, where R.sup.1, R.sup.2
and R.sup.3 are as defined above. Preferably, A is
methylenecarbonyl (--CH.sub.2CO--), amido (--CONR.sup.3--), or
ureido (--NR.sup.3CONR.sup.3--). Also, A can be a longer chain
moiety such as propanoyl, butanoyl or pentanoyl, or corresponding
derivative, wherein O is replaced by another value of X or the
chain is substituted with R.sup.1R.sup.2 or is heterogenous,
containing Y. Further, A can be a (C.sub.2-C.sub.6)alkylene chain,
a (C.sub.2-C.sub.6)alkylene chain substituted with R.sup.1R.sup.2
or can be heterogenous, containing Y. In certain cases, A can just
be a single bond.
[0123] In one preferred form of the invention, B is a nitrogen
atom, thereby presenting the possibility of an achiral backbone. B
can also be R.sup.3N.sup.+, where R.sup.3 is as defined above, or
CH.
[0124] In the preferred form of the invention, C is
--CR.sup.6R.sup.7--, but can also be a two carbon unit, i.e.,
--CHR.sup.6CHR.sup.7-- or --CR.sup.6R.sup.7CH.sub.2--, where
R.sup.6 and R.sup.7 are as defined above. R.sup.6 and R.sup.7 also
can be heteroaryl groups such as, for example, pyrrolyl, furyl,
thienyl, imidazolyl, pyridyl, pyrimidinyl, indolyl, or can be taken
together to complete an alicyclic system such as, for example,
1,2-cyclobutanediyl, 1,2-cyclopentanediyl or
1,2-cyclohexanediyl.
[0125] In the preferred form of the invention, E in the monomer
synthon is COOH or an activated derivative thereof, and G in the
oligomer is --CONR.sup.3-- (amide). As defined above, E may also be
CSOH, SOOH, SO.sub.2OH or an activated derivative thereof, whereby
G in the oligomer becomes --CSNR.sup.3--, --SONR.sup.3-- and
--SO.sub.2NR.sup.3--, (thioamide, sulfinamide or sulfonamide,
respectively). The G group can be in either orientation, e.g., for
amide --CONR.sub.3-- or --R.sub.3NCO--. The activation may, for
example, be achieved using an acid anhydride or an active ester
derivative, wherein hydrogen in the groups represented by E is
replaced by a leaving group suited for generating the growing
backbone.
[0126] The amino acids which form the backbone may be identical or
different. We have found that those based on 2-aminoethylglycine
are especially well suited to the purpose of the invention.
[0127] In some cases it may be of interest to attach ligands at
either terminus (Q, I) to modulate the binding characteristics of
the PNAS. Representative ligands include DNA intercalators, which
improve dsDNA binding or basic groups, such as lysine or
polylysine, which strengthen the binding of the PNA due to
electrostatic interaction. To decrease electrostatic repulsion
charged groups such as carboxyl and sulfo groups could be used. The
design of the synthons further allows such other moieties to be
located on non-terminal positions. Oligonucleotides and/or
oligonucleoside can be covalently bound to terminal positions Q or
I to form chimeras containing PNA portions and oligonucleotide
and/or oligonucleoside portions. Nucleosides and/or nucleotides
(mono, di or tri-phosphates) also can be attached to the terminal
positions.
[0128] In a further aspect of the invention, the PNA oligomers are
conjugated to low molecular weight effector ligands such as ligands
having nuclease activity or alkylating activity or reporter ligands
(fluorescent, spin labels, radioactive, protein recognition
ligands, for example, biotin or haptens). In a further aspect of
the invention, the PNAs are conjugated to peptides or proteins,
where the peptides have signaling activity and the proteins are,
for example, enzymes, transcription factors or antibodies. Also,
the PNAs can be attached to water-soluble or water-insoluble
polymers. In another aspect of the invention, the PNAs are
conjugated to oligonucleotides or carbohydrates. When warranted, a
PNA oligomer can be synthesized onto some moiety (e.g., a peptide
chain, reporter, intercalator or other type of ligand-containing
group) attached to a solid support.
[0129] Such conjugates can be used for gene modulation (e.g., gene
targeted drugs), for diagnostics, for biotechnology, and for
scientific purposes.
[0130] As a further aspect of the invention, PNAs can be used to
target RNA and ssDNA to produce both antisense-type gene regulating
moieties and hybridization probes for the identification and
purification of nucleic acids. Furthermore, the PNAs can be
modified in such a way that they form triple helices with dsDNA.
Reagents that bind sequence-specifically to dsDNA have applications
as gene targeted drugs. These are foreseen as extremely useful
drugs for treating diseases like cancer, AIDS and other virus
infections, and may also prove effective for treatment of some
genetic diseases. Furthermore, these reagents may be used for
research and in diagnostics for detection and isolation of specific
nucleic acids.
[0131] Triple helix formation, wherein an oligonucleotide is
triplexed to a dsDNA, is believed to be the only means known in the
art for sequence-specific recognition of dsDNA. However, triple
helix formation is largely limited to recognition of
homopurine-homopyrimidine sequences. Triplexing with strand
displacement using PNAS of this invention is superior to
oligonucleotide-dsDNA triple helix recognition in that it may allow
for recognition of any sequence by use of the four natural
bases.
[0132] Gene targeted drugs are designed with a nucleobase sequence
(containing 10-20 units) complementary to the regulatory region
(the promoter) of the target gene. Upon administration, the drug
binds to the promoter and blocks access thereto by RNA polymerase.
Consequently, no mRNA, and thus no gene product (protein), is
produced. If the target is within a vital gene for a virus, no
viable virus particles will be produced. Alternatively, if the
target is downstream from the promoter, RNA polymerase will
terminate at this position, thus forming a truncated mRNA/protein
which is nonfunctional.
[0133] Sequence-specific recognition of ssDNA by base complementary
hybridization can be exploited to target specific genes and
viruses. In this case, the target sequence is contained in the mRNA
such that binding of the drug to the target hinders the action of
ribosomes and, consequently, translation of the mRNA into protein.
The peptide nucleic acids of the invention are superior to prior
reagents in that they have significantly higher affinity for
complementary ssDNA. Also while a charged species such as a lysine
moiety can be added, PNAs possess no charge, they are water soluble
(which should facilitate cellular uptake), and they contain amides
of non-biological amino acids (which should make them biostable and
resistant to enzymatic degradation by, for example, proteases).
Further, they can triplex with the mRNA.
[0134] The PNA compounds used in this invention can be synthesized
by the methodologies disclosed in WO 92/20702, WO/92/20703 and the
foregoing United States patent application bearing attorney docket
ISIS-1017. Monomer synthons according to those disclosures are
coupled using standard protocols to give the desired PNA oligomeric
sequences. The synthesis of additional synthetic monomers are given
by example 33 through 45 of this specification.
[0135] Binding of PNA compounds to double-stranded DNA accompanied
by strand displacement is shown in illustrative examples in this
specification by both enzymatic and chemical probing. Our
illustrative examples show that the PNA compounds bind via
Watson-Crick binding to their complementary strand and extrudes the
other strand in a virtually single-stranded conformation.
[0136] Using the PNA compound to locally unwind the DNA duplex and
effect strand displacement can render the displaced DNA strand
sensitive to cleavage (e.g., cleavage by S.sub.1 nuclease). This
gives or induces high yield double strand DNA breaks at the site of
PNA binding. PNA-directed and PNA-provoked double strand DNA
cleavage by S.sub.1 nuclease is especially efficient when two
adjacent PNA sites are targeted, yielding quanti-tative conversion
of the target site into double-strand breaks.
[0137] As an illustrative example of use as an artificial
restriction enzyme, the targets are cloned within a pUC19
polylinker and the plasmids are linearized with the Cfr10I
restriction enzyme. In the linear DNA, the resulting polylinker
region is in the middle (1.33 kb from one end and 1.36 kb from the
other). A pT10 plasmid carrying a known sequence is then inserted
in the unique BamHl site in the polylinker. This is complexed with
a complementary PNA compound and subjected to treatment with
S.sub.1 nuclease. Upon nuclease treatment, a significant fraction
of DNA is cut, as shown by gel electrophoresis of the resulting
fragments. Cross reactivity experiments show that the targeting is
sequence-specific; only corresponding PNAs mediated cutting of the
targets. The yield of digested molecules is high and one can reach
the quantitative digestion with increasing exposure to
nuclease.
[0138] The results of such treatment demonstrate a novel method for
sequence-selective cleavage of double-stranded DNA. If two closely
positioned homo-pyrimidine stretches can be identified (as for
example 7-10 bases, preferably on opposite strands), this can be
done by synthesizing pairs of PNAs complementary to two parts of
this DNA sequence and separated by several base pairs. If this
strand displacement binding mode is extended to include PNAs
recognizing DNA sequences containing thymine and cytosine, this
strategy allows targeting and specific cleavage of any desired
sequence of 10-20 base pairs. In effecting such cleavage, the high
stability of PNA-DNA complexes consisting of two PNA strands (one
"Watson-Crick" and one "Hoogsteen") and one DNA strand must be
considered, as must the sequence of the "Hoogsteen-like"
strand.
[0139] We have found that complexes between homopyrimidine PNA and
complementary oligonucleotides show 2:1 stoichiometry. Further we
have found that the thermal stability of triplexes between
cytosine-containing homopyrimidine PNA and complementary
oligonucleotides is strongly pH dependent, indicating the
involvement of C.sup.+G-C Hoogsteen hydrogen bonding in
(PNA).sub.2/DNA triplexes. Further, while we do not wish to be
bound by any particular theory, we believe that the strand
displacement binding of homopyrimidine PNA to dsDNA is dependent on
the formation of such (PNA).sub.2/DNA triplexes with the
complementary strand of the dsDNA target. It is our present belief
that formation of the strand displacement complex proceeds via a
DNA/PNA Watson-Crick duplex which is subsequently stabilized by a
Hoogsteen bound second strand having C.sup.+-G Hoogsteen hydrogen
bonds.
[0140] In further studies of triplexing and strand displacing, we
utilized a cytosine containing homopyrimidine PNA
(T.sub.2CT.sub.2CT.sub.4-LysNH.s- ub.2; SEQ ID NO:1) and studied
its binding to a dimeric target. Stability of the complex was
probed by both dimethylsulfate (DMS) probing and KMnO.sub.4
probing. In these studies, we found that triple strand formation
involves Hoogsteen hydrogen bonding. This was evidenced by our
showing that the N7 atoms of guanines of the target participate in
base pairing and, thus, are protected from reaction with DMS upon
triplexing binding of the PNA. A model of this triplexing binding
is shown in FIGS. 8 and 9.
[0141] We have also used electron microscopy to confirm strand
displacement. In these examples, a PNA-DNA complex was formed with
a target. Full occupancy of the target resulted in a strand
displacement loop of 90-100 bases, as detected by electron
microscopy. The DNA molecules carry an open region in the form of
an "eye". In all cases, one of the two strands in the open region
is thicker than the other one and has the same thickness as the
normal DNA duplex. The thicker strand corresponds to the strand
covered by PNA while the thinner strand corresponds to the
displaced strand. In control experiments carried out without added
PNA, the displaced structures are not observed.
[0142] Discrimination of PNA compounds in binding to targets was
also observed utilizing labeled DNA fragments that were challenged
with various PNA compounds. We show that PNA compounds bind with
high preference to the complementary target. Binding decreases with
increasing mismatches in the targets. Binding to a target
containing one mismatch was weaker than for a fully complementary
target, while no binding was observed with a two-mismatch
target.
[0143] We have further found that PNA compounds truly mimic DNA in
terms of base pair specific hybridization to complementary strands
of oligonucleotides as measured by thermal stability of complexes.
We have shown that the formation of PNA/DNA duplexes exhibits a
decrease in entropy almost identical to that for the formation of
DNA/DNA duplexes and that like the formation of a DNA/DNA it is
strongly enthalpy driven. From this we deduce that single stranded
PNA must have the same degree of base stacking as single stranded
DNA and thus appears to be highly structured. Further we have found
that the rate of hybridization of PNA/RNA duplexes is at least as
fast as that for 2'-O-methyl RNA/RNA or DNA/DNA duplex formation
again supporting our findings that single stranded PNA is at least
as prestructured for duplex formation as is DNA or RNA.
[0144] We have further found that in contrast to DNA or RNA, PNA
compounds may bind to complementary DNA or RNA in one of two
orientations, a parallel orientation or an anti-parallel
orientation. We have further found that the PNA compounds prefer
the anti-parallel orientation (wherein the amino end of the PNA
strand is complementary to the 3'-end of the nucleic acid) over the
parallel orientation ((wherein the amino end of the PNA strand is
complementary to the 5'-end of the nucleic acid).
[0145] In our studies we have shown that homo-pyrimidine PNA binds
strongly to ssDNA, dsDNA or RNA with 2:1 stoichiometry (triplexing)
and effects stable strand displacement complexes with dsDNA.
Further, purine-pyrimidine PNA binds to Watson-Crick complementary
oligonucleotides, either RNA or DNA, with 1:1 stoichiometry.
Utilizing these findings, one embodiment of this invention is
directed to use of PNA compounds as effective inhibitors of the
elongation activity of RNA and DNA polymerases. As contrasted to
oligonucleotides, this allows use of PNA compounds to target genes
not only at promoter or regulatory regions of the genome but to
other regions as well. Furthermore, only decamer targets are
required for efficient binding of the PNA to dsDNA. Consequently,
it is easier to identify suitable PNA targets as compared to
oligonucleotide triple helix targets.
[0146] We have further found that PNA compounds have certain
effects on transcription in both prokaryotic and eukaryotic
systems. Transcription by RNA polymerases T.sub.3 or T.sub.7 of PNA
T.sub.10-LysNH.sub.2 (SEQ ID NO:2) and
T.sub.2CT.sub.2CT.sub.4-LysNH.sub.2 (SEQ ID NO:1) bound to dsDNA
targets A.sub.10 (SEQ ID NO:3), A.sub.5GA.sub.4 (SEQ ID NO:4) and
A.sub.2GA.sub.2GA, (SEQ ID NO:5) positioned downstream from T.sub.3
or T.sub.7 promoters in pBluescriptKS.sup.+ plasmids were studied.
As shown in our illustrative examples below, transcription
elongation is arrested at the site of PNA binding to the template
strand, whereas only a marginal effect is observed at the site of
PNA binding to the non-template strand. With PNA
T.sub.10-LysNH.sub.2 (SEQ ID NO:2), transcription arrest occurs at
the first base of the PNA binding site, while arrest with PNA
T.sub.5CT.sub.4-LysNH.sub.2 (SEQ ID NO:6) takes place 2-3 bases
inside the PNA binding site. In the case of PNA
T.sub.2CT.sub.2CT.sub.4-LysNH.sub.2 (SEQ ID NO:1), arrest is less
efficient and occurs at the last 1-3 bases of the binding site.
[0147] Transcription arrest has also been shown for PNAs
T.sub.5-LysNH.sub.2 (SEQ ID NO:7) and T.sub.8-LysNH.sub.2, although
at lower efficiency compared to longer PNA compounds. These results
show that efficient transcription elongation arrest can be obtained
by PNA targeting of the transcribed strand, and that "read through"
by the polymerase takes place in a sequence dependent manner.
[0148] Transcription arrest in eukaryotic systems has further been
shown for PNA compounds. For these studies plasmids were
constructed with the CMV IE 1 promoter driving the transcription of
viral DNA sequences. The viral DNA sequences containing homopurine
target sites for the PNAs were cloned downstream of the CMV IE 1
promoter. The plasmids were incubated with PNA oligomers under
various conditions then added to eukaryotic nuclear extracts to
initiate in vitro transcription. Transcripts were specifically
truncated at the site of PNA binding. This effect was dependent
upon hybridization of the PNA to the template strand, i.e., the
inhibition of transcription was sequence specific. Binding affinity
was increased by either lowering the ionic strength or pH of the
binding buffer. These results are consistent for PNA binding in
which homopyrimidine PNA's invade duplex DNA binding the
complementary DNA strand, while displacing the non-complementary
strand. A second PNA then interacts with the PNA/DNA duplex forming
a stable triple stranded structure which is capable of blocking RNA
polymerase II and terminating transcription thus specifically
inhibiting eukaryotic gene expression. This use of PNA compounds to
inhibit eukaryotic gene expression renders the PNA compounds useful
for antigene therapeutics.
[0149] In a further embodiment of this invention, we have found
that PNA compounds also can be used as sequence specific gene
activators and synthetic transcription factors. Transcription
initiation by RNA polymerase involves the sequence specific
recognition of the double-stranded DNA promoter either by the
polymerase itself or by auxiliary transcription factors.
Subsequently a transcription initiation open complex is formed in
which about 12 base pairs of the DNA helix are melted. This exposes
the bases of the template strand for base pairing with the RNA
strand being synthesized. It has been shown that an E coli phage
T.sub.7 RNA polymerase can utilize synthetic "RNA/DNA bubble
duplex" complexes containing an RNA/DNA duplex and a
single-stranded DNA D-loop for transcription elongation. We have
found that homopyrimidine PNAs also form D-loop structures when
binding to complimentary double-stranded DNA by strand
displacement. We believe that these structures behave like RNA/DNA
open complex structures and are recognized by RNA polymerase.
[0150] In the illustrative examples below, we show that E. Coli RNA
polymerase does indeed bind to PNA/dsDNA strand displacement
complexes and initiates RNA transcription therefrom. The results of
these examples further suggest that a single-stranded DNA loop is a
major structural determinant for RNA polymerase upon transcription
initiation and elongation. The results of these examples also have
implications for elucidating the mechanism of action of RNA
polymerase.
[0151] In further embodiments of the invention chimera strands are
formed between PNAs and either RNA or DNA. Such chimeric strands
can then in the same manner as the "homo" PNA strands described
above. Such chimeric structures thus will be used, as described
above, for binding, duplexing, triplexing and the like. In one
particularly preferred use, either a PNA strand or a PNA containing
chimera strand will be used to bind to or otherwise modulate
proteins in cells. Such proteins will include transcription factors
and other regulatory proteins.
[0152] The chimeric structure between PNAs and DNA or RNA are used
in place of or in addition to a normal PNA strand to effect
duplexing, triplexing, nucleic acid binding or protein binding. The
RNA or DNA nucleic acid portion of such chimeric structures include
nucleic acid connected via phosphodiester, phosphorothioate,
phosphorodithioate, alkyl phosphonate, hydrogen phosphonate,
phosphotriester, phosphoramidite and other like phosphorus
linkages. They further can include other substitutions such as
substitution at the 2' position of a ribose sugar. Particularly
preferred are 2'-deoxy-2'-fluoro since they increase affinity of
the nucleic acid portion of the chimera to other nucleic acids and
2'-O-alkyl, particularly 2'-O-propyl, 2'-O-allyl and the like since
they confer nuclease resistance to the nucleic acid strand.
[0153] The following examples are given to illustrate the
invention. These examples are given for illustrative purposes and
are not meant to be limiting.
EXAMPLE 1
[0154] General Method for the Synthesis of PNA Oligomers
[0155] PNA oligomeric compounds were prepared generally in
accordance with the methods disclosed by WO 92/20702, WO 92/20703
and the foregoing United States patent application bearing attorney
docket ISIS-1017. Other PNA monomers are prepared as per Examples
34-46 below. Briefly, benzyhydrylamine resin (initially loaded 0.28
mmol/gm with Boc-L-Lys(2-chlorobenzyloxycarbonyl)) was swollen in
DMF and an excess of a monomer to be coupled was added, followed by
dicyclohexylcarbodiimide (0.15M in 50% DMF in dichloromethane). The
Boc deprotection was accomplished by trifluoroacetic acid
treatment. The progress of the coupling reactions was monitored by
qualitative or quantitative ninhydrin analysis. The PNA was
released from the resin using anhydrous HF or trifluoromethyl
sulfonic acid under standard conditions. The products were purified
using HPLC with acetonitrile-water (0.1% TFA) gradient and
structure confirmed by fast atom bombardment mass spectrometry.
[0156] Representative sequences synthesized by these methods
include the following as well as other sequences noted in the
various examples:
1 H-T.sub.10LysNH.sub.2 (SEQ ID NO: 2) H-T.sub.4CT.sub.5LysNH.sub.2
(SEQ ID NO: 8) H-T.sub.2CT.sub.2CT.sub.4LysNH.sub.2 (SEQ ID NO: 1)
H-T.sub.4CT.sub.2CT.sub.2LysNH.sub.2 (SEQ ID NO: 9)
H-TGTACGTCACAACTA-NH.sub.2 (SEQ ID NO: 10) H-CCTTCCCTT-NH.sub.2
(SEQ ID NO: 11) H-TTCCCTTCC-NH.sub.2 (SEQ ID NO: 12)
H-TAGTTATCTCTATCT-NH.sub.2 (SEQ ID NO: 13)
H-TGTACGTCACAACTA-NH.sub.2 (SEQ ID NO: 14) H-GCACAGCC-LYS-NH.sub.2
(SEQ ID NO: 15) H-TTTTCTTTT-NH.sub.2 (SEQ ID NO: 16)
H-TTTTTTTTTCCCCCCC-NH.sub.2 (SEQ ID NO: 17)
H-CCCCCCCTTTTTTTTT-NH.sub.2 (SEQ ID NO: 18) H-CCTCCTTCCC-NH.sub.2
(SEQ ID NO: 19) H-TTCTCTCTCT-NH.sub.2 (SEQ ID NO: 20)
H-TTTTTCTCTCTCTCT-NH.sub.2 (SEQ ID NO: 21)
H-CCCCCACCACTTCCCCTCTC-(Lys).sub.9NH.sub.2 (SEQ ID NO: 22)
H-CTTATATTCCGTCATCGCTC-Lys-NH.sub.2 (SEQ ID NO: 23)
H-CTGTCTCCATCCTCTTCACT-NH.sub.2 (SEQ ID NO: 24)
H-TATTCCGTCATCGCTCCTCA-Lys-NH.sub.2 (SEQ ID NO: 25)
H-CCCCCACCACTTCCCCTCTC-NH.sub.2 (SEQ ID NO: 26)
H-CTGCTGCCTCTGTCTCAGGT-LysNH.sub.2 (SEQ ID NO: 27)
H-T.sub.4-(.beta.-alanine)C-T.sub.5-LysNH.sub.2 (SEQ ID NO: 28)
H-T.sub.4-(.beta.-alanine)T-T.sub.5-LysNH.sub.2 (SEQ ID NO: 29)
[0157] The PNA's are written from amino to the carboxyterminal.
LysNH.sub.2 designates a lysine amide is attached to the PNA and
NH.sub.2 indicates a free c-terminal carboxamide without
lysine.
EXAMPLE 2
[0158] Site-Specific S.sub.1 Nuclease Digestion of the pT10 Plasmid
Linearized with Cfr10I Restriction Enzyme and Complexed with PNA (a
Double-Stranded Target)
[0159] PNA H-T.sub.10-LysNH.sub.2 (SEQ ID NO:2) was synthesized as
described in Example 1. A pT10 plasmid was prepared from the pUC19
plasmid by inserting the dA.sub.10/dT10 (SEQ ID NO:3) sequence into
the BamH1 site of the polylinker as per the method of Egholm, M et
al., J.A.C.S. 1992 114:1895-1897. The pT10 plasmid was linearized
with Cfr10I restriction enzyme in the unique site. To form complex
with the PNA, about 0.1 .mu.g of the linearized plasmid was
incubated with 2 o.u./ml of PNA in 3 .mu.l of the TE buffer (10 mM
Tris-HCl; 1 mM EDTA, pH 7.4) at 37.degree. C. To perform the
S.sub.1 nuclease reaction, 10 .mu.l of Na-Acetate buffer (33 mM
NaAc; 50 mM NaCl; 10 mM ZnSO.sub.4; 0.5% of glycerol, pH 4.6) and
145 units of S.sub.1 nuclease (Sigma) were added and incubated for
various time periods at room temperature. The reaction was
terminated by adding 1 .mu.l of 0.5 M EDTA and cooling to
-20.degree. C. Electrophoresis was performed in 1% agarose gel in
the TBE buffer with subsequent staining with ethidium bromide. The
results of this example are shown in FIG. 1 wherein the size of the
markers are shown as are the incubation time periods. In FIG. 8
Lane 1: control--preincubation of DNA with PNA was performed under
conditions unfavorable for complex formation (200 mM of NaCl). Lane
2: the reference band obtained by digestion with BamHI restriction
enzyme. Lane 3: reference bands obtained by the digestion with Bgll
restriction enzyme. Lanes 4, 5: the results of S.sub.1 nuclease
digestion for different times of DNA-PNA complex.
[0160] As is shown in this example, targets were cloned within a
pUC19 polylinker. The plasmids were linearized with the Cfr10I
restriction enzyme. In the linear DNA, the obtained polylinker
region is in the middle (1.33 kb from one end and 1.36 kb from the
other). Utilizing a direct approach, a pT10 plasmid carrying the
sequence dA.sub.10dT.sub.10 (SEQ ID NO:31) was inserted in the
unique BamHl site in the polyIinker. This was complexed with PNA
H-T.sub.10-LysNH.sub.2 (SEQ ID NO:2) and subjected to treatment
with S.sub.1 nuclease. After treatment with 145 units of S.sub.1
nuclease at room temperature a significant fraction of DNA was cut.
The mobility by gel electrophoresis of the resulting fragments was
very close to the mobility of fragments obtained by cutting with
restriction enzyme BamH1. Similar results were obtained for
H-T.sub.5CT.sub.4-LysNH.sub.2 (SEQ ID NO:6) and
H-T.sub.2CT.sub.2CT-LysNH- .sub.2 (SEQ ID NO:51) PNAs and
corresponding plasmids carrying the
d(A.sub.5GA.sub.4)/d(T.sub.4CT.sub.5) (SEQ ID NO:4) and
d(A.sub.4GA.sub.2GA.sub.2)/d(T.sub.2CT.sub.2CT.sub.4) (SEQ ID
NO:44) inserts. Cross reactivity experiments showed that the
targeting was sequence specific: only corresponding PNAs mediated
cutting of the targets. It was noted that two very week bands are
seen in lanes 4, 5, which correspond to lengths 2.32 and 0.98 kb.
These bands are due to weak binding of PNA-TIO to the intrinsic
pUC19 site d(TTGT3)/d(A3CAT), which is 0.37 kb apart from the
Cfr10I restriction site. This is consistent with the results of
Example 3 above that shows that the introduction of mismatches
dramatically decreases the affinity of PNA to DNA.
[0161] From this example it is believed that the enzyme first
digests the displaced strand, then to some extent enlarges the gap
after which the opposite strand becomes a substrate for the enzyme.
As a result, the double-stranded break is created. The yield of
digested molecules is high and one can reach the quantitative
digestion with increasing exposure to S.sub.1 nuclease (data not
shown). However, while the two fragments are poorly resolved after
digestion with BamHI restriction enzyme, the PNA-mediated digestion
with the S.sub.1 nuclease leads to the clear-cut doublet. While not
wishing to be bound by theory, we believe this probably reflects a
widening of the gap by the S.sub.1 nuclease as well as digestion
from the ends of the CfrlOI site. With longer treatment the
downward shift of the doublet becomes noticeable (see lane 5).
Thus, quantitative digestion is clearly accompanied by a truncation
of the fragments (by about hundred base pairs). To obtain further
precision of cutting comparable with that exhibited by natural
restriction enzymes the original approach is modified as per
Example 4 below.
[0162] Alternate Site-Specific S.sub.1 Nuclease Digestion of
Plasmid with PNA)
[0163] As an alternative target, the insert:
2 5'-A.sub.5GA.sub.4GTCGACA.sub.5GA.sub.4-3' (SEQ ID NO: 30)
3'-T.sub.5CT.sub.4CAGCTGT.sub.5CT.sub.4-5'
[0164] was cloned into the Sal1 site of the pUC19 plasmid. This
plasmid, which contained two binding sites for PNA
H-T.sub.5CT.sub.4-LysNH.sub.2 (SEQ ID NO:6) separated by six base
pairs, was designated pT9CT9C. Strand displacement in the two
T.sub.5CT.sub.4 sites led to opening of the entire region,
including the sequence GTCGAC/GACGAC, providing a substrate for the
S.sub.1 nuclease in both strands. As control, a pT9C-5 plasmid was
used, which carried only one T.sub.5CT.sub.4 insert cloned into the
Sal1 site of the pUC19 plasmid.
EXAMPLE 3
[0165] Site-Specific S.sub.1 Nuclease Digestion of the pT9C Plasmid
Linearized by CfrlOl Restriction Enzyme and Complexed with PNA
H-T.sub.5CT.sub.4-LysNH.sub.2
[0166] This example illustrates the use of one molecule of PNA
hybridized to a target DNA to define a restriction site.
[0167] PNA 1, 2 and 3 were synthesized as Example 1 and the results
are shown in FIG. 2. A pT9C plasmid carried the insert:
3 5'-A.sub.5GA.sub.4GTCGACA.sub.5GA.sub.4-3' (SEQ ID NO: 30)
3'-T.sub.5CT.sub.4CAGCTGT.sub.5CT.sub.4-5',
[0168] cloned in the Sal1 site of the pUC19 polylinker. The pT9C-5
plasmid carried the single insert A.sub.5GA.sub.4/CT.sub.5 cloned
in the same site. The PNA-DNA complexes were prepared as described
in Example 2 with the only difference being the duration of the
incubation was 2 hours. Digestion by 30 units of the S.sub.1
nuclease was performed in the same buffer as described in Example 2
with two exceptions, lanes 2 and 4. In lane 2, 15 units of the
enzyme were used, whereas in lane 4 200 mM NaCl and 1 mM of
ZnSO.sub.4 were added to the buffer. Lane 1: the reference band
obtained by digestion with the BamHI restriction enzyme of the
pUC19 plasmid linearized by the CfrlOl restriction enzyme. Lanes
2-4 site-specific digestion by the S.sub.1 nuclease of the
linearized pT9C plasmid complexed with PNA
H-T.sub.5CT.sub.4-LysNH.sub.3 (SEQ ID NO:6). Lanes 5-7: various
controls. Lane 8: the same experiment as in lane 3, but using PNA
T.sub.10 (SEQ ID NO:2) instead of T.sub.4CT.sub.5 (SEQ ID NO:8).
Lane 9: the same experiment as in lane 3, but using PNA
T.sub.2CT.sub.2CT.sub.4 (SEQ ID NO:1) instead of T.sub.4CT.sub.5
(SEQ ID NO:8). Lane 10: the same experiment as in lane 3, but using
the pT9C-5 plasmid instead of the pT9C. In the header PNA
T.sub.5CT.sub.4 (SEQ ID NO:6) is labelled as 1, T.sub.10 (SEQ ID
NO:2) as 2 and T.sub.2CT.sub.2CT.sub.4 (SEQ ID NO:1) as 3.
[0169] This example show that subjecting the pT9CT9C plasmid,
linearized by the Cfr10I restriction enzyme and complexed with PNA
H-T.sub.5CT.sub.4-LysNH.sub.2 (SEQ ID NO:6), to 30 units of the
S.sub.1 nuclease results in full conversion of the full-length DNA
molecules into half-length fragments. The bands observed by gel
electrophoresis have the same position and width as the band
created by the BamHI restriction enzyme. Sequence specificity of
this artificial "restriction enzyme" was confirmed by the very weak
digestion of the pT9C plasmid in case of complexing with PNA
H-T.sub.10-LysNH.sub.2 (SEQ ID NO:2) and
H-T.sub.2CT.sub.2CT.sub.4-LysNH.sub.2 (SEQ ID NO:1) under
conditions which result in quantitative cutting of the plasmid in
the presence of PNA H-T.sub.5CT.sub.4-LysNH2 (SEQ ID NO:6) (see
lanes 8, 9). Moreover, under the much milder S.sub.1 nuclease
treatment necessary to generate the data for the pT9CT9C, the yield
of double-stranded breaks in the pT9C-5 plasmid was extremely low
(lane 10).
EXAMPLE 4
[0170] Site Specific S.sub.1 Nuclease Cleavage of the Plasmids
pT9C-5, pT9CT9C and pT9CA9GKS (Linearized with Sca1) Targeted by
PNA T.sub.4CT.sub.5-LysNH.sub.2
[0171] This example illustrates the use of two molecules of PNA
bound to opposite strands of a DNA target. A further plasmid
pT9CA9GKS was produced by cloning the insert
GTCGACA.sub.5GA.sub.4GTCGACT.sub.4CT.sub.5- GTCGAC (SEQ ID NO:32)
into pUC19 at the Sal I site and linearizing with Sca I restriction
enzyme. The linearized plasmid has two hybridization sites for PNA
H-T.sub.4CT.sub.5-Lys NH.sub.2 (SEQ ID NO:8) on opposite strands
spaced by six base pairs. The protocol of Example 3 was use except
the samples were treated using 1 U/.mu.l of S.sub.1 and 15 min
incubation at 37.degree. C. The results are shown in FIG. 3 Lanes
1-4: pT9C-5; lanes 5-8: pT9CT9C; lanes 9-12: pT9CA9GKS. Lanes 1, 5
& 9: no PNA; lanes 2, 6 & 10: 50 .mu.M; lanes 3, 7, 11: 500
uM; lanes 4, 8, 12: 5 mM.
EXAMPLE 5
[0172] Use of PNA/Nuclease to Cut Selectively Large DNA
Molecule
[0173] The standard strain lambda cI indl ts857 Sam7 (New England
Biolab) was used. The PNA double target was T.sub.7N.sub.6T.sub.7
(SEQ ID NO:33) which upon cleavage should give rise to a 6.1 kb
fragment. The PNA was a PNA (T.sub.7).sub.2 and the conditions were
as follows: 0.1 .mu.g DNA and 1-20 D units of PNA were incubated in
5 .mu.l TE buffer for 10 min at 37.degree. C. 5 .mu.l buffer (33 mM
NaAc, 30 mM NaCl, 10 .mu.M ZnSO.sub.4, pH 4.5) and 20 U mung bean
nuclease were added and the mixture was incubated for 5 min at room
temperature. Upon analysis by electrophoresis in 0.5% agarose in
TBE buffer, fragments of sizes 6 kb and 42 kb were observed using
lambda.times.Hind III or Sal I as size markers. Without additions
of PNA no bands were seen.
[0174] This example demonstrates the ability of PNA/nuclease to cut
selectively a large DNA molecule (by comparison to the much smaller
plasmids used in Examples 1 to 3).
EXAMPLE 6
[0175] Binding of PNAs-T.sub.10/T.sub.9C/T.sub.8C.sub.2 to
Double-Stranded DNA Targets A.sub.10/A.sub.9G/A.sub.8G.sub.2
[0176] A mixture of 200 cps .sup.32P-labeled EcoRI-PvuII fragment
(the large fragment labeled at the 3'-end of the EcoRI site) of the
indicated plasmid, 0.5 .mu.g carrier calf thymus DNA, and 300 ng
PNA in 100 .mu.l buffer (200 mM NaCl, 50 mM Na-acetate, pH 4.5, 1
mM ZnSO.sub.4) was incubated at 37.degree. C. for 120 min. A 50
unit portion of nuclease S.sub.1 was added and incubated at
20.degree. C. for 5 min. The reaction was stopped by addition of 3
.mu.l 0.5 M EDTA and the DNA was precipitated by addition of 250
.mu.l 2% potassium acetate in ethanol. The DNA was analyzed by
electrophoresis in 10% polyacrylamide sequencing gels and the
radiolabeled DNA bands visualized by autoradiography.
[0177] The target plasmids were prepared by cloning of the
appropriate oligonucleotides into pUC19. Target A.sub.10:
oligonucleotides GATCCA.sub.10G (SEQ ID NO:34) & GATCCT.sub.10G
(SEQ ID NO:35) cloned into the BamHI site (plasmid designated
pT10). Target A.sub.5GA.sub.4: oligonucleotides
TCGACT.sub.4CT.sub.5G (SEQ ID NO:36) & TCGACA.sub.5GA.sub.4G
(SEQ ID NO:37) cloned into the SalI site (plasmid pT9C). Target
A.sub.2GA.sub.2GA.sub.4: oligonucleotides
CA.sub.2GA.sub.2GA.sub.4CTGCA (SEQ ID NO:38) and
GT.sub.4CT.sub.2CT.sub.2- CTGCA (SEQ ID NO:39) into the PstI site
(plasmid pT8C2). The results are shown in FIG. 4. The positions of
the targets in the gel are indicated by bars to the left. A/G is an
A+G sequence ladder of target P10.
EXAMPLE 7
[0178] Inhibition of Restriction Enzyme Cleavage by PNA (FIG.
4)
[0179] A 2 .mu.g portion of plasmid pT10 was mixed with the
indicated amount of PNA-T.sub.10 (SEQ ID NO:2) in 20 .mu.l TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) and incubated at
37.degree. C. for 120 min. 2 .mu.l 10.times. buffer (10 mM
Tris-HCl, pH 7.5, 10 mM, MgCl.sub.2, 50 mM NaCl, 1 mM DTT). PvuII
(2 units) and BamHI (2 units) were added and the incubation was
continued for 60 min. The DNA was analyzed by gel electrophoresis
in 5% polyacrylamide and the DNA was visualized by ethidium bromide
staining. This gel is illustrated in FIG. 5.
EXAMPLE 8
[0180] Kinetics of PNA-T.sub.10--dsDNA Strand Displacement Complex
Formation
[0181] A mixture of 200 cps .sup.32P-labeled EcoRI-PvuII fragment
of pT10 (the large fragment labeled at the 3'-end of the EcoRI
site), 0.5 .mu.g carrier calf thymus DNA, and 300 ng of
PNA-T.sub.10-LysNH.sub.2 (SEQ ID NO:2) in 100 .mu.l buffer (200 mM
NaCl, 50 mM Na-acetate, pH 4.5, 1 mM ZnSO.sub.4) were incubated at
37.degree. C. At the times indicated, 50 U of S.sub.1 nuclease was
added to each of 7 samples and incubation was continued for 5 min
at 20.degree. C. The DNA was then precipitated by addition of 250
.mu.l 2% K-acetate in ethanol and analyzed by electrophoresis in a
10% polyacrylamide sequencing gel. The amount of strand
displacement complex was calculated from the intensity of the
S.sub.1-cleavage at the target sequence, as measured by
densitometric scanning of autoradiographs. The results are
illustrated in FIG. 6.
EXAMPLE 9
[0182] Stability of PNA-dsDNA Complexes
[0183] A mixture of 200 cps .sup.32P-pT10 fragment, 0.5 .mu.g calf
thymus DNA and 300 ng of the desired PNA (either
T.sub.10-LysNH.sub.2 (SEQ ID NO:2), T.sub.8-LysNH.sub.2 (SEQ ID
NO:40) or T.sub.6-LysNH.sub.2 (SEQ ID NO:7)) was incubated in 100
.mu.l 200 mM NaCl, 50 mM Na-acetate, pH 4.5, 1 mM ZnSO.sub.4 for 60
min at 37.degree. C. A 2 .mu.g portion of oligonucleotide
GATCCA.sub.10G (SEQ ID NO:34) was added and each sample was heated
for 10 min at the temperature indicated, cooled in ice for 10 min
and warmed to 20.degree. C. A 50 U portion of S.sub.1 nuclease was
added and the samples treated and analyzed and the results
quantified as is illustrated in FIG. 7.
EXAMPLE 10
[0184] Biological Stability of PNA
[0185] A mixture of PNA-T.sub.5 (SEQ ID NO:41) (10 .mu.g) and a
control, "normal" peptide (10 .mu.g) in 40 .mu.l 50 mM Tris-HCl, pH
7.4 was treated with varying amounts of peptidase from porcine
intestinal mucosa or protease from Streptomyces caespitosus for 10
min at 37.degree. C. The amount of PNA and peptide was determined
by HPLC analysis (reversed phase C-18 column: 0-60% acetonitrile,
0.1% trifluoroacetic acid).
[0186] At peptidase/protease concentrations where complete
degradation of the peptide was observed (no HPLC peak) the PNA was
still intact.
EXAMPLE 11
[0187] Inhibition of Gene Expression
[0188] A preferred assay to test the ability of peptide nucleic
acids to inhibit expression of the E2 mRNA of papillomavirus is
based on the well-documented transactivation properties of E2.
Spalholtz, et al., J. Virol., 1987, 61, 2128-2137. A reporter
plasmid (E2RECAT) was constructed to contain the E2 responsive
element, which functions as an E2 dependent enhancer. E2RECAT also
contains the SV40 early promoter, an early polyadenylation signal,
and the chloramphenicol acetyl transferase gene (CAT). Within the
context of this plasmid, CAT expression is dependent upon
expression of E2. The dependence of CAT expression on the presence
of E2 has been tested by transfection of this plasmid into C127
cells transformed by BPV-1, uninfected C127 cells and C127 cells
cotransfected with E2RECAT and an E2 expression vector.
[0189] A. Inhibition of BPV-1 E2 Expression
[0190] BPV-1 transformed C127 cells are plated in 12 well plates.
Twenty four hours prior to transfection with E2RE1, cells are
pretreated by addition of antisense PNAs to the growth medium at
final concentrations of 5, 15 and 30 mM. The next day cells are
transfected with 10 .mu.g of E2RE1CAT by calcium phosphate
precipitation. Ten micrograms of E2RE1CAT and 10 .mu.g of carrier
DNA (PUC 19) are mixed with 62 .mu.l of 2 M CaCl.sub.2 in a final
volume of 250 .mu.l of H.sub.2O, followed by addition of 250 .mu.l
of 2.times.HBSP (1.5 mM Na.sub.2PO.sub.2. 10 mM KCl, 280 mM NaCl,
12 mM glucose and 50 mM HEPES, pH 7.0) and incubated at room
temperature for 30 minutes. One hundred microliters of this
solution is added to each test well and allowed to incubate for 4
hours at 37.degree. C. After incubation, cells are glycerol shocked
for 1 minute at room temperature with 15% glycerol in 0.75 mM
Na.sub.2PO.sub.2, 5 mM KCl, 140 mM NaCl, 6 mM glucose and 25 mM
HEPES, pH 7.0. After shocking, cells are washed 2 times with serum
free DMEM and fed with DMEM containing 10% fetal bovine serum and
antisense oligonucleotide at the original concentration. Forty
eight hours after transfection cells are harvested and assayed for
CAT activity.
[0191] For determination of CAT activity, cells are washed 2 times
with phosphate buffered saline and collected by scraping. Cells are
resuspended in 100 .mu.l of 250 mM Tris-HCl, pH 8.0 and disrupted
by freeze-thawing 3 times. Twenty four microliters of cell extract
is used for each assay. For each assay the following are mixed
together in an 1.5 ml Eppendorf tube and incubated at 37.degree. C.
for one hour: 25 .mu.l of cell extract, 5 .mu.l of 4 mM acetyl
coenzyme A, 18 .mu.l H.sub.2O and 1 .mu.l .sup.14C-chloramphenicol,
40-60 mCi/mM. After incubation, chloramphenicol (acetylated and
nonacetylated forms) is extracted with ethyl acetate and evaporated
to dryness. Samples are resuspended in 25 .mu.l of ethyl acetate,
spotted onto a TLC plate and chromatographed in chloroform:methanol
(19:1). Chromatographs are analyzed by autoradiography. Spots
corresponding to acetylated and nonacetylated
.sup.14C-chloramphenicol are excised from the TLC plate and counted
by liquid scintillation for quantitation of CAT activity. Peptide
nucleic acids that depress CAT activity in a dose dependent fashion
are considered positives.
[0192] B. Inhibition of HPV E2 Expression
[0193] The assay for inhibition of human papillomavirus (HPV) E2 by
peptide nucleic acids is essentially the same as that for BPV-1 E2.
For HPV assays appropriate HPVs are co-transfected into either CV-1
or A431 cells with PSV2NEO using the calcium phosphate method
described above. Cells which take up DNA are selected for by
culturing in media containing the antibiotic G418. G418-resistant
cells are then analyzed for HPV DNA and RNA. Cells expressing E2
are used as target cells for antisense studies. For each PNA, cells
are pretreated as above, transfected with E2RE1CAT, and analyzed
for CAT activity as above. Peptide nucleic acids are considered to
have a positive effect if they can depress CAT activity in a dose
dependent fashion.
EXAMPLE 12
[0194] Triplexing of PNA to Nucleic Acids--Probing Protocols
[0195] Probing was effected in 100.mu. buffer (S1: 100 mM NaCl, 1
mM ZnSO.sub.4, 50 mM NaAc, pH 4.5; KMnO.sub.4/dimethyl sulphate
(DMS): 10 mM Na-cacodylate, 1 mM EDTA, pH 7.0 or as other wise
noted) containing about 200 cps .sup.32P-labeled DNA fragment, 0.5
.mu.g calf thymus DNA and the desired amount of PNA. Following a
preincubation for 60 min at 37.degree. C., the probing reagent was
added and the incubation was continued at room temperature. The
reactions were terminated by the addition of a stop-buffer. The DNA
was precipitated by addition of 200 .mu.L 2% KAc in 96% EtOH and
was analyzed by electrophoresis in 10% polyacrylamide sequencing
gels. Radioactive DNA bands were visualized by autoradiography
using amplifying screens and Agfa curix RPA X-ray films exposed at
-70.degree. C.
[0196] Probing conditions were: S1: 0.5 U/.mu.l, 5 min. stopped
with 3 .mu.l 1 M EDTA; KMnO.sub.4: 1 mM, 15 sec. stopped with 50
.mu.l 1M 6-mercaptoethanol, 1.5 M NaAc, pH 7.0; DMS: 1% DMS, 15
sec., stopped as for KMnO.sub.4 probing. Samples probed with DMS or
KMnO.sub.4 were treated with piperidine (0.5 M, 90.degree. C., 20
min.) prior to gel analysis.
EXAMPLE 13
[0197] Enzymatic and Chemical Probing of the Binding of PNA
T.sub.2CT.sub.2CT.sub.4-LysNH.sub.2 to pT8C2A8G2
[0198] For this example the 264 bp EcorI/PvuII fragment of
pT8C2A8G2 that was 3'-.sup.32P-end-labeled at the EcorI site was
used. Probing was effected as per the protocols of Example 16. The
results are shown in FIG. 10. Lane 1 is a control in S1 buffer
without S.sub.1 nuclease and without PNA. Lanes 2-5: S1 probing,
lanes 6-9: KMnO.sub.4 probing and lanes 10-13: DMS probing. The
following concentrations of PNA were used: 0 .mu.M (lanes 2, 6
& 10), 0.25 .mu.M (lanes 3, 7 & 11), 2.5 .mu.M (lanes 4, 8
& 12) or 25 .mu.M (lanes 5, 9 & 13). Lanes S are A+G
sequence markers.
[0199] The plasmid pT8C2A8G2 contains two targets for PNA
T.sub.2CT.sub.2CT.sub.4 (SEQ ID NO:1) in opposite orientation.
Therefore both KMnO.sub.4 (thymines of the pyrimidine strand) and
DMS (guanines of the purine strand) probing was done on the same
DNA fragment. Probing with single strand specific nuclease,
S.sub.1, verified that upon binding of PNA, the pyrimidine strand
but not the purine strand of the PNA targets, became susceptible to
attack by S.sub.1 (FIG. 10, lanes 2-5). Interestingly, however, in
the absence of PNA S.sub.1 attacks the purine strand (lane 2). This
is undoubtedly due to the formation of an intramolecular triple
helix (H-DNA), a well characterized feature of such
polypurine/pyrimidine mirror repeats. It is also observed that in
the presence of PNA, the S.sub.1 sensitivity extends into the
linker between the two PNA targets. This can be explained if both
PNA targets are occupied, thereby forming a combined loop where the
DNA region between the targets is single-stranded. This type of
binding further illustrates that PNA can be used for directed
double strand DNA cleavage by S.sub.1.
[0200] The KMnO.sub.4 probing of the PNA-dsDNA complex reveals that
all thymines of the target are oxidized by KMnO.sub.4 (FIG. 13,
lanes 8-9). Concomitantly (in terms of PNA concentration) with the
occurrence of KMnO.sub.4 susceptibility, virtually full protection
against reaction with DMS is observed at the two guanines of the
opposite PNA target (see FIG. 10, lanes 12-13). These results show
that displacement of the pyrimidine strand of the DNA target is
accompanied by protection of the major groove upon binding of PNA
supporting the model in which two PNA strands are participating in
the PNA-DNA complex forming PNA DNA-DNA triplex.
EXAMPLE 14
[0201] Chemical Probing of the Binding of PNA
T.sub.2CT.sub.2CT.sub.4-LysN- H.sub.2 to pA8G2
[0202] For this example, the 248 bp EcoRI/PvuII fragment of pA8G2
was used that was .sup.32P-end-labeled (5'-labeling, lanes 1-5) or
(3'-labeling, lanes 6-9)) at the EcoRI site. Probing was effected
as per the protocols of Example 12. The results are shown in FIG.
11 wherein lane 1 is a control in S1 buffer without S.sub.1
nuclease and without PNA. Lanes 2-5: KMnO.sub.4 probing and lanes
6-9: DMS probing. The following concentrations of PNA were used: 0
.mu.M (lanes 2 & 6), 0.25 .mu.M (lanes 3 & 7), 2.5 .mu.M
(lanes 4 & 8) or 25 .mu.M (lanes 5 & 9). Lanes S are A+G
sequence markers.
[0203] In this example, in order to assure that the results were
not an artifact of the dimeric target, we performed a similar
experiment to that of Example 13 except a plasmid (pA8G2) having
only a single PNA target was used. As is seen in FIG. 14, virtually
identical results to those with the dimeric target of Example 13
were obtained with KMnO.sub.4 and DMS probing of the single
target.
EXAMPLE 15
[0204] Effect of pH and Ionic Strength of the Binding of PNA
T.sub.2CT.sub.2CT.sub.4-LysNH.sub.2 to pT8C2A8G2
[0205] For this example the 264 bp EcorI/PvuII fragment of
pT8C2A8G2 was used that was 3'-.sup.32P-end-labeled at the EcorI
site. Probing was effected as per the protocols of Example 12. The
results are shown in FIG. 12 wherein lanes 1 & 9 are controls
without PNA. Lanes 1-7 are KMnO.sub.4 probing while lanes 9-15 are
DMS probing. The samples of lanes 2-7 & 9-15 contained 5 .mu.M
PNA. The samples of lanes 3, 5, 7, 11, 13 & 15 contained 100 mM
NaCl in the 10 mM Na phosphate, 1 mM EDTA buffer. The pH of the
buffer was 7.5 (lanes 2, 3 & 10, 11); 6.5 (lanes 4, 5 & 12,
13) or 5.5 (lanes 6, 7 & 14, 15). Lanes are A+G sequence
markers.
[0206] This example shows that the Hoogsteen hydrogen bonding of
cytosine to guanine requires protonation of N3 of cytosine and
consequently interactions involving C.sup.+-G Hoogsteen base
pairing are very sensitive to the acidity of the medium. This has
been studied extensively by others with DNA triple helices, and can
also be seen with (PNA).sub.2/DNA triplexes using
oligonucleotides.
[0207] The results presented in FIG. 12 further show that the
strand displacement binding of PNA T.sub.2CT.sub.2CT, (SEQ ID NO:1)
to the target of pT8C2A8G2 is also sensitive to pH. Very little
binding is observed at pH 7.5 (lanes 2 & 10) compared to pH 6.5
(lanes 4 & 12) and 5.5 (lanes 6 & 14). Full consistency is
also seen between the KMnO.sub.4 and DMS probing results showing
clear correlation between strand displacement and Hoogsteen type
binding of the PNA. We have previously shown that medium ionic
strength (50-100 mM Na.sup.+) inhibits strand displacement binding
of PNA to double-stranded DNA. The results presented in FIG. 15
(lanes 3, 5, 7) are fully consistent with this. There is no reason
from physio-chemical considerations that binding of PNA to DNA as a
conventional PNA DNA-DNA triple helix should be strongly salt
dependent. Therefore, such complexes could be envisaged under salt
conditions that disfavor strand displacement. However, we see no
evidence that this be the case since no DMS footprint is observed
under "high-salt" conditions (FIG. 12, lanes 11, 13 & 15).
[0208] From Examples 13, 14 and 15 as well as other of the Examples
of this specification, we conclude that sequence specific binding
of PNA to double-stranded DNA involves PNA DNA-DNA triplexes
employing conventional Hoogsteen and Watson-Crick base pair
hydrogen bonding for recognition.
EXAMPLE 16
[0209] Electron Microscopy
[0210] A pA98 plasmid containing an A.sub.98/T.sub.98 (SEQ ID
NO:42) insert in the PstI site of the polylinker of a pUC19 plasmid
was used. 0.1 .mu.g of the pA98 plasmid linearized by the Cfr10I I
restriction enzyme was incubated with 0.15 .mu.g of PNA
H-T.sub.10-LysNH.sub.2 at 37.degree. C. for 3 hours in 10 .mu.L of
the TE buffer (10 mM of Tris-HCl, 1 mM of Na.sub.3EDTA, pH 8.0).
The complex was diluted with buffer containing 10 mM of Tris-HCl,
10 mM of NaCl, pH 7.5 to the final DNA concentration of 0.2-0.5
.mu.g/mL and absorbed to the surface of glow discharged in
tripropylamine carbon grid for 2 minutes as described in Duochet,
J., Ultrastruct. Res., 1971 35, 147-167. The grid was stained in
0.1-0.5% water solution of uranyl acetate for 10-15 sec and dried.
The sample was shadowed with Pt/C (95/5) and studied in a Philips
400 electron microscope at accelerating voltage of 40 kV. The
length of the DNA molecules were measured and a histogram was
plotted as described by A. V. Kurakin in Micron Microscopica Acta,
22, 213-221 (1991). The length of unwound region was measured by
its thicker strand, for which the number of base pairs per unit
length was assumed to be the same as for the DNA duplex. For
analysis by use of a histogram, the molecules were oriented in such
a manner that the left boundary of the loop was closer to the end
of the molecule.
[0211] The electron microscopy of the PNA-DNA complex was carried
out on the A.sub.98/T.sub.98 (SEQ ID NO:42) target contained within
the supercoiled plasmid, pA98, linearized with the Cfr10I
restriction enzyme, and challenged with a PNA
H-T.sub.10-LysNH.sub.2 (SEQ ID NO:2). Full occupancy of the target
results in a strand displacement loop of 90-100 bases was detected
as is shown in FIG. 13. This histogram plot is illustrated in FIG.
14. As is seen in FIG. 13, the DNA molecules carry an open region
in the form of "an eye". In all cases one of the two strands in the
open region was thicker than the other one and has the same
thickness as the normal DNA duplex. The thicker strand corresponds
to the A-strand covered by PNA, while the thinner strand
corresponds to the displaced T-strand. The positions of two branch
points of the loops were 47.1% and 51.2%, which within the error of
EM-measurements (0.5%), coincides with the ends of the A98/T98
insert (47.5% and 51.0%). The average size of the loop was 4.1%,
i.e., 114 bp, which also agrees well with the length of the insert
(the standard error is 17 bp). In control experiments carried out
without added PNA, the eye-like structures were not observed.
EXAMPLE 17
[0212] Unwinding of Closed Circular DNA with PNA
[0213] Relaxed circular DNA was prepared by treating ordinary
supercoiled plasmid DNA with an extract containing DNA
topoisomerase I, as described by V. I. Lyamichev, et. al., J.
Biomolec. Struct. Dynamics, 3, 327-338 (1985). DNA-PNA complexes
were obtained by incubation of 1-2 .mu.g of DNA in 5-20 .mu.L TE
with 0.4 optical units./mL of PNA for 4 hours at 20-22.degree. C.
This corresponded to about 10 times molar excess of PNA to its
potential binding sites. Agarose (1.5%) gel electrophoresis was
performed in the TAE buffer containing 1 .mu.g/mL of chloroquine at
10.degree. C. for 15 hours at 1.5 V/cm. Two-dimensional gel
electrophoresis was preformed in the first direction (from top to
bottom) in the TAE buffer and in the second direction (from left to
right) in the same buffer with addition of 1 .mu.g/mL of
chloroquine.
[0214] In this example, the efficiency of PNA T10 incorporating
into the DNA duplex and displacing the DNA T-strand in solution was
shown by unwinding of closed circular DNA by PNA. The pA98 plasmid
(Example 16) was prepared in the form of relaxed circles (rcDNA).
It was expected that complexing with PNA would unwind about 10
turns of the DNA duplex. Because of topological constraints (the
two strands in rcDNA were closed and therefore topologically
linked), this unwinding would make the rcDNA molecules behave as if
they were positively supercoiled by 10 superturns. This would
manifest itself in an increase of electrophoretic mobility of the
complex as compared with control rcDNA preincubated in the same
buffer without PNA. This increase in electrophoretic mobility is
shown in FIG. 15 that shows that after incubation with PNA in the
low-salt TE buffer for 4 hours at 20.degree. C., virtually all
rcDNA molecules significantly increased their mobility and moved as
if they were highly supercoiled. Two-dimensional gel
electrophoresis gels, FIG. 16, shows that these molecules behave
actually as positively supercoiled ones. This technique is used to
resolve individual topoisomers. Using a sample with a wide
distribution of topoisomers as reference, it is clearly seen FIG.
16 that on the average 8-10 turns of the double helix are released
upon binding of PNA. These results indicate that in the TE buffer
PNA occupied all available binding sites on the DNA duplex
efficiently displacing the DNA T-strand in the A98/T98 (SEQ ID
NO:42) insert. When NaCl is added to the incubation buffer, the
degree of conversion of rcDNA into rapidly moving species
dramatically decreased within a narrow range between 20 and 40 mM
of NaCl as seen in FIG. 17, panel A. This emphasizes a strong
sensitivity of the PNA incorporation to the salt concentration.
However, after being formed at low salt, the complex showed
remarkable stability and tolerated increasing salt concentrations
up to at least 500 mM of NaCl (FIG. 20, panel B, lane 4). The
complex could be dissociated by alkali treatment (FIG. 20, panel B,
lane 5).
EXAMPLE 18
[0215] Inhibition of RNA Polymerase T.sub.3 Transcription
Elongation by PNA
[0216] The complex, between PNA T.sub.10-LysNH.sub.2 (SEQ ID NO:2)
(1 or 10 uM) and pAIOKS (pBluescriptKS.sup.+ in which the
d(A.sub.10) target is cloned into the BamH1 site, analogous to
pTlO) (100 ng) cleaved with restriction enzyme PvuII, XbaI, or
BamH1, was formed by incubation in 14 .mu.L 10 mM Tris-HCl, 1 mM
EDTA, (pH 7.4) buffer for 60 min. at 37.degree. C. Subsequently 4
.mu.L 5.times. concentrated polymerase buffer (0.2 M Tris-HCl, pH
8.0, 125 mM NaCl, 40 mM MgCl.sub.2, 10 mM spermidine) was added
together with 15 U T.sub.3 RNA polymerase and ATP (10 mM), CTP (10
mM), GTP (10 mM), UTP (1 mM) and .sup.32P-UTP (0.1 .mu.Ci), and the
incubation was continued for 10 min. Following ethanol
precipitation, the RNA was analyzed by electrophoresis in
sequencing gels and the radiolabeled bands were visualized by
autoradiography using intensifying screens. The results are shown
in FIG. 18: Lane 1: pTIOKS was cut with PvuH and no PNA was
present. Lane 2: pTIOKS was cut with BamHl, and no PNA was present.
Lane 3: pTIOKS was cut with XbaI, no PNA. Lane 4: as in lane 1 but
in the presence of PNA (1 uM). Lane 5: as lane 3+PNA (1 uM). Lane
6: as lane 1+PNA (10 uM). Lane 7: as lane 3+PNA (10 uM).
[0217] As is seen in FIG. 18, In the absence of PNA the expected
run-off transcript is observed (lanes 1, 2 & 3). When the
preformed complex between PNA T.sub.10-LysNH.sub.2 (SEQ ID NO:2)
and the template was used, a transcript which corresponds in length
to transcription arrest at the encounter of the PNA, i.e., one
nucleotide longer than the run-off at the BamH1 site (lane 3), is
produced (lanes 4-7). This shows that transcription elongation by
RNA polymerase T.sub.3 is effectively blocked by PNA bound to the
template strand. If PNA was bound to the non-template strand, no
transcript corresponding to blockage by the PNA was observed.
Similar results were also obtained using RNA polymerase T.sub.7
(data not shown). In this experiment the PNA/DNA complexes were
formed in low salt buffer prior to addition of the buffer required
for enzyme action since binding of PNA to dsDNA is inhibited by
elevated (>50 mM Na.sup.+) salt concentrations. Once formed, the
complexes are exceedingly stable to these conditions.
EXAMPLE 19
[0218] Inhibition of Taq DNA Polymerase Primer Extension by PNA
[0219] A mixture of 100 500 ng Pvull cleaved plasmid pAIOKS, 0.1 ug
M13 reverse primer and 1 ug of PNA T.sub.10-LysNH.sub.2 (SEQ ID
NO:2) in 10 ul buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM
MgCl.sub.2, 0.1 mg/ml gelatine) was incubated for 5 min at
90.degree. C. and then for 60 min at 37.degree. C. Subsequently, 1
U of Taq polymerase, 1 ul dCTP (100 UM), dGTP (100 UM), dTTP (100
uM) and .sup.32P-dATP (1 uCi) were added and the incubation
continued for 5 min at 20.degree. C. Following the addition of 2 ul
dATP, dCTP, dGTP, dTTP (1 mM each), the sample was incubated for 15
min at 60.degree. C. The DNA was precipitated with ethanol and the
samples treated as described for Example 18. The results are shown
in FIG. 19: Lane 1: pTIOKS was cut with PvuII and no PNA was
present. Lane 2: same as lane one with PNA present. Lane 3: no PNA
and pTIOKS was cut with BamHl.
[0220] As is shown in this example, in a primer extension
experiment using a pre-formed complex between PNA
T.sub.10-LysNH.sub.2 (SEQ ID NO:2), a single-stranded target, and a
primer downstream from the target sequence, a product corresponding
in length to blockage at the PNA was produced by Taq DNA polymerase
(FIG. 19, lane 2). No product was detected in the absence of PNA
(lane 3). Similar results were obtained using the large fragment
(Klenow fragment) of E. Coli DNA polymerase (data not shown). Thus
demonstrates elongation by DNA polymerases is blocked by PNA.
EXAMPLE 20
[0221] Inhibition of Transcription by PNA
[0222] The PNA oligomers T.sub.10-LysNH.sub.2 (SEQ ID NO:2),
T.sub.5CT.sub.4-LysNH.sub.2 (SEQ ID NO:6) and
T.sub.2CT.sub.2CT.sub.4-Lys- NH.sub.2 (SEQ ID NO:1) were
synthesized as described in Example 1. Plasmids containing the
target sequences were obtained by cloning of the appropriate
oligonucleotides into the vector pBluescripKS.sup.+. To obtain
pT10KS and pA10KS, 16-mers 5'-TCGACT.sub.4CT.sub.5G (SEQ ID NO:36)
and 5'-GATCCA.sub.10G (SEQ ID NO:34) were cloned into the BamH1
site, and clones containing the insert in either orientation were
isolated. pT9CA9GKS was obtained by cloning
5'-TCGACT.sub.4CT.sub.5G (SEQ ID NO:36) and
5'-TCGACA.sub.5GA.sub.4G (SEQ ID NO:37) into the SalI site and
pT8C2KS and pA8G2KS were obtained by cloning
5'-GT.sub.4CT.sub.2CT.sub.2C- TGCA (SEQ ID NO:39) AND
5'-GA.sub.2GA.sub.2GA.sub.4CTGCA (SEQ ID NO:38) into the PstI site.
E. coli JM103 was used as host in all cases, and transformations
and isolation of clones were done by standard techniques. Plasmids
were purified by buoyant density centrifugation in CsCl gradients
and characterized by dideoxy sequencing.
[0223] Inhibition of RNA Polymerase Transcription Elongation by
PNA
[0224] The complex between the desired PNA and the desired DNA (100
ng) cleaved with the desired restriction enzyme was formed by
incubation in 14 .mu.L 10 mM Tris-HCl, 1 mM EDTA, (pH 7.4) buffer
for 60 min at 37.degree. C. Subsequently 4 .mu.L 5.times.
concentrated polymerase buffer (0.2 M Tris-HCl, pH 8.0, 125 mM
NaCl, 40 mM MgCl.sub.2 10 mM spermidine) was added together with 15
U of RNA polymerase and ATP (10 mM), CTP (10 mM), UTP (1 mM) and
.sup.32P-UTP (0.1 .mu.Ci). The incubation was continued for 10 min.
Following ethanol precipitation, the RNA was analyzed by
electrophoresis in polyacrylamide sequencing gels, and radiolabeled
RNA bands were visualized by autoradiography (using Agfa Curix RPI
X-ray films and intensifying screens). Quantitation was performed
by densitometric scanning using a Molecular Dynamics laser scanner
and the ImageQuant.TM. software.
[0225] FIG. 20 illustrates certain concentration dependence between
complexes of pA10KS cleaved with XbaI and the following
concentrations of PNA T.sub.10-LsyNH.sub.2 (SEQ ID NO:2): 0, 0.2,
0.35, 0.7, 1, 1.4, 1.75, 2.1, 2.8, 3.5, 4.3 or 5 .mu.M (lanes
1-12). The complexes were formed by incubation in TE buffer for 60
min. at 37.degree. C. The buffer was adjusted to transcription
conditions and following the addition of NTPs, .sup.32P-UTP and
T.sub.3 RNA polymerase, transcription was allowed to proceed for 5
min. at 37.degree. C. The transcripts were analyzed by
electrophoresis in 10% polyacrylamide/7M urea gels and visualized
by autoradiography.
[0226] The results shown in FIG. 21 are similar to those of FIG. 20
except that the plasmid pA8H2KS was cleaved with BamH1 and PNA
T.sub.2CT.sub.2CT.sub.4-LysNH.sub.2 (SEQ ID NO:1) at the following
concentrations: 0, 0.6, 1.2, 2.4, 3.6, 4.8, 6, 7.2, 9.6, 14.4 or
16.7 .mu.M (lanes 1-12).
[0227] FIG. 22 illustrates a quantitative representation of the
results shown in FIGS. 20 and 21. In this figure the following
symbols, respectively, are utilized, squares: pA10KS.times.XbaI and
PNA T.sub.10-LysNH.sub.2 (SEQ ID NO:2); circles: pA8GKS.times.BamH1
and PNA T.sub.2CT.sub.2CT.sub.4-LysNH2 (SEQ ID NO:1). closed
squares: relative amount of the truncated transcript; closed
circles: total amount of transcript.
[0228] This examples illustrates that the phage T.sub.3 and
T.sub.7RNA polymerases can be used as a model system for very
efficient and robust transcription. In this example, a template was
constructed by cloning the appropriate PNA targets into the
polylinker of the BluescriptKS.sup.+ plasmid. The target was clone
into the BamHl site in both orientations, thus allowing for four
transcription experiments to be performed using either the
constructs and either the T.sub.3- or the T.sub.7-promoter. The
results presented in FIGS. 20, 21 and 22 show the dose dependent
inhibition of the transcription of pA10KS (FIG. 20) or pA8G2KS
(FIG. 21) by PNA T.sub.10-LysNH.sub.2 (SEQ ID NO:2) or
T.sub.2CT.sub.2CT.sub.4-LysN- H.sub.2 (SEQ ID NO:1) using T.sub.3
RNA polymerase. It is observed that as the concentration is raised
the amount of full length transcript is decreased and the amount of
truncated product is increased suggesting that sequence specific
transcription arrest is taking place. However, the total amount of
transcript also decreases indicating that general inhibition of
transcription also occurs.
EXAMPLE 21
[0229] Sequence Specificity of the Transcription Elongation Arrest
by PNA T.sub.10-LysNH.sub.2 and PNA T.sub.2CT.sub.2CT-LysNH.sub.2
using T.sub.3 or T.sub.7 RNA Polymerase
[0230] Samples identical to those used for Example 20 were analyzed
by electrophoresis in a polyacrylamide sequencing gel. The results
are shown in FIG. 23 where, Lanes 1-4: pA10KS/T.sub.3 RNA
polymerase; lanes 5-8: pT10KS/T.sub.7 RNA polymerase; lanes 9-12:
pA8G2KS/T.sub.3 RNA polymerase; lanes 13-16: pT8C2KST.sub.7 RNA
polymerase. The plasmid used in the samples of lanes 1-3 was
cleaved with Xba1, that in lanes 6-8 with Pst1, that in lanes 9-11
with BamH1 and that in lanes 14-16 with HindIII. Lanes 1, 8, 9
& 16 are controls without PNA. Lanes 4, 5, 12 & 13 are
controls cleaved proximal to the PNA target site i.e., with BamH1
(lanes 4 & 5) or Pst1 (lanes 12 & 13). Samples of lanes 2,
3, 6 & 7 were preincubated with PNA T.sub.10-LysNH.sub.2 (SEQ
ID NO:2), and those of lanes 10, 11, 14 & 15 were preincubated
with PNA T.sub.2CT.sub.2CT.sub.4-- LysNH.sub.2 (SEQ ID NO:1).
[0231] In this example, in order to determine the position of
transcription arrest, RNA transcripts were analyzed on high
resolution sequencing gels. The results show that a specific RNA
transcript is produced by T.sub.3-RNA polymerase in the presence of
PNA-T.sub.10 (SEQ ID NO:2) that is one nucleotide longer than the
run-off transcript produced using a template (BamH1 cleaved) ending
one nucleotide in front of the PNA target (FIG. 23, lanes 2-4).
Thus transcription proceeds to, but does not include, the first
nucleotide involved in hydrogen bonding with the PNA. The results
obtained with T.sub.7-RNA polymerase are similar to those with
T.sub.3-RNA polymerase (FIG. 23, lanes 5-8). Interestingly,
however, in this case the transcript is less homogeneous in length
than the transcript obtained with the T.sub.3-polymerase, and the
lengths of the transcripts indicate that the polymerase is able to
transcribe into the PNA target. These results show that this
polymerase is able to some extent to displace the bound PNA during
transcription, although the polymerase-PNA encounter may sometimes
result in chain termination. Using mixed A/G PNA targets gave the
results shown in FIG. 23, lanes 9-16. It is noteworthy that
transcription arrest with an A.sub.4GA.sub.5 (SEQ ID NO:43) target
and a T.sub.4CT.sub.5-LysNH.sub.2 (SEQ ID NO:8) PNA occurs one to
two nucleotides inside the target, while transcription arrest with
an A.sub.4GA.sub.2GA.sub.2 (SEQ ID NO:44) target and a
T.sub.4CT.sub.2CT.sub.2-LysNH.sub.2 (SEQ ID NO:9) PNA is much less
efficient (as compared to the results of Example 22) and occurs at
the end of the target (FIG. 23, lanes 10, 11, 14 & 15).
EXAMPLE 22
[0232] Transcription Arrest in pT9CA9GKS Plasmid
[0233] In FIG. 24 the effect of PNA T.sub.10-LysNH.sub.2 (SEQ ID
NO:2) (lanes 1, 2, 4 & 5) on T.sub.7 RNA polymerase
transcription of pT10KS (lanes 1-3) and pA10KS (lanes 4 & 5),
and of PNA T5CT.sub.4-LysNH.sub.2 (SEQ ID NO:6) (lanes 6, 7, 10
& 11) on T.sub.3 or T.sub.7 RNA polymerase transcription of
pT9CA9GKS (lanes 6-11) are shown. The plasmid of the samples in
lanes 1, 2, 4-7, 10 & 11 were cut with PvuII while BamHl was
used for the sample of lane 3, and SalI was used for the samples of
lanes 8 & 9. The PNA concentration was 5 .mu.M.
[0234] As is shown in this example, experiments using the pT9CA9GKS
plasmid and T.sub.3 or T.sub.7 RNA polymerase showed (FIG. 26,
lanes 5-11) that transcription arrest at this PNA target occurred
2-3 bases inside the target. Employing a gel-retardation assay, it
was ascertained that the complexes between PNAs
T.sub.10-LysNH.sub.2 (SEQ ID NO:2), T.sub.4CT.sub.5-LysNH.sub.2
(SEQ ID NO:8) or T.sub.4CT.sub.2CT.sub.2-LysN- H.sub.2 (SEQ ID
NO:9) with their complimentary dsDNA targets are stable for at
least 15 min when transferred from the TE-buffer to the
transcription assay-buffer, thus eliminating the possibility that
the PNA was dissociating spontaneously during transcription. These
results indicate that an increase in the G-content of the target,
and thus in the C-content of the PNA decreases the efficiency of
transcriptional arrest due to read-through. In all cases it is also
observed that PNAs at higher concentrations result in an unspecific
inhibition of transcription by T.sub.3 or T.sub.7 RNA polymerase.
We cannot say if this effect is due to an inhibitory effect of the
PNA directly at the polymerase, or if it is caused by binding of
the PNA to other sites on the DNA template, e.g., the promoter.
EXAMPLE 23
Sequence Specificity of the Transcription Elongation Arrest by PNA
T.sub.10-LysNH.sub.2
[0235] In this example, template versus non-template binding of the
PNA was examined. The experiments were performed essentially as
described for Example 21. The results are shown in FIG. 25 using
plasmid pA10KS (a & c) or pT10KS (b) cleaved with PvuII (lanes
1 & 4), Xba1 (lanes 2 & 5) or BamH1 (lane 3). PNA
T.sub.10-LysNH.sub.2 (SEQ ID NO:2) was present at 3.3 .mu.M in the
samples of lanes 4 & 5. Panel c is a longer exposure of panel
a.
[0236] As is shown in FIG. 25, if the PNA is bound to the
non-template strand transcription is virtually not arrested (FIG.
25, FIG. 24: lanes 1-5). A very weak band corresponding to arrest
at the end of the PNA target can be detected upon longer exposure
of the autoradiogram (FIG. 25, panel c, lanes 4, 5). These findings
are similar to results reported with DNA templates site
specifically modified with covalent psoralen adducts.
EXAMPLE 24
[0237] Sequence Specificity of the Transcription Elongation Arrest
by PNA T.sub.6, T.sub.7 or T.sub.10-LysNH.sub.2 Using
pA10KS/T.sub.3 RNA Polymerase
[0238] In this example transcription arrest with PNAs shorter than
10 mers was examined. For the results shown in FIG. 26, the plasmid
was cleaved with XbaI in all cases and preincubated with PNA
T.sub.6-LysNH.sub.2 (SEQ ID NO:7) (.mu.M, lane 2),
T.sub.7-LysNH.sub.2 (SEQ ID NO:45) (.mu.M, lane 3) or
T.sub.10-LysNH.sub.2 (SEQ ID NO:2) (.mu.M, lane 4). Lane 1 is a
control without PNA.
[0239] The results seen in FIG. 26 show that an 8-mer and even a
6-mer PNA, although less efficiently, are able to arrest
transcription by T.sub.3-RNA polymerase. The results also show that
the arrest is occurring with the PNA bound at the far end of the
target indicating that the PNA is binding to the 10-mer target in a
floating mode and that the RNA polymerase is "pushing" the PNA.
EXAMPLE 25
[0240] Transcription Initiation from PNA-DNA Displacement Loops
[0241] Peptide nucleic acid (PNA) form (PNA).sub.2/DNA, DNA
triplex-D-loop structure upon binding to complimentary
double-stranded DNA as shown schematically in FIG. 27. If two
adjacent PNA sites are present in cis or in trans (wherein cis is
where two PNA bind adjacent to each other on the same strand and
trans is where the two PNA bind on opposite strands), structures of
the type shown in FIG. 27b,c are formed. In this example, since
these structures resemble transcription elongation loops we tested
if E. coli RNA polymerase is able to recognize and bind to such
PNA/DNA loops.
[0242] The three plasmids pT9C, pT9CT9C (pUC19 derivatives) and
pT9CA9GKS (Bluescript derivative) were used for this in vitro
transcription experiments. Restriction fragments were isolated by
digestion with PvuII and on polyacrylamide gels resulting in the
fragments shown in FIG. 28. In addition, the isolated PvuII
fragment from pKST9C was restricted with XbaI or SacI before PNA
hybridization to obtain a shorter transcript from the
`PNA-promoter`. PNA-DNA complexes were formed by combining 0.3,
.mu. PNA (50 OD) with DNA fragments in 10 mM Tris-HCl pH 8.0 and
0.1 mM EDTA in a total volume of 25 .mu.L for 1 hour at 37.degree.
C. The transcription were initiated by addition of 50-100 mM E.
coli RNA Polymerase holoenzyme, T3 and T7. The reaction mix
contained a final concentration of: 40 mM Tris-HCl pH 8.0, 120 mM
KCl, 5 mM MgCl.sub.2, 0.1 mM DTT and 1 mM of ATP, CTP, GTP and 0.1,
mM of UTP and .sup.32P UTP. The complete mixture of 30 ul were
incubated at 37.degree. for 20 minutes followed by ethanol
precipitation. The RNA transcripts were analyzed on 8% denaturing
polyacrylamide gels, and visualized by autoradiography. The RNAse H
experiment was done by hybridization of a complimentary
oligonucleotide to the mRNA to synthesize a double-stranded target
for RNAse H.
EXAMPLE 26
[0243] Cis Competition Between the PNA Promoter and lacUV5
[0244] A PvuII fragment including a single or a triple PNA binding
site together with the lacUV5 promoter was isolated. The DNA was
incubated with increasing amounts of PNA for 1 hour. The
transcriptions were performed as described in Example 25. As
indicated by the gel-shifts shown in FIG. 29, a slower migrating
complex between the target DNA and the RNA polymerase is formed
only if a PNA binding to this target is formed is prebound to the
DNA.
EXAMPLE 27
[0245] RNA Polymerase Footprinting
[0246] In this example DNase I footprinting experiments are
undertaken to define where on the DNA fragment binding takes place.
DNA fragments of the length shown in FIG. 30 are labeled in the 3'
end with klenow polymerase and .sup.32P-dXTP. The PNA-DNA complexes
are formed as described in Example 26 above. The RNA
Polymerase-PNA/DNA complexes are formed in a reaction buffer as
described for Example 26 with the addition of 2 ug/ml calf Thymus
DNA in a total volume of 100 ul. After 15 min. incubation at
37.degree. C. the samples are digested with 0.03 .mu.L DNAse (1
mg/ml) for 3 min followed by ethanol precipitation and analyzing by
8% denaturing PAGE. Expected results of this example are also shown
in FIG. 29. In the presence of PNA but in the absence of RNA
polymerase only a weak footprint corresponding to PNA binding to
the target is observed. However, upon addition of E. coli RNA
polymerase a clear footprint is observed.
EXAMPLE 28
[0247] Gel-Shift Assay
[0248] PNA-DNA complexes are formed as for the footprint experiment
in Example 27. After 15 min at 37.degree. C. incubation the samples
are analyzed on 5% PAGE. The gel-shift and DNaseI footprinting
results should demonstrate that E. coli RNA polymerase binds to a
PNA/dsDNA strand displacement loop. The binding should differ
distinctly from that seen for RNA polymerase in the initiation and
in the elongation complex. Binding in case of the PNA/DNA complex
likely will be confined to the DNA loop and will not extend much
into the surrounding double-stranded DNA.
[0249] Although the (PNA).sub.2/DNA, DNA triplex-D-loop
structurally does resemble an RNA transcription elongation loop, it
differs in one important aspect; the PNA does not contain a
3'-hydroxyl group to be used as an elongation substrate for RNA
polymerase. Nevertheless, PNA dependent transcription is observed
from a DNA molecule containing a PNA target. Furthermore, the
length of the resulting transcript should corresponds to a run-off
transcript initiated at the bound PNA, as exemplified in FIG. 27a.
The transcription should be more efficient if a double PNA target
is used giving rise to a loop of approximately 30 bases (FIG. 27b)
in the cis configuration, and approximately 16 bases in the trans
configuration (FIG. 27c). In the latter case, transcripts of two
distinct sizes should be produced (FIG. 27c), which in length
corresponds to initiation at both PNA targets and proceeding in
opposite directions (FIG. 27c).
[0250] Two experiments will be undertaken to estimate the strength
of the PNA dependent transcription initiation. In one experiment,
both the PNA target and the strong CAP independent UV5 promoter are
present on the same DNA fragment. Upon titration with PNA the full
run-off transcript from the UV5 promoter is inhibited, while a new
transcript corresponding to transcription arrest at the PNA site
appears. A very faint band of a length corresponding to
transcription initiated at the PNA site is also observed. However,
when a DNA fragment containing a triple PNA binding site is used,
transcription from this "PNA-promoter" should be able to compete
fully with the UV5 promoter. As the PNA concentration is increased,
transcription from the UV5 promoter decreases with concomitant
increase in the amounts of two transcripts that correspond in
length to the products expected for transcription in either from
the "PNA-promoter". An analogous experiment should be performed in
trans using a mixture of a UV5 containing DNA fragment and either
of the DNA fragments containing the single or the cis or trans
double PNA targets. The results of these experiments should confirm
that a single PNA decamer target is not able to compete with the
UV5 promoter whereas both of the dimeric targets competes very
efficiently. While we do not want to be bound by theory, the
results should suggest that a single-stranded DNA loop is a major
structural determinant for RNA polymerase upon transcription
initiation and elongation.
EXAMPLE 29
[0251] Site Specific Termination of In Vitro Transcription in
Eukaryotic Nuclear Extracts
[0252] PNA TTTTCTTTT-NH.sub.2 (SEQ ID NO:16), TTCCCTTCC-LysNH.sub.2
(SEQ ID NO:12), CCCCCCCTTTTTTTT-NH.sub.2, (SEQ ID NO:18),
TTCTCTCTCT-NH.sub.2 (SEQ ID NO:20) and CCTCCTTCCC-NH.sub.2 (SEQ ID
NO:19) were synthesized as described in Example 1. Phosphodiester
oligonucleotides were synthesized using standard protocols as for
example Vickers, T. et. al., Nucleic Acids Research, 1991, 19,
3359-3368. Plasmids were constructed with the CMV IE 1 promoter
driving the transcription of viral DNA sequences. The plasmid pIE72
is a CMV IE cDNA clone which was constructed as described by
Stenberg, M. et. al., J. Virol., 1993, 64, 1556-1565. pCH495 was
constructed by ligating the 495 bp SalI/EcoRI fragment from the
plasmid pBH10 (see Hahn, B., et. al., Nature, 1984, 312, 166-169)
into the vector pUC-CMV, prepared by digestion with the same two
enzymes. pCIN was constructed by ligating a 463 bp PvuII fragment
from the plasmid pSVCC3 (see Depeto, A. S. and Stenberg, R. M., J.
Virol., 1989, 63, 1232-1238), a genomic CMV clone, into the vector
pCEP-4 (Invitrogen) linearized at the PvuII site. pCINr was
constructed in the same manner, however the insert is cloned in the
opposite orientation. The plasmids are illustrated in FIG. 31. All
plasmids were constructed with the CMV IE1 promoter (open box)
driving the transcription of sequences containing homopurine sites
for targeting with homopyrimidine PNA. pCH495 contains a 495 bp
SalI/EcoRI fragment from pBH10 (3738 to 4233 of the HIV-1 genome).
The plasmid pCIN contains a 463 bp PvuII fragment subcloned from
the plasmid pSVCC3, a genomic CMV clone. pCINr contains the same
fragment in the opposite orientation. pIE72 is a CMV IE1 cDNA
clone. The filled boxes represent the transcribed region of each
linearized plasmid. The expected transcript length is shown at the
3' end of each. The sequence of the PNA target site is also shown
along with the position relative to the end of the transcript.
[0253] In vitro transcription. 100 ng/ul of linearized plasmid was
pre-incubated with PNA at various concentrations in a volume of 10
ul for 3 hours at room temperature in KCl buffered with 10 mM
HEPES, pH 6.8. For studies on pH dependence, KCl was buffered with
10 mM TrisOAc, pH 5.1 or 10 mM Tris-HCl, pH 7.9. KCl ranged in
concentration from 0.5 to 100 mM. Following the pre-incubation, 2
ul of the PNA bound plasmid was transcribed in HeLa nuclear
extracts (Promega) following the manufacturer's protocol. The
transcribed RNA's were precipitated then separated by
electrophoresis through 5% denaturing acrylamide gels, run 2 hours
at 15 W. Gels were then dried and exposed to film. Bands on the
exposed film were quantitated using a Molecular Dynamics laser
densitometer.
[0254] A PNA with the sequence TTTTCTTTT-NH.sub.2 (SEQ ID NO:16)
and a non homologous PNA control were pre-incubated with the 200 ng
of EcoRI linerized plasmid pCH495 at the indicated concentrations
for 3 hours at 22.degree. C. in 10 ul of 10 .mu.M KCl, pH 6.8
(potassium salt was used in all binding buffers because it is the
most compatable with the transcription extract). Following the
pre-incubation, the PNA bound plasmid was transcribed in HeLa cell
nuclear lystates (Promega) containing .sup.32P GTP. The transcribed
RNA product was then electrophoresed on a 5% acrylamide gel with
50% w/v urea, which was then dried and exposed to film. The
expected size of the full length run off transcript and the PNA
truncated transcript are shown in FIG. 31. The concentration at
which 50% of the transcript was truncated was determined to be
approximately 80 uM by quantitating the bands from the exposed film
using a Molecular Dynamics densitometer. Results are shown in FIG.
32. Linearization of the plasmid with EcoRI and PvuII provides
transcription templates with expected run-off transcripts of 495
(lane1) and 190 (lane 2) nucleotides respectively. In the presence
of the complementary PNA, the EcoRI linearized plasmid yields two
distinct transcripts, one corresponding to the run-off transcript
and a second truncated transcript with a molecular weight
consistent with the 373 nucleotide product expected if truncation
occurred at the PNA binding site. The relative proportion of the
truncated nucleotide product increases with increasing
concentration of PNA in the pre-incubation. When a DNA
oligonucleotide of the same sequence (5'-TTTTCTTTT-3') was used in
place of the PNA no truncated product was observed (data not
shown). The experiment was repeated for the other PNA target sites
shown in FIG. 31. These results are shown in Table I and are given
as IVT.sub.50; the PNA concentration at which 50% of the transcript
is specifically truncated. All pre-incubations were carried in 10
mM KCl at pH 5.1, 6.8, or 7.9. In all cases, the amount of
transcript, both full length and truncated, decreases with
increasing PNA concentration, suggesting a non-specific inhibition
of transcription by PNA. This effect seems to be become more
pronounced as the percentage of cytosine residues in the PNA
increases. For example, at a PNA concentration of 1 mM, the total
yield of transcript is reduced approximately 63 percent in the
presence of PNA TTTTCTTTT-NH.sub.2 (SEQ ID NO:16), 84 percent in
the presence of TTCTCTCTCT-NH.sub.2 (SEQ ID NO:20) and completely
in the presence of CCCCCCCTTTTTTTTT-NH.sub.2 (SEQ ID NO:18)
(compare the 1 mM lanes in FIG. 32 and FIG. 33. Direct addition of
PNA to transcription extracts did not result in the production of
any truncated product (data not shown).
4TABLE I Summary of in vitro transcription activity for PNA
oligomers. PNA plasmid pH 5.1 pH 6.8 pH 7.9 TTTTCTTTT-NH.sub.2
pCH495 58 .mu.M 76 .mu.M >500 .mu.M TTCTCTCTCT-NH.sub.2 pIE72 29
.mu.M 103 .mu.M >1 mM CCCCCCCTTTTTTTTT-NH.sub.2 pCIN, 30 .mu.M
124 .mu.M >1 mM pCINr CCTCCTTCCC-NH.sub.2 pIE72 >1 mM none
none
EXAMPLE 30
[0255] Effect of Target Site Orientation on PNA Mediated
Termination of In Vitro Transcription.
[0256] Plasmids pCIN and pCINr were linearized with HindIII, then
pre-incubated with PNA of the sequence CCCCCCCTTTTTTTTT-NH.sub.2
(SEQ ID NO:18) at the concentrations shown in FIG. 33 under the
conditions described for Example 29. Following the IVT a truncated
product was clearly observed for pCIN, where the PNA is bound to
the template strand. However, for pCINr, where the PNA is bound to
the non-transcribed strand, only a slight amount of truncated
product of the expected size is observed. The plasmids pCIN and
PCINr contain a CMV gene fragment cloned in opposite orientations
under the transcriptional control of of the CMV promoter. pCIN
contains the binding site for the PNA CCCCCCCTTTTTTTTT-NH.sub.2
(SEQ ID NO:18) on the template strand, while pCINr contains the
same target site on the non-transcribed strand. The PNA was tested
for the ability to effect in vitro transcription as per Example 29.
The results are shown in FIG. 33. Both plasmids show non-specific
inhibition of in vitro transcription at the highest concentration
of PNA tested (1 mM). However, when the PNA concentration is
decreased to 100 uM, approximately one half of the RNA produced
corresponds to the size expected for the PNA truncated product (209
nucleotides) with the pCIN template. In contrast, pCINr shows only
a faint truncated product of the correct size at the same
concentration.
EXAMPLE 31
[0257] Effect of Ionic Strength and pH on PNA Mediated Termination
of Transcription.
[0258] 25 uM PNA TTTTCTTTT-NH.sub.2 (SEQ ID NO:16) was
pre-incubated three hours with pCH495 at pH 6.8 in 0.5 to 100 mM
KCl buffer. Following the pre-incubation the linearized plasmid was
transcribed as detailed in Example 29. The results are shown in
FIG. 34 with the percent PNA truncated product indicated at the
bottom of each lane. The proportion of the truncated product
relative to full length transcript is increased as the salt
concentration is decreased.
[0259] The same PNA and plasmid template were used to determine the
effects of pH on the ability of PNA to effect transcription. PNA at
a concentration of 10 or 100 uM, was pre-incubated (as described in
Example 29) with the template in 10 mM KCl buffer at pH 5.1, 6.8,
or 7.9 then transcribed in HeLa cell extracts. The resultant
transcription products are shown in FIG. 35 with the percent
truncated product shown below each lane. At the highest pH tested,
7.9, little specifically truncated product is observed even at a
PNA concentration of 100 uM. A slight decrease in pH to 6.8 results
in a dramatic increase in the amount of truncated message produced
at the 100 uM PNA concentration, although no truncated message is
observed at a PNA concentration of 10 uM. However, when the pH is
decreased to 5.1, a small amount of the truncated product is
observed even at the lower PNA concentration and is increased at
the higher PNA concentration.
[0260] The experiments were also performed with the other PNA's.
The IVT.sub.50 for each is listed in Table I, above. The effect of
pH on PNA binding seems to become more enhanced as the percentage
of cytosine residues in the PNA oligomer increases, probably due to
enhanced binding upon cytosine protonation. At pH 5.1, one PNA,
CCTCCTTCCC-NH.sub.2 (SEQ ID NO:19), showed only a small amount of
truncated product at the highest concentration tested. At higher pH
no truncated product was observed at all. Higher PNA concentrations
were not tested since they resulted in complete non-specific
inhibition of transcription. This non-specific effect was generally
more pronounced in the oligomers with larger cytosine/thymidine
ratios.
[0261] The ability of PNA to inhibit transcription in vitro
supports it use as an antigene therapeutic agent. In using PNA as
an antigene agent both pH and ionic barriers would be considered.
The effects of pH are mitigated by targeting adenosine rich
regions, avoiding dG/dC duplexes. Alternatively, modified residues,
such as methyl cytosine could facilitate binding. In this example
inhibition of in vitro transcription was most efficient at ionic
strengths less than those typically found in cells. Therefore, it
is possible that PNA will strand invade under physiological
conditions if given sufficient time to overcome kinetic barriers.
Further to enhance the rate of strand invasion metabolically active
regions of the genome would be targeted.
EXAMPLE 32
[0262] In Vitro Binding of PNA to Target
[0263] The target for in vitro binding of duplex DNA was prepared
by annealing two 50 base DNA oligonucleotides in which the binding
site for PNA TTTTCTTTT-NH.sub.2 (SEQ ID NO:16) is centered
(5'-AAACAGGGCA GGAAACAGCA TATTTTCTTT TAAAATTAGC AGGAAGATGG-3' and
5'-CCATCTTCC TGCTAATTTT AAAAGAAAAT ATGCTGTTTC CTGCCCTGTTT-3' (SEQ
ID NO:46)). The target was .sup.32P end labeled, then incubated at
roughly 500 nM with various concentrations of PNA in the buffers
described above for 4 hours at room temperature. PNA bound duplex
was seperated from free by electrophoresis on a 5% native
acrylamide gel in TBE. The gel was then dried and exposed on a
Molecular Dynamics PhosphoImager. The K.sub.d is given as the PNA
concentration at which one half of the labeled target is bound. Gel
shifts of PNA against DNA oligonucleotide complement were carried
out in the same buffers using an end labeled DNA oligonucleotide
with the sequence 5-AAAAGAAAA-3', which was present in each
hybridization mix at 1 nM. Bound DNA oligonucleotide was seperated
from free by electrophoresis through a 12% native acrylamide gel in
TBE.
[0264] Following the above protocols, the in vitro binding of PNA
to DNA was assessed by gel mobility shift assay. The 50 base pair
duplex DNA target for the PNA TTTTCTTTT-NH.sub.2 (SEQ ID NO:16) was
prepared with the binding site for PNA TTTTCTTTT-NH.sub.2 (SEQ ID
NO:16) centered in the fragment. The DNA complement
(5'-AAAAGAAAA-3') was synthesized and end labeled. PNA was
incubated with the either the single strand complement or duplex
DNA target in 20 mM KCl buffer at pH 5.1, 6.8, or 7.9. PNA bound
DNA was then seperated from free on a native acrylamide gel. FIG.
36a shows that PNA binding to both the single stranded and duplex
target is effected by the pH of the binding buffer. Binding
affinity (K.sub.d, the PNA concentration at which one half of the
target is shifted in mobility) to both single stranded and duplex
DNA complement was determined by the gel shift assay. As the pH is
decreased affinity increases. PNA affinity for the single stranded
target is much greater than for the duplex target, presumably due
to the stability of the DNA duplex in 20 mM KCl. Other experiments
to determine the binding affinity of other PNA oligomers showed
that the effect of pH on binding was more pronounced with cytosine
rich PNA's (data not shown).
[0265] Binding of PNA to target was also utilized to study the
effects of salt concentration on PNA binding by incubating PNA
TTTTCTTTT-NH.sub.2 (SEQ ID NO:16) with it's single stranded or
duplex target in buffers containing 1, 10, or 100 mM KCl at pH 6.8.
The results are shown in FIG. 36b. While the salt concentration
does have a large effect on the ability of PNA to bind duplex DNA,
there is little salt effect on the binding to single stranded
complement. Similar results were obtained when PNA
TTCCCTTCC-LysNH.sub.2 (SEQ ID NO:12) was tested (data not
shown).
EXAMPLE 33
[0266] PNA-DNA Base Pair Recognition
[0267] To test if PNA containing all four natural nucleobases is a
true DNA mimic in terms of base pair specific hybridization to
complementary oligonucleotides a pentadecamer PNA was designed to
contain an almost equal number of pyrimidines and purines yet
having no more than two purines or pyrimidines juxtaposed. It
contains an equal number of thymines and cytosines and a single
guanine. Furthermore, the sequence of the pentadecamer is
non-selfcomplementary and contains a GTCA sequence at the center.
By measuring the thermal stability of complexes between the PNA
pentadecamer and the Watson-Crick complementary oligonucleotide, as
well as 12 other oligonucleotides each having a single base
mismatch at one of the four center PNA nucleobases, information
about PNA-DNA base pair recognition was obtained. Finally, the PNA
was designed with no pyrimidine stretches so as to strongly
disfavor--if not prohibit--triplex formation. The thermal stability
measured as the melting temperature, T.sub.m, of complexes between
the pentadecamer, H-TGTACGTCACAACTA-NH.sub.- 2 (SEQ ID NO:10) and
the complementary deoxyoligonucleotide 3'-ACATGCAGTGTTGAT (SEQ ID
NO:47) (termed anti-parallel orientation: amino terminal of the PNA
complementary to the 3'-end of the oligonucleotide) was
69.5.degree. C. (see Table II), whereas the T.sub.m of the
corresponding PNA-DNA complex in parallel orientation was
56.1.degree. C. The T.sub.m for the PNA-RNA complexes was 72.3 and
51.2.degree. C., respectively (see Table II). The orientation
preference was further settled by using two decamer PNAs, and
hybridizing these to complementary oligonucleotides in both
orientations. The anti-parallel orientation was preferred in all
cases (see Table II). Furthermore, it is noteworthy that virtually
identical T.sub.ms were obtained regardless of which strand is the
PNA and which is the DNA (see Table II, 2nd & 3rd row). The
results also show that the presence of a terminal lysine amide (add
to certain PNAs to reduce aggregation of oligo thymine PNA) does
not influence the preferred orientation of the PNA relative to the
DNA.
5TABLE II Melting temperatures T.sub.m(.degree. C.) for PNA/DNA,
PNA/RNA, DNA/DNA and DNA/RNA complexes..sup.a 1st strand
sequence.sup.b TGTACGTCACAACTA.sup.c GTAGATCACT.sup.d
AGTCATCTAC.sup.d DNA: DNA 53.3 33.5 33.5 DNA:RNA 50.6 nd nd PNA:DNA
56.1 38.0 38.0 (parallel) PNA:DNA 69.5 51.0 49.0 (anti- parallel)
PNA:RNA 51.2 nd nd (parallel) PNA:RNA 72.3 nd nd (anti- parallel)
.sup.aAbsorbance vs. temperature curves were measured at 260 nm in
100 mM NaCl, 10 mM Na-phosphate, 0.1 mM EDTA, pH 7. T.sub.m, the
temperature at which half of the molecules are hybridized was
obtained by fitting triplicate melting curved at 4 .mu.M of each
strand to a modified two state model with linear sloping baseline.
.sup.bWritten 5'-3' for oligonucleotides and N-- to C-- terminal
for PNA. .sup.cThe PNA terminates in a carboxamide. .sup.dThe PNA
terminates in a lysine amide.
[0268] When a Watson-Crick base pair mismatch was introduced in the
oligonucleotide at any position facing the four middle PNA
nucleobases (GTCA) in the pentadecamer a large increase in T.sub.m
(8-20.degree. C., FIG. 37, was observed, thereby providing
compelling evidence that PNA-DNA recognition takes place by
Watson-Crick base pairing, i.e., A-T and G-C base pairing.
Qualitatively similar results were obtained using oligonucleotides
in the parallel orientation. Shown in FIG. 37 are the effect of
base pair mismatches on the thermal stability of PNA/DNA complexes.
Thermal stability of complexes between PNA,
H-TGTACGTCACAACTA-NH.sub.2 (SEQ ID NO:10) and the thirteen
oligonucleotides 3'-d(ACATGXYZVGTTGAT), in which X, Y, Z, V=C, A,
G, T for the case where the PNA and DNA sequences are complementary
are shown. In each of the twelve other oligonucleotides, three of
the bases (X, Y, Z, V) were complementary while the fourth was one
of the three non-complementary nucleobases. For example, when (X=T,
Y-G, Z=A) then V=A or C or G etc. Thus each of the twelve
oligonucleotides contains one of the twelve possible base pair
mismatches relative to the PNA pentadecamer. The T.sub.m of these
complexes are displayed as solid bars in FIG. 37. For comparison,
the results of similar experiments performed with DNA/DNA duplexes
are displayed (hatched bars). Hybridizations were performed in 10
mM Na-phosphate, pH 7.0, 150 mM NaCl, 1 mM MgCl.sub.2.
[0269] For comparison we also measured the thermal stabilities of
the corresponding DNA/DNA duplexes as shown in FIG. 38.
.DELTA.T.sub.m values for mismatches are shown in FIG. 38.
Hybridizations were as for FIG. 37.
[0270] It is noteworthy that for virtually all base pair
mismatched, the decrease in stability is greater for the PNA/DNA
complex than for the DNA/DNA complex (see FIG. 38), thereby
indicating that the sequence discrimination is, if anything, more
efficient for PNA recognizing DNA than for DNA recognizing DNA.
[0271] The unambiguous evidence for Watson-Crick base pairing
suggest that these PNA/DNA complexes are duplexes rather than the
(PNA).sub.2/DNA triplexes previously observed with homo-pyrimidine
PNA. This conclusion was confirmed by titration experiments using
.sup.32P-labeled oligonucleotides in a gel retardation assay.
Complete complex formation was observed at a 1:1 stoichiometry of
[PNA] to [DNA] as shown in FIG. 39. Similar results were obtained
using circular dichroism for the detection of complex formation
(data not shown). In this example titration was by gel-shift of the
binding of PNA H-TGTACGTCACAACTA-NH.sub- .2 (SEQ ID NO:10) to the
5'-endlabeled oligonucleotide 3'-d(ACATGCAGTGTTGAT) (SEQ ID NO:47).
The oligonucleotide was labeled with .sup.32P at the 5'-end using
standard techniques.sup.22. The oligonucleotide (1 nmole, 103 cpm)
was incubated with various amounts of PNA (0-5 nmol) in 10.mu. 10
mM Tris-HCl, 1 mM EDTA, pH 7.4 for 1 hour at 37.degree. C. The
samples were analyzed by electrophoresis in 20% polyacrylamide gels
(TBE buffer=89 mM Tris-borate, pH 8.3, 1 mM EDTA) and the
radiolabeled DNA visualized by autoradiography. Concentrations of
oligonucleotides and PNA were measured photometrically. Similar
results were obtained using the complementary oligonucleotide of
reversed polarity (5'-d(ACATGCAGTGTTGAT) (SEQ ID NO:52).
EXAMPLE 34
[0272] Structure of the PNA/DNA Duplex
[0273] The T.sub.m data (Table II above) show that PNA, in contrast
to DNA or RNA, may bind to complementary DNA or RNA in either
orientation, although the antiparallel orientation is preferred.
Since the PNA backbone is achiral, the orientation does not by
itself impose any steric constraints on the winding of the helix,
i.e., left or right handed helicity, and computer modelling also
indicates that both binding orientations are possible.
[0274] Information on secondary structure may be obtained from
circular dichroism (CD) measurements since these are sensitive to
the base pair geometry in the helix. FIG. 40 shows the CD spectra
of the PNA/DNA, PNA/RNA, DNA/DNA and DNA/RNA duplexes. Shown in
FIG. 40 is the circular dichroism spectra of PNA/DNA (a:
antiparallel; b: parallel), PNA/RNA (c: antiparallel; d: parallel),
DNA/DNA (e) and DNA/RNA (f) complexes. The complexes were formed by
mixing equal molar amounts of the two complements in distilled
H.sub.2O. Circular dichroism spectra were recorded on a Jasco 700
instrument at room temperature using an optical path of 1 mm. All
measurements were averaged ten times and smoothed. All spectra are
largely similar suggesting that PNA/DNA and PNA/RNA duplexes are
right handed helices with a base pair geometry not drastically
different from that found in a B- or an A-DNA helix. However, it is
interesting that the CD-spectra, and thus the structure, of PNA/DNA
(or PNA/RNA) duplexes are distinctly different when parallel and
anti-parallel complexes are compared. The reference DNA/DNA duplex
would be expected to adopt a B-like helix while the RNA/DNA duplex
would be expected to adopt a more A-like helix, but the CD-spectra
of neither of these short duplexes show typical B- or A-like
features. Thus it is not possible to conclude from these results if
the PNA/DNA or PNA/RNA helices are preferentially A- or B-like.
EXAMPLE 35
[0275] Thermodynamics of the PNA/DNA Duplex Formation
[0276] Thermodynamics parameters for hybridization can be extracted
from thermal stability measurements when these are performed at
varying concentrations of the complexes, or from the shape of the
thermal denaturation curves. Both methods were used to determine
.DELTA.H*, .DELTA.S* and .DELTA.G* for formation of the PNA/RNA,
DNA/RNA, PNA/DNA and DNA/DNA duplexes (Table III). It is remarkable
that the decrease in entropy is almost identical for the formation
of DNA/DNA and PNA/DNA duplexes and that both reactions are
strongly enthalpy driven.
6TABLE III Thermodynamic parameters for the formation of PNA/DNA,
PNA/RNA, DNA/RNA and DNA/DNA duplexes with the sequence
TGTACGTCACAACTA present in the PNA strand strand.sup.a. DNA:RNA
PNA:RNA DNA:DNA PNA:DNA .DELTA.H (kcal/mol).sup.b -94.0 -109.7
-91.5 -103.5 .DELTA.S (eu).sup.b -264.9 -291.4 -253.9 -276.4
.DELTA.G.sub.3.sup.b -11.8 -19.3 -12.7 -17.7 T.sub.M (c, 8
.mu.M).sup.b 49.8 72.3 53.6 68.9 .DELTA.H (kcal/mol).sup.c -128.9
-128.5 -105.3 -106.6 .DELTA.S (eu).sup.c -372.8 -345.9 -296.2
-285.8 .DELTA.G.sub.37 (kcal/ -13.3 -21.2 -13.4 -8.0 mol).sup.c
T.sub.M (c, 8 .mu.M).sup.c 50.1 72.2 53.5 68.8 .DELTA.H
(kcal/mol).sup.d -111.5 -119.1 -98.4 -105.0 .DELTA.S (eu).sup.d
-318.9 -318.7 -275.1 -281.1 .DELTA.G.sub.37 (kcal/ -12.6 -20.2
-13.1 -17.9 mol).sup.d T.sub.M (C, 8 .mu.M).sup.d 50.0 72.2 53.5
68.8 .sup.aMeasured in 100 mM BaCl, 10 mM Na-phosphate, 0.1 mM
EDTA, pH 7.0. .sup.bObtained by fitting melting curves to a
modified two state model with linear sloping baselines.
.sup.cObtained from linear plots of 1/T.sub.M versus log
(concentration). .sup.dTemperature independent parameters
calculated as the average of the two methods described above.
[0277] The finding that .DELTA.H* and .DELTA.S* are similar for the
formation of PNA/DNA and DNA/DNA duplexes, combined with the
finding that the PNA/DNA and RNA/DNA duplex structures have similar
stacking according to the CD results, indicates that single
stranded PNA must have much the same degree of base stacking as
single stranded DNA, and thus appears to be highly structured. The
kinetics of PNA/RNA duplex formation was also measured, and the
results show that the rate of hybridization is at least as fast as
that for 2'O-Me-RNA/RNA or DNA/DNA duplex formation (Table IV).
Again fully consistent with the suggestion that the single stranded
PNA is at least as prestructured for duplex formation as is DNA (or
RNA).
7TABLE IV Equilibrium and rate constants for duplexes. Duplex [Na+]
(M)T (.degree. C.) K.sub.D.sup.a k.sub.1 (M.sup.-1s.sup.-1).sup.a,b
PNA/RNA.sup.c 0.1 37 5 .times. 10.sup.-11 2 .times. 10.sup.6
2'-O--Me/RNA.sup.c 0.1 37 5 .times. 10.sup.-11 2 .times. 10.sup.5
DNA/RNA.sup.c 0.1 37 2 .times. 10.sup.-9 -- DNA/DNA.sup.d 0.2 39 --
3 .times. 10.sup.5 .sup.aThe buffer was 100 mM NaCl, 10 mM
phospphate, 1 mM EDTA, pH 7.0, and the dissociation constant was
determined from gel-shift experiments ussing a .sup.32P-labeled
RNA. The rate was measured in experiments which were stopped by
adding 50-fold excess of unlabeled RNA followed by rapid freezing.
.sup.bThe rate constants were determined from plots of the
pseudo-first order rate constant versus the reagent concentration,
which in all cases were much higher than the concentration of the
labeled RNA target. .sup.cThe sequence of the reagent was: N-(or
5')-TGTACGTCACAACTA-C-terminal (or 3'), and the sequence of the RNA
target: 5'-TAGTTGTGACGTACA-3. 2'-O--Me is the 2'-O-methyl RNA
derivative. .sup.dResults taken from Tibanyenda et. al., Eur. J.
Biochem. 1984, 139, 19. The sequence was: 5'-CAACTTGATATTAATA.
[0278] The following chemical synthesis are shown in Schemes I and
II.
[0279] scheme I
EXAMPLE 36
MonoBoc-diethylenetriamine dibydrochloride (1)
[0280] A solution of t-butyl-p-nitrophenyl carbonate (10 g; 0.0418
mole) in CHCl.sub.3 (400 ml) was added to a solution of
diethylenetriamine (45 ml; 0.0417 mole) in CHCl.sub.3 (250 ml) at
0.degree. C. over a period of 3 h. The reaction mixture was stirred
overnight at room temperature. The precipitate that appeared was
filtered and washed in CHCl.sub.3. The solvent was evaporated,
first under reduced pressure with a water-aspirator, then with an
oil-pump (0.05 mmHg; 50.degree. C.). The residue was dissolved in a
mixture ethylacetate (50 ml)/H.sub.2O (50 ml) and the solution was
acidified to pH 4 with HCl 4N, extracted with ethylacetate
(3.times.50 ml). The aqueous solution was adjusted to pH 9 with
NaOH 2N and extracted with ethylacetate (3.times.50 ml). The
aqueous phase was adjusted to pH 11.5 and extracted with
ethylacetate (10.times.50 ml). The combined organic phases of the
last extraction were evaporated under reduced pressure and the
resulting oil was dissolved in water (50 ml) and acidified to pH 5.
Evaporation of water yielded a slightly yellow solid, which was
thoroughly washed with ether (yield: 6.41 g; 55%). .sup.1H-NMR
(D.sub.2O): .delta. (ppm): 1.4 (s, 9H); 3.0 (t, 2H); 3.3 (s broad,
4H); 3.4 (t, 2H)
EXAMPLE 37
Boc-, Z-diethylenetriamine hydrochloride (2)
[0281] To a solution of 1 (5.5 g; 19.9 mmoles) in dioxane (50
ml)/water (50 ml) adjusted at pH 11 was added a solution of
benzylnitrophenyl carbonate (5.44 g; 19.9 moles) in dioxane (50 ml)
at 0.degree. C. over a period of 1 h, while maintaining the pH at
11 with NaOH 2N. The reaction mixture was then stirred at room
temperature for 1.5 h. Subsequently, ethylacetate (100 ml) was
added and the reaction mixture was cooled at 0.degree. C. and
acidified to pH 4 with HCl 4N. A precipitate appeared in the
organic phase. The two phases were separated and the organic phase
filtered. This (addition of ethylacetate, separating the two phases
and filtering the organic phase) was repeated 3 times to give 3.05
g of white solid. An additional 1.38 g was obtained by joining all
the organic phases and adding ether (Yield: 4.427 g; 59%).
.sup.1H-NMR (DMSO d6): .delta. (ppm): 1.5 (s, 9H); 3.0 (m, 4H); 3.4
(m, 4H); 5.1 (s, 2H); 7.4 (s, 5H); 7.1 (m, 1H); 7.6 (m, 1H). MS
FAB+: M+1: 338.2
EXAMPLE 38
[0282] Coupling of 2 with N-1-carboxymethylthymine
[0283] Compound 2 (4 g; 10.7 mmoles), N-1-carboxymethylthymine
(1.97 g; 10.7 mmoles), DhbtOH (1.75 g; 10.7 mmoles) and DIEA (1.2
ml; 12 mmoles) were dissolved in a mixture of CH.sub.2Cl.sub.2 (50
ml)/DMF (50 ml). After cooling at 0.degree. C., DCC (2.2 g; 10.7
mmoles) was added and the reaction mixture was stirred at 0.degree.
C. for 1/2 h and 2.5 h at room temperature. DCU was filtered and
washed with CH.sub.2Cl.sub.2 (2.times.100 ml). The combined organic
phases were washed with NaHCO.sub.3 1M (3.times.100 ml), KHSO.sub.4
1M (3.times.100 ml), H.sub.2O (2.times.100 ml), dried over
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure.
The resulting oil was crystallized in chloroform/ether to yield
4.04 g (75%) of product. .sup.1H-NMR (CDCl.sub.3); .delta. (ppm);
1.4 (s, 9H); 1.9 (s, 3H); 3.4 (m, 8H); 4.4 (d, 2H); 5.1 (d, 2H);
5.5 (m, 1H); 5.9 (m, 1H); 6.9 (s, 1H); 7.4 (s, 5H); 9.0 (s, 1H).
MSFAB+: M+1; 504.
EXAMPLE 39
[0284] Hydrogenolysis of 3 to 4
[0285] Compound 3 (4 g; 7.9 mmoles) was dissolved in MeOH (150 ml).
At 0.degree. C., 10% Pd/C (1.4 g) was added. The reaction mixture
was hydrogenated at 0.degree. C. for 1 h, filtered through Celite
and the solvent removed under reduced pressure to give 3.0 g (100%)
of 4. .sup.1H-NMR (CDCl.sub.3); .delta. (ppm); 1.4 (s, 9H); 1.9 (s,
3H); 3.0-3.8 (m); 4.6 (d, 2H); 6.9 (s, 1H); 7.3 (s, 5H); 9.0 (s,
1H). MSFAB+:M+1: 370.2
EXAMPLE 40
N-(bensyl acetate)-glycine ethyl ester hydrochloride (5)
[0286] At room temperature, benzyl bromo acetate (5.7 ml; 36
mmoles) was added over a period of 10 min. to a solution of glycine
ethyl ester hydrochloride (5 g; 36 mmoles) and triethylamine (10.43
ml; 0.072 moles) dissolved in absolute EtOH (100 ml). After 4 days
at room temperature, the solvent was removed under reduced
pressure. The residue was dissolved in ethylacetate (50
ml)/H.sub.2O) (25 ml). After separation of the two phases, the
organic phase was thoroughly washed with water (8.times.25 ml).
After evaporation of the solvent, the resulting oil was dissolved
in ether (20 ml)/water (30 ml) and acidified to pH 4.5. After
separation of the two phases, the aqueous phase was concentrated
and the resulting oil crystallized in cold ether to yield 4 g (39%)
of the product. .sup.1H-NMR (CDCl.sub.3); .delta. (ppm); 1.3 (t,
3H); 4.1 (d, 4H); 4.4 (q, 2H); 5.3 (d, 2H); 5.5 (m, 1H); 7.4 (s,
5H). MS FAB+; M+1: 252.1
EXAMPLE 41
[0287] Coupling of 5 with N-1-carboxymethylthymine to give 6
[0288] Compound 5 (2.8 g; 9.7 mmoles), N-1-carboxymethylthymine
(1.79 g; 9.7 mmoles), DhbtOH (1.6 g; 9.7 mmoles) and DIEA (1.7 ml;
10 mmoles) were dissolved in a mixture of CH.sub.2Cl.sub.2 (30
ml)/DMF (30 ml). After cooling at 0.degree. C., DCC (2.0 g; 9.7
mmoles) was added and the reaction mixture was stirred at 0.degree.
C. for 1/2 h and 4 h at room temperature. The DCU was filtered and
washed with CH.sub.2Cl.sub.2 (120 ml). The combined organic phases
were washed with NaHCO.sub.3 1M (3.times.60 ml), KHSO.sub.4 1M
(3.times.60 ml), H.sub.2O (2.times.60 ml), dried over
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure.
The resulting oil was crystallized in chloroform (60 ml)/petroleum
ether (120 ml) to yield 3.235 g (80%) of 6. .sup.1H-NMR (DMSO d6):
6 (ppm); 1.3 (dt, 3H); 1.9 (s, 3H); 4.2 (m, 4H); 4.4 (d, 2H); 5.3
(d, 2H); 7.2 (s, 1H); 7.4 (s, 5H); 11.3 (s, 1H). MS FAB+: M+1:
418.1
EXAMPLE 42
[0289] Hydrogenolysis of 6 to 7
[0290] Compound 6 (4 g; 9.6 mmoles) was dissolved in MeOH (100
ml)/DMF (25 ml). At 0.degree. C., 10% Pd/C (1.6 g) was added. The
reaction mixture was hydrogenated at 0.degree. C. for 1H, filtered
through Celite and the solvent removed under reduced pressure to
give 3.12 g (99%) of 7. .sup.1H-NMR (DMSO d6): .delta. (ppm); 1.3
(dt, 3H); 1.9 (s, 3H); 4.0-4.5 (m); 4.9 (s, 2H); 7.2 (s, 1H); 11.3
(s, 1H). MS FAB+: M+1: 328.1
EXAMPLE 43
[0291] Coupling of 4 and 7 to give 8
[0292] Compound 4 (2.8 g; 7.6 mmoles), 7 (2.5 g; 7.6 mmoles),
DhbtOH (1.24 g; 7.6 mmoles) were dissolved in a mixture of
CH.sub.2Cl.sub.2 (50 ml)/DMF (50 ml). After cooling at 0.degree.
C., DCC (1.56 g; 7.6 mmoles) was added and the reaction mixture was
stirred at 0.degree. C. for 1/2 h and overnight at room
temperature. The DCU was filtered and washed with CH.sub.2Cl.sub.2
(250 ml). The combined organic phases were washed with NaHCO.sub.3
1M (3.times.100 ml), KHSO.sub.4 1M (3.times.100 ml), H.sub.2O
(2.times.100 ml), dried over Na.sub.2SO.sub.4, filtered and
evaporated under reduce pressure. The resulting oil was
crystallized in chloroform (30 ml)/petroleum ether (60 ml). This
material was recrystallized in chloroform (30 ml)/petroleum ether
(25 ml) to give 0.608 g (12%) of 8. MS FAB+: M+1: 418.1
EXAMPLE 44
[0293] Hydrolysis of 8 to Give 9
[0294] Compound 8 (25 mg, 0.037 mmoles) was dissolved in absolute
ethanol (5 ml). Then NaOH 1M (1 ml) was added. The reaction mixture
was stirred for 35 min. at room temperature and after cooling,
neutralized with Dowex 50W (ca. 1 g). After filtration and
evaporation of the solvent, the acid 9 was obtained as a white
solid, yield 18 mg (82%).
[0295] Scheme IX
EXAMPLE 45
[0296] Alanine Bensyl Ester (10)
[0297] Thionylchloride (9.8 ml, 135 mmol, 1.2 eqv) was added
dropwise during 15 min. to benzyl alcohol (210 ml) stirred under
nitrogen and cooled to -10.degree. C. Alanine (10.0 g, 112 mmol,
1.0 eqv) was added in portions during 10 min. The reaction mixture
was heated overnight at 60-70.degree. C. The reaction mixture was
evaporated In vacuo and the residue was dissolved in water (150
ml). The pH was adjusted to 1-2 by addition of 4N HCl (aq.) and the
aqueous phase was extracted with dichloromethane (2.times.200 ml).
The organic phase was washed with hydrochloric acid (pH 1,
1.times.50 ml). The aqueous phases were collected, alkalinized (to
pH 9-10) by addition of 2N NaOH (aq.) and subsequently extracted
with dichloromethane (2.times.200 ml and 1.times.100 ml--alkaline
extract). The alkaline extract was dried (Na.sub.2SO.sub.4),
filtered and evaporated under reduced pressure affording 13.39 g
(67%) of the title compound as a colorless oil. The product was
characterized as its hydrochloride: The product (0.50 g, 2.8 mmol)
was dissolved in ether (5 ml) and HCl in ether (2 ml) was added.
The precipitate was collected by filtration and washed with ether
(2.times.5 ml) yielding 0.57 g (95%) of white crystalline alanine
benzyl ester hydrochloride. .sup.1H-NMR (D.sub.2O/TMS): .delta.
1.48 (unresolved d, 3H, Me); 4.15 (unresolved q, 1H, CH); 5.20 (s,
2H, CH.sub.2); 7.38 (br s, 5H, Ph). .sup.13C-NMR (D.sub.2O/TMS):
.delta. 14.7 (Me); 48.4 (CH); 68.1 (CH.sub.2); 128.1, 128.5, 134.3
(Ph); 170.1 (C.dbd.O). MS(FAB+) m/z (%): 180 (100, M-HCl+H). Anal.
Calcd. for C.sub.10H.sub.14ClNO.sub.2: C, 55.69; H, 6.54; N, 6.49.
Found: C, 55.60; H, 6.56; N, 6.40.
EXAMPLE 46
N-(2-Boc-aminoethyl)alanine benzyl ester hydrochloride (11)
[0298] Alanine benzyl ester (10, 8.82 g, 49 mmol, 1 eq) was
dissolved in MeOH (100 ml) and glacial acetic acid (10.34 g, 172
mmol, 3.5 eq) was added. The mixture was stirred at 0.degree. C.
under nitrogen and sodium cyanoborohydride (10.82 g, 172 mmol, 3.5
eq) was added. Boc-aminoacetaldehyde (15.67 g, 98 mmol, 2 eq) in
MeOH (200 ml) was added dropwise during 2 h. The reaction mixture
was stirred at 0.degree. C. for 35 min. and then at 4-5.degree. C.
overnight. Water (300 ml) was added to the reaction mixture and the
pH was adjusted to 9 by addition of solid sodium carbonate. The
saturated solution was filtered and subsequently extracted with
ether (3.times.500 ml). The organic phase was washed with a 1:1
mixture of saturated aqueous solutions of sodium chloride and
sodium bicarbonate (1.times.450 ml), dried (Na.sub.2SO.sub.4),
filtered and evaporated under reduced pressure to give 22.60 g of
crude N-(2-Boc-amino-ethyl)alanine benzyl ester as a slightly
golden oil. The crude product was dissolved in dry ether (500 ml)
and stirred at 0.degree. C. HCl in ether (60 ml) was then added to
the solution, and the resulting precipitate was collected by
filtration and washed with cold dry ether (3.times.20 ml) affording
7.21 g (41%) of 11 as white crystals. A second crop of 2.12 g (12%)
could be collected as a white semicrystalline material by keeping
the mother liquor in the freezer overnight. This second crop was
slightly less pure than the first crop. .sup.1H-NMR (D.sub.2O/TMS):
.delta. 1.36 (s, 9H, Boc); 1.52 (d, J=7.2 Hz, 3H, Me); 3.16 (m, 2H,
NCH.sub.2); 3.35 (m, 2H, NCH.sub.2); 4.17 (q, J=7.2 Hz, 1H, CH);
5.26 (s, 2H, CH.sub.2Ph); 7.40 (s, 5H, Ph). .sup.13C-NMR
(D.sub.2O/TMS): .delta. 13.5 (Me); 27.3 (Box); 36.4 (CH); 45.5
(NCH.sub.2); 55.2 (NCH.sub.2); 68.4 (CH.sub.2Ph); 128.3, 128.7,
128.8 (Ph). MS(FAB+) m/z (%): 323 (100, M-HCl+H) Anal. Calcd for
C.sub.17H.sub.27CIN.sub.2O.sub.4: C, 56.90; H, 7.58; N, 7.81; Cl,
9.88. Found C, 55.11; H, 7.52; N, 7.93; Cl, 10.45.
EXAMPLE 47
N-(2-Boc-aminoethyl)-N-(1-thyminylacetyl)alanine benzyl ester
(12)
[0299] N-(2-Boc-aminoethyl)alanine benzyl ester hydrochloride (11,
3.00 g, 8.4 mmol, 1.0 eq) was dissolved in dry dichloromethane (50
ml) and stirred at 0.degree. C. under nitrogen. DIEA (1.5 ml, 8.4
mmol, 1.0 eq), DHBtOH (1.50 g, 9.2 mmol, 1.1 eq) in dichloromethane
(30 ml), 1-thyminylacetic acid (1.69 g, 9.2 mmol, 1.1 eq) in
dichloromethane (30 ml) and DCC (2.07 g, 10.0 mmol, 1.2 eq) in
dichloromethane (40 ml) were added in that order. The reaction
mixture was stirred at 0.degree. C. for 1 h and then at 4-5.degree.
C. overnight. The precipitated DCU was filtered off and washed with
dichloromethane (3.times.15 ml). The filtrate was washed with
saturated aqueous sodium bicarbonate (3.times.150 ml) diluted to
five times its volume, saturated aqueous potassium hydrogen sulfate
(2.times.150 ml) diluted to three times its volume, saturated
aqueous sodium chloride (1.times.150 ml), dried (Na.sub.2SO.sub.4),
filtered and finally evaporated under reduced pressure yielding
3.83 g of crude product as a pink glassy foam. The crude product
was chromatographed (silica, 0.063-0.200 mm) using
MeOH/dichloromethane as the eluent (3/97, v/v until the first
fraction appeared then 5/95, v/v) affording 2.87 g (70%) of 12 a
white glassy foam. 12 was isolated as mixture of two conformers due
to restricted rotation around the amide bond. Consequently, some of
the signals in the NMR spectra were split into a major and minor
component. The values provided below are for the major component.
.sup.1H-NMR (CDCl.sub.3/TMS): .delta. 1.44 (s, 9H, Boc); 1.52 (d,
J=7.1 Hz, 3H, ala-Me); 3.31 (m, 2H, NCH.sub.2); 3.44 (m, 1H,
NCH.sub.2); 3.55 (m, 1H, NCH.sub.2); 4.34 (q, J=7.3 Hz, 1H, CH);
4.53 (s, 2H, acetyl-CH.sub.2); 5.16 (s, 2H, CH.sub.2Ph); 6.90 (s,
1H, thymine-H-6); 7.35 (m, 5H, Ph). .sup.13C-NMR (CDCl.sub.3/TMS):
.delta. 12.2 (thymine-Me); 14.3 (ala-Me); 28.3 (Boc); 39.3 (CH);
46.0 (acetyl-CH.sub.2); 48.0 (NCH.sub.2); 55.6 (NCH.sub.2); 67.2
(CH2Ph) 79.8 (Boc); 110.5 (thymine-C-5); 128.0, 128.3, 128.5,
128.6, 135.1 (Ph); 140.8 (thymine-C-6); 150.9 (thymine-C-2); 155.8
(Boc-C.dbd.O); 164.1 (thymine-C-4); 166.8 (acetyl-CH.sub.2); 171.3
(ester-C.dbd.O). MS(FAB+) m/z (%): 489 (64, M+H). Anal. Calcd for
C.sub.24H.sub.32N.sub.4O.sub.7: C, 59.01; H, 6.60; N, 11.47. Found:
C, 58.55; H, 6.65; N, 11.16.
EXAMPLE 48
N-(2-Boc-aminoethyl)-N-(1-thyminylacetyl)alanine (13)
[0300] N-(2-Boc-aminoethyl)-N-(1-thyminylacetyl)alanine benzyl
ester (12, 2.02 g, 4.1 mmol, 1 eq) was dissolved in MeOH (100 ml)
and stirred at 0.degree. C. under nitrogen. 10% palladium on
activated carbon (1.7 g) was added and the mixture was hydrogenated
at atmospheric pressure and 0.degree. C. for 1 h at which time
hydrogen uptake had ceased (91 ml, 4.1 mmol, 1 eq had been
consumed). The reaction mixture was filtered through celite which
was washed thoroughly with MeOH. The collected filtrates were
evaporated giving 1.65 g (100%) of 13 as a white glassy foam. 13
was isolated as a mixture of two conformers due to restricted
rotation around the amide bond. Consequently, some of the signals
in the NMR spectra were split into a major and minor component. The
values provided below are for the major component. .sup.1H-NMR
(DMSO-d.sub.6/TMS): .delta. 1.42 (d, J=7.1 Hz, 3H, ala-Me); 1.46
(s, 9H, Boc); 3.26 (m, 2H, NCH.sub.2); 3.42 (m, 2H, NCH.sub.2);
4.39 (q, J=7.0 Hz, 1H, CH); 4.69 (s, 2H, acetyl-CH.sub.2); 7.00 (s,
1H, thymine-H-6); 11.35 (s, 1H, COOH). .sup.13C-NMR
(DMSO-d.sub.6/TMS): .delta. 12.0 (thymine-Me); 14.8 (ala-Me); 28.3
(Boc); 33.6 (CH); 45.7 (acetyl-CH.sub.2); 48.0 (NCH.sub.2); 54.8
(NCH.sub.2); 78.1 (Boc); 108.1 (thymine-C-5); 142.2 (thymine-C-6);
151.0 (thymine-C-2); 155.8 (Boc-C.dbd.O); 164.4 (thymine-C-4) 166.9
(acetyl-CH.sub.2); 172.6 (acid-C.dbd.O). MS(FAB-) m/z (%): 397
(100, M-H); 398 (21, M-H+1); 399 (5, M-H+2). Anal. Calcd for
C.sub.17H.sub.26N.sub.4O.sub.7: C, 51.25; H, 6.58; N, 14.06.
EXAMPLE 49
[0301] Chimera Macromolecule Having Peptide Nucleic Acids Section
Attaching to 3' Terminus of a 2'-Deoxy Phosphorothioate
oligonucleotide Section
[0302] A first section of peptide nucleic acids is prepared as per
PCT patent application PCT/EP/01219. The peptide nucleic acids are
prepared from the C terminus towards the N terminus using monomers
having protected amine groups. Following completion of the peptide
region, the terminal amine blocking group is removed and the
resulting amine reacted with a
3'-C-(formyl)-2',3'-dideoxy-5'-trityl nucleotide as prepared as per
the procedure of Vasseur, et. al., J. Am. Chem. Soc. 1992, 114,
4006. The condensation of the amine with the aldehyde moiety of the
C-formyl nucleoside is effected as per the conditions of the
Vasseur, ibid., to yield an intermediate oxime linkage. The oxime
linkage is reduced under reductive alkylation conditions of
Vasseur, ibid., with HCHO/NaBH.sub.3CN/AcOH to yield the nucleoside
connected to the peptide nucleic acid via an methyl alkylated amine
linkage. An internal 2'-deoxy phosphorothioate nucleotide region is
then continued from this nucleoside as per standard automatated DNA
synthetic protocols (see oligonucleotide synthesis, a practic
approach, M. J. Gait ed, IRL Press, 1984).
EXAMPLE 50
[0303] Chimera Macromolecule Having Peptide Nucleic Acids Section
Attaching to 5' Terminus of a Phosphorothioate Oligonucleotide
Section
[0304] A phosphorothioate oligonucleotide is prepare in the
standard manner on a solid support as per standard protocols (see
Oligonucleotides and Analogues, A Practical Approach, F. Eckstein
Ed., IRL Press, 1991. The dimethoxytrityl blocking group on that
nucleotide is removed in the standard manner. Peptide synthesis for
the peptide region is commenced by reaction of the carboxyl end of
the first peptide nucleic acid of this region with the 5' hydroxy
of the last nucleotide of the DNA region. Coupling is effected via
DEA in pyridine to form an ester linkage between the peptide and
the nucleoside. Peptide synthesis is then continued in the manner
of patent application PCT/EP/01219 to complete the peptide nucleic
acid region.
EXAMPLE 51
[0305] Double-Stranded Duplex Structures that Include Chimera Stand
and Triple-stranded Triplex structures That Include Chimera
Strand
[0306] Duplex and triplex structures will be formed with the
chimera strands of Examples 42 and 43 as per the protocols of other
of the above examples. Duplex structures can include duplexes
between a PNA-RNA or PNA-DNA strand and a RNA strand, a PNA-RNA or
PNA-DNA strand and a DNA strand, a PNA-RNA or PNA-DNA strand and a
PNA strand or a PNA-RNA or PNA-DNA strand and a further chimeric
PNA-DNA or PNA-RNA strand. Triplex structures can include a PNA
containing chimeric strands triplexing with dsDNA or with a
double-stranded PNA construct. Further triplex structures can
include two of the PNA containing chimera triplexing with a single
DNA or RNA strand. Additional triplex structures can include a
single PNA containing chimera plus a PNA triplexing with an
additional PNA containing chimera, an additional PNA strand or a
DNA or RNA strand.
EXAMPLE 52
[0307] Binding Between Single Strand PNA Containing Chimera and
Transcription Factor or Other Protein
[0308] A PNA containing chimeric strand will be used to bind to or
otherwise modulate single-stranded DNA, double-stranded DNA, RNA, a
transcription factor or other protein. In use of a PNA containing
chimera, part of the binding between the chimera and the
transcription factor or other protein will be binding between the
sugar-phosphate backbone of the DNA or RNA portion of the chimera
and hydrogen bonding between the ligands, e.g., nucleobases, of the
PNA portion of the chimera. Binding to the sugar-phosphate backbone
includes binding to phosphodiester linkages, phosphorothioate
linkages or other linkgages that may be used as the bacbone of the
DNA or RNA. In other instances, bonding can include hydrophobic
contacts between hydrophobic groups on the ligands, including
nucleobases, of the PNA or the nucleobases of the nucleic acid
portion of the chimera with like hydrophobic groups on proteins
that are being bound. Such hydrophobic groups on the chimeric
strand include the methyl groups on thymine nucleobases.
EXAMPLE 53
[0309] PNA Dimer (detT-idaT) Having "Reversed" Monomer 4
[0310] A. MonoBoc-diethylenetriamine dihydrochloride.
[0311] A solution of t-butyl-p-nitrophenyl carbonate (log; 0.0418
mole) in CHCl.sub.3 (400 ml) was added to a solution of
diethylenetriamine (45 ml; 0.417 mole) in CHCl.sub.3 (250 ml) at
0.degree. C. over a period of 3 h. Then, the reaction mixture was
stirred overnight at room temperature.
[0312] The precipitate that appeared was filtered and washed with
CHCl.sub.3. The solvent was evaporated, first under reduced
pressure with a water-aspirator, then with an oil-pump (0.05 mmHg;
50.degree. C.).
[0313] The residue was dissolved in a mixture of ethyl acetate (50
ml) and H.sub.2O (50 ml). The solution was acidified to pH 4 with
4N HCl and extracted with ethyl acetate (3.times.50 ml). The
aqueous solution was adjusted to pH 9 with 2N NaOH and extracted
with ethyl acetate (3.times.50 ml). Then, the aqueous phase was
adjusted to pH 11.5 and extracted with ethyl acetate (10.times.50
ml). The combined organic phases of the last extraction were
evaporated under reduced pressure and the resulting oil was
dissolved in water (50 ml) and acidified to pH 5. Evaporation of
water yielded a slightly yellow solid, which was thoroughly washed
with ether (yield: 6.41 g; 55%).
[0314] .sup.1H-NMR (D.sub.2O): .delta. (ppm): 1.4 (s,9H); 3.0
(t,2H); 3.3 (s broad, 4H); 3.4 (t, 2H)
[0315] B. Boc-, Z-diethylenetriamine hydrochloride.
[0316] To a solution containing the product of Example 53A (5.5 g:
19.9 moles) in dioxane (50 ml)/water (50 ml) adjusted at pH 11 was
added a solution of benzyl-nitrophenyl carbonate (5.44 g; 19.9
mmoles) in dioxane (50 ml) at 0.degree. C. over a period of 70 min,
while maintaining the pH at 11 with 2N NaOH. The reaction mixture
was then stirred at room temperature for 1.5 h.
[0317] Subsequently, ethyl acetate (100 ml) was added and the
reaction mixture was cooled at 0.degree. C. and acidified to pH 4
with 4N HCl. A precipitate appeared in the organic phase. The two
phases were separated and the organic phase filtered.
[0318] These operations (adding ethyl acetate, separating the two
phases and filtering the organic phase) were repeated 3 times. A
white solid (3.05 g) was collected this way. An additional 1.38 g
was obtained by joining all the organic phases and adding ether.
(Yield: 4.427 g; 59%)
[0319] .sup.1H-NMR (DMSO d6): .delta. (ppm): 1.5 (s,9H); 3.0
(m,4H); 3.4 (m,4H); 5.1 (s,2H); 7.4 (s,5H); 7.1 (m,1H); 7.6 (m,1H).
MS FAB+: M+1: 338.2
[0320] C. Coupling with N-1-carboxymethylthymine.
[0321] The product of Example 53B (4 g; 10.7 mmoles),
N-1-carboxymethylthymine (1.97 g; 10.7 mmoles). DhbtOH (1.75 g;
10.7 mmoles) and DIEA (1.9 ml; 11.2 mmoles) were dissolved in a
mixture of CH.sub.2Cl.sub.2 (50 ml)/DMF (50 ml). After cooling at
0.degree. C., DCC (2.2 g; 10.7 mmoles) was added and the reaction
mixture was stirred at 0.degree. C. for 0.5 h and 2.5 h at room
temperature. Then, DCU was filtered and washed with
CH.sub.2Cl.sub.2 (2.times.100 ml). The combined organic phases were
washed with 1M NaHCO.sub.3 (3.times.100 ml), 1M KHSO.sub.4
(3.times.100 ml), H.sub.2O (2.times.100 ml), dried over
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure.
The resulting oil was crystallized in chloroform/ether. (Yield:
4.04 g; 75%)
[0322] .sup.1H-NMR (CDCl.sub.3): .delta. (ppm): 1.4 (s,9H); 1.9
(s,3H); 3.4 (m, 8H); 4.4 (d,2H); 5.1 (d,2H); 5.5 (m,1H); 5.9
(m,1H); 6.9 (s,1H): 7.4 (s,5H); 9.0 (s,1H). MS FAB+: M+1: 504
[0323] D. Hydrogenolysis
[0324] The product of Example 53C (4 g; 7.9 mmoles) was dissolved
in MeOH (150 ml). At 0.degree. C., 10% Pd/C (1.4 g) was added. The
reaction mixture was hydrogenated at 0.degree. C. for 1 h, filtered
through Celite and the solvent removed under reduced pressure to
give 4. (Yield: 3.0 g; 100%)
[0325] .sup.1H-NMR (CDCl.sub.3): .delta. (ppm): 1.4 (s,9H); 1.9
(s,3H); 3.0-3.8 (m); 4.6 (d,2H); 6.9 (s,1H); 7.3 (s,5H); 9.0
(s,1H). MS FAB+: M+1: 370.2
[0326] E. N-(benzyl acetate)-glycine ethyl ester hydrochloride.
[0327] At room temperature, benzyl bromoacetate (5.7 ml; 36 mmoles)
was added over a period of 10 min to a solution of glycine ethyl
ester hydrochloride (5 g; 36 mmoles) and triethylamine (10.43 ml;
0.072 moles) dissolved in absolute ethanol (100 ml). After 4 days
at room temperature, the solvent was removed under reduced
pressure. The residue was dissolved in ethyl acetate (50
ml)/H.sub.2O (25 ml). After separation of the two phases, the
organic phase was thoroughly washed with water (8.25 ml). After
evaporation of the solvent, the resulting oil was dissolved in
ether (20 ml)/water (30 ml) and acidified to pH 4.5. After
separation of the two phases, the aqueous phase was concentrated
and the resulting oil crystallized in cold ether. (Yield 4 g;
39%)
[0328] .sup.1H-NMR (CDCl.sub.3): .delta. (ppm): 1.3 (t,3H); 4.1 (d,
4H); 4.4 (q,2H); 5.3 (d,2H); 5.5 (m,1H); 7.4 (s,5H). MS FAB+: M+1:
252.1
[0329] E. Coupling N-1-carboxymethylthymine.
[0330] The product of Example 53D (2.8 g; 9.7 mmoles),
N-1-carboxymethylthymine (1.79 g; 9.7 mmoles), DhbtOH (1.6 g; 9.7
mmoles) and DIEA (1.7 ml; 10 mmoles) were dissolved in a mixture of
CH.sub.2Cl.sub.2 (30 ml)/DMF (30 ml). After cooling at 0.degree.
C., DCC (2.0 g; 9.7 mmoles) was added and the reaction mixture was
stirred at 0.degree. C. for 0.5 h and 4 h at room temperature. Then
DCU was filtered and wash with CH.sub.2Cl.sub.2 (120 ml). The
combined organic phases were washed with 1M NaHCO.sub.3 (3.times.60
ml), 1M KHSO.sub.4 (3.times.60 ml), H.sub.2O (2.times.60 ml), dried
over Na.sub.2SO.sub.4, filtered and evaporated under reduced
pressure. The resulting oil was crystallized in chloroform (60
ml)/petroleum ether (120 ml). (Yield: 3.235 g; 80%)
[0331] .sup.1H-NMR (DMSO d6): .delta. (ppm): 1.3 (dt,3H); 1.9
(s,3H); 4.2 (m,4H); 4.4 (d,2H); 5.3 (d,2H); 7.4 (s,1H); 7.4 (s,
5H); 11.3 (s,1H). MS FAB+: M+1: 418.1
[0332] F. Hydrogenolysis
[0333] The product of Example 53E (4 g; 9.6 mmoles) was dissolved
in methanol (100 ml)/DMF (25 ml). At 0.degree. C., 10% Pd/C (1.6 g)
was added. The reaction mixture was hydrogenated at 0.degree. C.
for 1 h, filtered through Celite and the solvent removed under
reduced pressure. (Yield=3.12 g; 99%)
[0334] .sup.1H-NMR (DMSO d6): .delta. (ppm): 1.3 (dt,3H); 1.9
(s,3H); 4.0-4.5 (m); 4.9 (s,2H); 7.2 (s,1H); 11.3 (s,1H). MS FAB+:
M+1: 328.1
[0335] G. Coupling
[0336] The products of Examples 53D (2.8 g; 7.6 mmoles) and 53F
(2.5 g; 7.6 mmoles), and DhbtOH (1.24 g; 7.6 mmoles) were dissolved
in a mixture in a mixture of CH.sub.2Cl.sub.2 (50 ml)/DMF (50 ml).
After cooling at 0.degree. C., DCC (1.56 g; 7.6 mmoles) was added
and the reaction mixture was stirred at 0.degree. C. for 0.5 h and
overnight at room temperature. Then, DCU was filtered and washed
with CH.sub.2Cl.sub.2 (250 ml). The combined organic phases were
washed with 1M NaHCO.sub.3 (3.times.100 ml), 1M KHSO.sub.4
(3.times.100 ml), H.sub.2O (2.times.100 ml), dried over
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure.
The resulting oil was crystallized in chloroform (30 ml)/petroleum
ether (60 ml). This material was recrystallized in chloroform (30
ml)/petroleum ether (25 ml) to give a pure compound. (Yield: 0.60
g; 12%)
[0337] MS FAB+: M+1: 418.1
[0338] H. Hydrolysis
[0339] The product of Example 53G (529 mg; 0.8 mmoles) was
dissolved in absolute ethanol (100 ml). Then NaOH 1M (20 ml) was
added. The reaction mixture was stirred for 30 min at room
temperature and after cooling, neutralized with Dowex 50W (about 13
g). After filtration, washing with H.sub.2O, ethanol and
evaporation of the solvent, the residue was suspended in ether and
filtered. The acid product was obtained as a white solid, pure in
HPLC (Yield: 450 mg: 86%)
[0340] MS FAB+: 673 (M+Na.sup.+); 617 (M Na.sup.+-tBu); 595
(M+1-tBu); 573 (M Na.sup.+-Boc); 551 (M+1-Boc)
EXAMPLE 5
[0341] PNA Oligomer Having "Reversed" Monomer
[0342] The dimer prepared in Example 53 was incorporated into PNA
H-TT(detT-idaT)CCTCTC-LysNH.sub.2 (SEQ ID NO:53) generally in
accordance with Example 1. The melting temperatures, T.sub.m, of
complexes between this PNA and decamers 5'd(AAAAGGAGAG) (SEQ ID
NO:54) and 5'd(GAGAGGAAAA) (SEQ ID NO:55) were 55.degree. C. and
43.5.degree. C., respectively, at pH 7. By comparision, the T.sub.m
of complexes between PNA H-TTTCCTCTC-LysNH.sub.2 (SEQ ID NO:56),
prepared generally in accordance with Example 1, and these decamers
was 58.5.degree. C. and 40.5.degree. C., respectively, at pH 7.
[0343] Those skilled in the art will appreciate that numerous
changes and modifications may be made to the preferred embodiments
of the invention and that such changes and modifications may be
made without departing from the spirit of the invention. It is
therefore intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
Sequence CWU 1
1
89 1 11 PRT Artificial Sequence Novel Sequence 1 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 2 11 PRT Artificial Sequence
Novel Sequence 2 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10
3 20 DNA Artificial Sequence Novel Sequence 3 aaaaaaaaaa tttttttttt
20 4 20 DNA Artificial Sequence Novel Sequence 4 aaaaagaaaa
ttttcttttt 20 5 10 DNA Artificial Sequence Novel Sequence 5
aagaagaaaa 10 6 11 PRT Artificial Sequence Novel Sequence 6 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 7 7 PRT Artificial
Sequence Novel Sequence 7 Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 8 11 PRT
Artificial Sequence Novel Sequence 8 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Lys 1 5 10 9 11 PRT Artificial Sequence Novel Sequence
9 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 10 15 PRT
Artificial Sequence Novel Sequence 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 11 9 PRT Artificial
Sequence Novel Sequence 11 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
12 9 PRT Artificial Sequence Novel Sequence 12 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 13 15 PRT Artificial Sequence Novel Sequence 13
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 14 15 PRT Artificial Sequence Novel Sequence 14 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 15 9 PRT
Artificial Sequence Novel Sequence 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Lys 1 5 16 9 PRT Artificial Sequence Novel Sequence 16 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 17 16 PRT Artificial Sequence Novel
Sequence 17 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 18 16 PRT Artificial Sequence Novel Sequence 18
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 19 10 PRT Artificial Sequence Novel Sequence 19 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 20 10 PRT Artificial Sequence
Novel Sequence 20 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 21
15 PRT Artificial Sequence Novel Sequence 21 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 22 29 PRT
Artificial Sequence Novel Sequence 22 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Lys
Lys Lys Lys Lys Lys Lys Lys Lys 20 25 23 21 PRT Artificial Sequence
Novel Sequence 23 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Lys 20 24 20 PRT
Artificial Sequence Novel Sequence 24 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 25
21 PRT Artificial Sequence Novel Sequence 25 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Xaa Lys 20 26 20 PRT Artificial Sequence Novel Sequence 26 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa 20 27 21 PRT Artificial Sequence Novel Sequence 27
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Lys 20 28 12 PRT Artificial Sequence Novel
Sequence 28 Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10
29 12 PRT Artificial Sequence Novel Sequence 29 Xaa Xaa Xaa Xaa Ala
Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 30 26 DNA Artificial Sequence
Novel Sequence 30 aaaaagaaaa gtcgacaaaa agaaaa 26 31 20 DNA
Artificial Sequence Novel Sequence 31 aaaaaaaaaa tttttttttt 20 32
38 DNA Artificial Sequence Novel Sequence 32 gtcgacaaaa agaaaagtcg
acttttcttt ttgtcgac 38 33 20 DNA Artificial Sequence Novel Sequence
33 tttttttnnn nnnttttttt 20 34 16 DNA Artificial Sequence Novel
Sequence 34 gatccaaaaa aaaaag 16 35 16 DNA Artificial Sequence
Novel Sequence 35 gatccttttt tttttg 16 36 16 DNA Artificial
Sequence Novel Sequence 36 tcgacttttc tttttg 16 37 16 DNA
Artificial Sequence Novel Sequence 37 tcgacaaaaa gaaaag 16 38 16
DNA Artificial Sequence Novel Sequence 38 caagaagaaa actgca 16 39
16 DNA Artificial Sequence Novel Sequence 39 gttttcttct tctgca 16
40 9 PRT Artificial Sequence Novel Sequence 40 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Lys 1 5 41 5 PRT Artificial Sequence Novel Sequence 41
Xaa Xaa Xaa Xaa Xaa 1 5 42 196 DNA Artificial Sequence Novel
Sequence 42 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaatt
tttttttttt tttttttttt 120 tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt tttttttttt 180 tttttttttt tttttt 196 43 10
DNA Artificial Sequence Novel Sequence 43 aaaagaaaaa 10 44 20 DNA
Artificial Sequence Novel Sequence 44 aaaagaagaa ttcttctttt 20 45 8
PRT Artificial Sequence Novel Sequence 45 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Lys 1 5 46 50 DNA Artificial Sequence Novel Sequence 46
aaacagggca ggaaacagca tattttcttt taaaattagc aggaagatgg 50 47 15 DNA
Artificial Sequence Novel Sequence 47 tagttgtgac gtaca 15 48 11 PRT
Artificial Sequence Novel Sequence 48 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Lys 1 5 10 49 11 PRT Artificial Sequence Novel Sequence
49 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 50 16 DNA
Artificial Sequence Novel Sequence 50 caacttgata ttaata 16 51 8 PRT
Artificial Sequence Novel Sequence 51 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Lys 1 5 52 15 DNA Artificial Sequence Novel Sequence 52 acatgcagtg
ttgat 15 53 11 PRT Artificial Sequence Novel Sequence 53 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 54 10 DNA Artificial
Sequence Novel Sequence 54 aaaaggagag 10 55 10 DNA Artificial
Sequence Novel Sequence 55 gagaggaaaa 10 56 11 PRT Artificial
Sequence Novel Sequence 56 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Lys 1 5 10 57 10 DNA Artificial Sequence Novel Sequence 57
aagaagaaaa 10 58 10 DNA Artificial Sequence Novel Sequence 58
aaaaagaaaa 10 59 10 DNA Artificial Sequence Novel Sequence 59
aaaaaaaaaa 10 60 22 DNA Artificial Sequence Novel Sequence 60
ggatccaaaa aaaaaaggat cc 22 61 22 DNA Artificial Sequence Novel
Sequence 61 ggatcctttt ttttttggat cc 22 62 24 DNA Artificial
Sequence Novel Sequence 62 ctgcagaaga agaaaactgg gcag 24 63 22 DNA
Artificial Sequence Novel Sequence 63 ctgcagtttt cttcttctgc ag 22
64 10 DNA Artificial Sequence Novel Sequence 64 ttttcttctt 10 65 10
DNA Artificial Sequence Novel Sequence 65 aagaagaaaa 10 66 10 DNA
Artificial Sequence Novel Sequence 66 ttcttctttt 10 67 9 DNA
Artificial Sequence Novel Sequence 67 aaaagaaaa 9 68 16 DNA
Artificial Sequence Novel Sequence 68 gggggggaaa aaaaaa 16 69 10
DNA Artificial Sequence Novel Sequence 69 cccttcctcc 10 70 10 DNA
Artificial Sequence Novel Sequence 70 gggaaggagg 10 71 10 DNA
Artificial Sequence Novel Sequence 71 tctctctctt 10 72 10 DNA
Artificial Sequence Novel Sequence 72 agagagagaa 10 73 15 DNA
Artificial Sequence Novel Sequence 73 tgaacgtcac aacta 15 74 15 DNA
Artificial Sequence Novel Sequence 74 tagttgagtc gtaca 15 75 15 DNA
Artificial Sequence Novel Sequence 75 tgtacgtcac aacta 15 76 15 RNA
Artificial Sequence Novel Sequence 76 uaguugugac guaca 15 77 50 DNA
Artificial Sequence Novel Sequence 77 ccatcttcct gctaatttta
aaagaaaata tgctgtttcc tgccctgttt 50 78 9 DNA Artificial Sequence
Novel Sequence 78 aaaagaaaa 9 79 10 DNA Artificial Sequence Novel
Sequence 79 gtagatcact 10 80 10 DNA Artificial Sequence Novel
Sequence 80 agtcatctac 10 81 16 DNA Artificial Sequence Novel
Sequence 81 tgtacgtcac aactac 16 82 15 DNA Artificial Sequence
Novel Sequence 82 tagttgtgac gtaca 15 83 16 DNA Artificial Sequence
Novel Sequence 83 caacttgata ttaata 16 84 26 DNA Artificial
Sequence Novel Sequence 84 ttttccccct gtcgacccct cttttt 26 85 22
DNA Artificial Sequence Novel Sequence 85 cctaggtttt ttttttccta gg
22 86 9 DNA Artificial Sequence Novel Sequence 86 ttttctttt 9 87 16
DNA Artificial Sequence Novel Sequence 87 cccccccttt tttttt 16 88
16 DNA Artificial Sequence Novel Sequence 88 aaaaaaaaag gggggg 16
89 15 DNA Artificial Sequence Novel Sequence 89 acaugcagug uugau
15
* * * * *