U.S. patent application number 11/947705 was filed with the patent office on 2012-11-29 for oxocarbonamide peptide nucleic acids and methods of using same.
This patent application is currently assigned to LUMINEX CORPORATION. Invention is credited to James W. Jacobson, Ananda G. LUGADE.
Application Number | 20120302453 11/947705 |
Document ID | / |
Family ID | 39272510 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120302453 |
Kind Code |
A1 |
LUGADE; Ananda G. ; et
al. |
November 29, 2012 |
OXOCARBONAMIDE PEPTIDE NUCLEIC ACIDS AND METHODS OF USING SAME
Abstract
The present invention concerns oxocarbonamide peptide nucleic
acids (OxoPNAs). OxoPNAs provide increased stability, sensitivity,
and specificity as compared to their natural DNA and RNA
counterparts. The OxoPNA molecules of the present invention may be
employed in a wide range of applications, particularly in
applications involving hybridization. For example, OxoPNA probes
may be employed for the detection and functional analysis of
nucleic acid molecules, including miRNAs and other non-coding
RNAs.
Inventors: |
LUGADE; Ananda G.; (Austin,
TX) ; Jacobson; James W.; (Leander, TX) |
Assignee: |
LUMINEX CORPORATION
|
Family ID: |
39272510 |
Appl. No.: |
11/947705 |
Filed: |
November 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60868514 |
Dec 4, 2006 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/6.19;
436/501; 530/300 |
Current CPC
Class: |
C07K 14/003
20130101 |
Class at
Publication: |
506/9 ; 530/300;
436/501; 435/6.19 |
International
Class: |
C07K 2/00 20060101
C07K002/00; C40B 30/04 20060101 C40B030/04; C12Q 1/68 20060101
C12Q001/68; G01N 21/64 20060101 G01N021/64 |
Claims
1. An oxocarbonamide peptide nucleic acid having the formula (II):
##STR00011## wherein: n is an integer from 1 to 100; each L is
independently selected from the group consisting of nucleobases and
DNA intercalators; each Y is independently selected from the group
consisting of CH.sub.2CO, formula (IVa), formula (IVb), formula
(IVc), formula (IVd), formula (IVe), formula (IVf), formula (IVg),
and formula (IVh): ##STR00012## wherein each W is independently
selected from the group consisting of O and S, and M is selected
from the group consisting of no linker, benzene, substituted
benzene, formula (IVi), and formula (IVj): ##STR00013## and where
at least one Y is selected from the group consisting of formula
(IVa), formula (IVb), formula (IVc), formula (IVd), formula (IVe),
formula (IVf), formula (IVg), and formula (IVh); R' is selected
from the group consisting of hydrogen, alkyl, reporter ligands,
carboxylates, esters, alcohols, carbamides, aldehydes, amines,
amides, sulfur oxides, nitrogen oxides, and halides; and Z is
selected from the group consisting of CO.sub.2H, NH.sub.2, and
SH.
2. The oxocarbonamide peptide nucleic acid of claim 1, wherein n is
an integer from 5 to 30.
3. The oxocarbonamide peptide nucleic acid of claim 1, wherein at
least one Y is formula (IVa).
4. The oxocarbonamide peptide nucleic acid of claim 1, wherein at
least one Y is formula (IVe).
5. The oxocarbonamide peptide nucleic acid of claim 1, wherein at
least one Y is CH.sub.2CO.
6. The oxocarbonamide peptide nucleic acid of claim 1, wherein each
L is a naturally occurring nucleobase.
7. The oxocarbonamide peptide nucleic acid of claim 6, wherein the
naturally occurring nucleobase is selected from the group
consisting of adenine, thymine, guanine, and cytosine.
8. The oxocarbonamide peptide nucleic acid of claim 1, wherein at
least one L is a non-naturally occurring nucleobase.
9. The oxocarbonamide peptide nucleic acid of claim 1, wherein the
non-naturally occurring nucleobase is selected from the group
consisting of bromothymine, azaadenine, and azaguanine.
10. The oxocarbonamide peptide nucleic acid of claim 1, wherein R'
is an amine or amide.
11. The oxocarbonamide peptide nucleic acid of claim 1, further
defined as having the formula (IIa): ##STR00014## wherein W is O or
S.
12. The oxocarbonamide peptide nucleic acid of claim 11, wherein W
is O.
13-23. (canceled)
24. A method for detecting a target nucleic acid molecule,
comprising: (a) providing an oxocarbonamide peptide nucleic acid of
claim 1 comprising a sequence complementary to a sequence of a
target nucleic acid molecule; (b) contacting the oxocarbonamide
peptide nucleic acid with the target nucleic molecule under
conditions that allow the oxocarbonamide peptide nucleic acid to
hybridize with the target molecule; and (c) detecting the
hybridization.
25. The method of claim 24, wherein the target nucleic acid
molecule is a DNA molecule.
26. The method of claim 24, wherein the target nucleic acid
molecule is an RNA molecule.
27. The method of claim 26, wherein the RNA molecule is an miRNA
molecule.
28. The method of claim 24, wherein the oxocarbonamide peptide
nucleic acid is labeled.
29. The method of claim 24, wherein the target nucleic acid
molecule is labeled.
30. The method of claim 24, wherein the oxocarbonamide peptide
nucleic acid is covalently attached to a solid support.
31. The method of claim 30, wherein the solid support is a
microsphere.
32. The method of claim 31, wherein the microsphere is
fluorescently labeled.
33. A method for detecting one or more target nucleic acid
molecules in a multiplexed assay, comprising: (a) providing a
plurality of different oxocarbonamide peptide nucleic acids of
claim 1, wherein each different oxocarbonamide peptide nucleic acid
is covalently attached to a defined location on an array; (b)
contacting a sample comprising the one or more target nucleic acid
molecules with the array under conditions that allow the one or
more target nucleic acid molecules to hybridize to complementary
oxocarbonamide peptide nucleic acids on the array; and (c)
detecting the hybridization.
34. The method of claim 33, wherein the array comprises a plurality
of microspheres.
35. The method of claim 33, wherein the one or more target nucleic
acid molecules are miRNA molecules.
36. The method of claim 33, wherein the oxocarbonamide peptide
nucleic acids are between about 10 and about 60 nucleobases in
length.
37-40. (canceled)
Description
[0001] This application claims priority to U.S. Application No.
60/868,514, filed on Dec. 4, 2006, the entire disclosure of which
is incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to nucleic acid
probes useful in the detection and analysis of target nucleic acid
sequences. More particularly, the present invention concerns
nucleic acid probes wherein naturally occurring nucleobases or
other nucleobase-binding moieties are covalently bound to an
oxocarbonamide containing peptide backbone. In certain aspects, the
present invention concerns methods employing nucleic acid probes in
the detection and analysis of target nucleic acid sequences
including, for example, mRNAs, miRNAs, and siRNAs.
[0004] 2. Description of Related Art
[0005] A large number of small, non-coding RNAs have been
identified and designated as microRNAs (miRNAs) (Ke et al., 2003).
miRNAs have been shown to regulate gene expression at many levels,
representing a novel gene regulatory mechanism. Understanding this
RNA-based regulation will be useful to understand the complexity of
the genome in higher eukaryotes as well as understand the complex
gene regulatory networks.
[0006] miRNAs are 18-25 nucleotide (nt) RNAs that are processed
from longer endogenous hairpin transcripts by the enzymes Dicer and
Argonaute (Ambros et al., 2003, Grishok et al., 2001). To date more
than 4160 microRNAs have been identified in mammals, birds, fish,
worms, flies, plants, and viruses according to the miRNA registry
database release 9.0 in October 2006, hosted by Sanger Institute,
UK. Some miRNAs have multiple loci in the genome (Reinhart et al.,
2002) and may be arranged in tandem clusters (Lagos-Quintana et
al., 2001).
[0007] The first miRNAs to be discovered, lin-4 and let-7,
base-pair incompletely to repeated elements in the 3' untranslated
regions (UTRs) of other heterochrony genes, and regulate the
translation directly and negatively by antisense RNA-RNA
interaction (Lee et al., 1993; Reinhart et al., 2000). Some miRNAs
are thought to interact with target mRNAs by limited complementary
and suppressed translation as well (Lagos-Quintana et al., 2001;
Lee and Ambros, 2001). Perfect complementarity between miRNAs and
their target RNA may lead to target RNA degradation rather than
inhibit translation (Hutvagner and Zamore, 2002), which suggests
that the degree of complementarity determines function.
[0008] Several human diseases have been identified in which miRNAs
or their processing machinery might be implicated. One such disease
is spinal muscular atrophy (SMA), a pediatric neurodegenerative
disease caused by reduced protein levels or loss-of-function
mutations of the survival of motor neurons (SMN) gene (Paushkin et
al., 2002). Another disease linked to mi/siRNAs is fragile X mental
retardation (FXMR) caused by absence or mutations of the fragile X
mental retardation protein (FMRP) (Nelson et al., 2003). Poy et al.
(2004) concluded that miR-375 is a regulator of insulin secretion
and could constitute a novel pharmacological target for the
treatment of diabetes. Links between cancer and miRNAs have also
been described. For example, one study determined that two
different miRNA (miR15 and miR16) genes are clustered and located
within the deleted minimal region of the B-cell chronic lymphocytic
leukemia (B-CLL) tumor suppressor locus, and both genes are deleted
or down-regulated in the majority of CLL cases (Calin et al.,
2002).
[0009] RNA interference (RNAi), in which double-stranded RNA leads
to the degradation of any RNA that is homologous (Fire et al.,
1998), relies on a mechanism that probably evolved for protection
against viral attack and mobile genetic elements. One step in the
RNAi mechanism is the generation of short interfering RNAs
(siRNAs), double-stranded RNAs that are about 22 nt long. The
siRNAs lead to the degradation of homologous target RNA and the
production of more siRNAs against the same target RNA (Lipardi et
al., 2001; Zhang et al., 2002; Nykanen et al., 2001).
[0010] The involvement of short RNAs in gene regulation has
resulted in high interest among researchers in the discovery of
siRNAs, miRNAs, their targets and mechanism of action. However, the
detection and analysis of these small RNAs is not trivial. The size
and often low level of expression of miRNAs require the use of
sensitive analysis tools. The use of conventional quantitative
real-time PCR for monitoring expression of mature miRNAs is
excluded due to their small size. Most miRNA researchers use
Northern blot analysis combined with polyacrylamide gels to examine
expression of both the mature and pre-miRNAs (Reinhart et al.,
2000; Lagos-Quintana et al., 2001; Lee and Ambros, 2001). Primer
extension has also been used to detect the mature miRNA (Zeng and
Cullen, 2003). Disadvantages of all the gel-based assays (Northern
blotting, primer extension, RNase protection assays etc.) for
monitoring miRNA expression include low throughput and poor
sensitivity. Consequently, a large amount of total RNA per sample
is required for gel-based methods, which is not feasible when the
cell or tissue source is limited.
[0011] Microarrays are an alternative to Northern blot analysis for
analyzing miRNA expression. Krichevsky et al. (2003) used cDNA
microarrays to monitor the expression of miRNAs during neuronal
development; however, the mature miRNAs had to be separated from
the miRNA precursors using micro concentrators prior to microarray
hybridization. Liu et al (2004) developed a microarray for
expression profiling of 245 human and mouse miRNAs using 40-mer DNA
oligonucleotide capture probes. Thomson et al. (2004) described the
development of a oligonucleotide microarray platform for expression
profiling of 124 mammalian miRNAs using oligonucleotide capture
probes complementary to the mature microRNAs.
[0012] Although microarrays can provide high throughput, the
disadvantages of DNA-based oligonucleotide arrays may include: the
requirement of high concentrations of labeled input target RNA for
efficient hybridization and signal generation, low sensitivity for
rare and low-abundant miRNAs, and the necessity for post-array
validation using more sensitive assays.
[0013] A PCR-based approach has also been used to determine the
expression levels of mature miRNAs (Grad et al., 2003). However,
this method is cumbersome for routine miRNA expression profiling,
since it involves gel isolation of small RNAs and ligation to
linker oligonucleotides. Schmittgen et al. (2004) described an
alternative method to Northern blot analysis, in which real-time
PCR assays were used to quantify the expression of miRNA
precursors. The disadvantage of this method, however, is that it
only allows quantification of the precursor miRNAs, which does not
necessarily reflect the expression levels of mature miRNAs.
[0014] Many limitations of DNA probes for the detection of short
nucleotide targets have been overcome by using locked nucleic acid
(LNA) based probes or peptide nucleic acid (PNA) based probes. The
use of LNAs and PNAs in oligonucleotide probes has been shown to
increase sensitivity and selectivity for small RNA targets compared
to their DNA-probe counterparts (see e.g., Valoczi et al., 2004).
Nevertheless, additional compositions and methods are needed to
increase the sensitivity and specificity of oligonucleotide
sequences for the detection and analysis of miRNAs and other small
RNAs, as well as for use in disease diagnostics and for
antisense-based therapies.
[0015] The present invention addresses these needs by providing
novel oligonucleotide compositions for the accurate, sensitive, and
specific detection and functional analysis of miRNAs and other
non-coding RNAs. The compositions of the present invention will
also be useful as biomarkers for disease diagnostics as well as for
antisense-based intervention targeted against disease-associated
miRNAs and other non-coding RNAs.
SUMMARY OF THE INVENTION
[0016] The present invention provides a novel class of compounds
that bind complementary DNA and RNA strands. The compounds of the
invention generally comprise ligands linked to a oxocarbon acid
amide modified peptide backbone. Non-limiting examples of ligands
include thymine, cytosine, adenine, guanine, uracil, inosine,
5-methylcytosine, thiouracil, bromothymine, azaadenine, or
azaguanine. Representative oxocarbon acid amides
("oxocarbonamides") include deltic acid amide, thio-deltic acid
amide, squaric acid amide, thio-squaric acid amide, croconic acid
amide, thio-croconic acid amide, rhodizonic acid amide, and
thio-rhodizonic acid amide.
[0017] In one embodiment, the present invention provides
oxocarbonamide peptide nucleic acids having the formula (I):
##STR00001##
[0018] wherein:
[0019] n is at least 1;
[0020] each of L.sup.1-L.sup.n is independently selected from the
group consisting of heteroatom substituted aryls, naturally
occurring nucleobases, non-naturally occurring nucleobases,
nucleobase binding groups, hydrogen, hydroxy,
(C.sub.1-C.sub.4)alkanoyl, aromatic moieties, and reporter ligands,
wherein at least one of L.sup.1-L.sup.n is a naturally occurring
nucleobase, non-naturally occurring nucleobase, nucleobase binding
group, or DNA intercalator;
[0021] each of X.sup.1-X.sup.n is independently selected from the
group consisting of R.sub.1NH and NHR.sub.2, wherein R.sub.1 and
R.sub.2 are independently selected from the group consisting of H,
CH.sub.2NH.sub.2, (CH.sub.2).sub.(1-10)--NH.sub.2,
(CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.(1-10)NH.sub.2,
(CH.sub.2).sub.2(OCH.sub.2CH.sub.2).sub.(1-10)CO.sub.2H,
(CH.sub.2).sub.(1-10);
[0022] each of Y.sup.1-Y.sup.n is independently selected from the
group consisting of CH.sub.2CO, formula (IVa), formula (IVb),
formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula
(IVg), and formula (IVh), and where at least one Y is formula
(IVa), formula (Nb), formula (IVc), formula (Nd), formula (We),
formula (IVf), formula (IVg), and formula (IVh),
##STR00002##
wherein each W is independently selected from the group consisting
of O and S, and M is selected from the group consisting of no
linker, benzene, substituted benzene, formula (IVi), and formula
(IVj);
##STR00003##
[0023] Q is selected from the group consisting of CO.sub.2H,
CONR'R'', SO.sub.3H, NH.sub.2, SH, or SO.sub.2NR'R'' or an
activated derivative of CO.sub.2H or SO.sub.2H; and
[0024] Z is selected from the group consisting of CO.sub.2H,
CONR'R'', SO.sub.3H, NH.sub.2, SH, SO.sub.2NR'R'', NHR''R''', or
NR'''COR'''', where R', R'', R''', and R'''' are independently
selected from the group consisting of hydrogen, alkyl, amino
protecting groups, reporter ligands, heteroatom substituted acyls,
carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteratom
substituted alkyls, carbamides, aldehydes, amines, amides, sulfur
oxides, nitrogen oxides, halides, reporter ligands, intercalators,
chelators, peptides, proteins, carbohydrates, lipids, steroids,
oligonucleotides, and soluble and non-soluble polymers.
[0025] The substituted benzene may be substituted with, for
example, alkyl, amine, substituted amine, amide, branched amine,
PEG, etc.
[0026] In certain embodiments, the present invention provides
oxocarbonamide peptide nucleic acids having the formula (II):
##STR00004##
[0027] wherein:
[0028] each L is independently selected from the group consisting
of heteroatom substituted aryls, naturally occurring nucleobases,
non-naturally occurring nucleobases, nucleobase binding groups,
hydrogen, hydroxy, (C.sub.1-C.sub.4)alkanoyl, aromatic moieties,
and reporter ligands, wherein at least one of L.sup.1-L.sup.n is a
heteroatom substituted acyl, naturally occurring nucleobase,
non-naturally occurring nucleobase, nucleobase binding group, or
DNA intercalator;
[0029] each Y is independently selected from the group consisting
of CH.sub.2CO, formula (IVa), formula (IVb), formula (IVc), formula
(IVd), formula (IVe), formula (IVf), formula (IVg), and formula
(IVh), and where at least one Y is formula (IVa), formula (IVb),
formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula
(IVg), and formula (IVh);
[0030] R' is selected from the group consisting of hydrogen, alkyl,
amino protecting groups, reporter ligands, heteroatom substituted
acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl,
heteroatom substituted alkyl, heteroatom substituted acyl,
carbamides, aldehydes, amines, amides, sulfur oxides, nitrogen
oxides, halides, reporter ligands, intercalators, chelators,
peptides, proteins, carbohydrates, lipids, steroids,
oligonucleotides, and soluble and non-soluble polymers;
[0031] Z is selected from the group consisting of CO.sub.2H,
CONR'R'', SO.sub.3H, NH.sub.2, SH, SO.sub.2NR'R'', NHR''R''', or
NR'''COR'''', where R', R'', R''', and R'''' are independently
selected from the group consisting of hydrogen, alkyl, amino
protecting groups, reporter ligands, heteroatom substituted acyl,
carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom
substituted alkyl, heteroatom substituted acyl, carbamides,
aldehydes, amines, amides, sulfur oxides, nitrogen oxides, halides,
reporter ligands, intercalators, chelators, peptides, proteins,
carbohydrates, lipids, steroids, oligonucleotides, and soluble and
non-soluble polymers; and
[0032] n is an integer from 1 to 100.
[0033] In certain embodiments, the present invention provides an
oxocarbonamide peptide nucleic acid having the formula (II):
[0034] wherein:
[0035] n is an integer from 1 to 100;
[0036] each L is independently selected from the group consisting
of heteroatom substituted aryl, naturally occurring nucleobases,
non-naturally occurring nucleobases, nucleobase binding groups, and
DNA intercalators;
[0037] each Y is independently selected from the group consisting
of CH.sub.2CO, formula (IVa), formula (IVb), formula (IVc), formula
(IVd), formula (IVe), formula (IVf), formula (IVg), and formula
(IVh), and where at least one Y is formula (IVa), formula (IVb),
formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula
(IVg), and formula (IVh);
[0038] R' is selected from the group consisting of hydrogen, alkyl,
reporter ligands, heteroatom substituted acyl, carboxylates,
esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted
alkyl, carbamides, aldehydes, amines, amides, sulfur oxides,
nitrogen oxides, and halides; and
[0039] Z is selected from the group consisting of CO.sub.2H,
NH.sub.2, and SH.
[0040] In particular embodiments, the present invention provides an
oxocarbonamide peptide nucleic acid having the formula (IIa):
##STR00005##
[0041] wherein W is O or S;
[0042] each L is independently selected from the group consisting
of heteroatom substituted aryl, naturally occurring nucleobases,
non-naturally occurring nucleobases, nucleobase binding groups,
hydrogen, hydroxy, (C.sub.1-C.sub.4)alkanoyl, aromatic moieties,
and reporter ligands, wherein at least one of L.sup.1-L.sup.n is a
heteroatom substituted aryl, naturally occurring nucleobase,
non-naturally occurring nucleobase, nucleobase binding group, or
DNA intercalator;
[0043] R' is selected from the group consisting of hydrogen, alkyl,
amino protecting groups, reporter ligands, heteroatom substituted
acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl,
heteroatom substituted alkyl, carbamides, aldehydes, amines,
heteroatom substituted amide, sulfur oxides, nitrogen oxides,
halides, reporter ligands, intercalators, chelators, peptides,
proteins, carbohydrates, lipids, steroids, oligonucleotides, and
soluble and non-soluble polymers;
[0044] Z is selected from the group consisting of CO.sub.2H,
CONR'R'', SO.sub.3H, NH.sub.2, SH, SO.sub.2NR'R'', NHR''R''', or
NR'''COR'''', where R', R'', R''', and R'''' are independently
selected from the group consisting of hydrogen, alkyl, amino
protecting groups, reporter ligands, heteroatom substituted acyl,
carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom
substituted alkyl, carbamides, aldehydes, amines, amides, sulfur
oxides, nitrogen oxides, halides, reporter ligands, intercalators,
chelators, peptides, proteins, carbohydrates, lipids, steroids,
oligonucleotides, and soluble and non-soluble polymers; and
[0045] n is an integer from 1 to 100.
[0046] In certain embodiments, the present invention provides an
oxocarbonamide peptide nucleic acid having the formula (III):
##STR00006##
[0047] wherein:
[0048] each L is independently selected from the group consisting
of heteroatom substituted aryl, naturally occurring nucleobases,
non-naturally occurring nucleobases, nucleobase binding groups,
hydrogen, hydroxy, (C.sub.1-C.sub.4)alkanoyl, aromatic moieties,
and reporter ligands, wherein at least one of L.sup.1-L.sup.n is a
heteroatom substituted aryl, naturally occurring nucleobase,
non-naturally occurring nucleobase, nucleobase binding group, or
DNA intercalator;
[0049] each Y is independently selected from the group consisting
of CH.sub.2CO, formula (IVa), formula (IVb), formula (IVc), formula
(IVd), formula (IVe), formula (IVf), formula (IVg), and formula
(IVh), and where at least one Y is formula (IVa), formula (IVb),
formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula
(IVg), and formula (IVh);
[0050] R' is selected from the group consisting of hydrogen, alkyl,
amino protecting groups, reporter ligands, heteroatom substituted
acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl,
heteroatom substituted alkyl, carbamides, aldehydes, amines,
amides, sulfur oxides, nitrogen oxides, halides, reporter ligands,
intercalators, chelators, peptides, proteins, carbohydrates,
lipids, steroids, oligonucleotides, and soluble and non-soluble
polymers;
[0051] R.sub.3 is selected from the group consisting of H,
CH.sub.3, and cationic polymers;
[0052] Z is selected from the group consisting of CO.sub.2H,
CONR'R'', SO.sub.3H, NH.sub.2, SH, SO.sub.2NR'R'', NHR''R''', or
NR'''COR'''', 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,
oligonucleotides, and soluble and non-soluble polymers; and
[0053] n is an integer from 1 to 100. In certain aspects of the
invention, the cationic polymer is a branched amine such as, for
example, a polyamidoamine (PMAM) dendrimer or a polyethyleneimine
(PEI). In some aspects of the invention, the cationic polymer is a
polyammonium group (e.g., (CH.sub.2).sub.nNR.sub.3). Due to their
structure and charge, cationic polymers are useful nucleic acid
transfection agents and drug carriers.
[0054] In certain embodiments, the present invention provides an
oxocarbonamide peptide nucleic acid having the formula (III):
[0055] wherein:
[0056] n is an integer from 1 to 100;
[0057] each L is independently selected from the group consisting
of naturally occurring nucleobases, non-naturally occurring
nucleobases, nucleobase binding groups, and DNA intercalators;
[0058] each Y is independently selected from the group consisting
of CH.sub.2CO, formula (IVa), formula (IVb), formula (IVc), formula
(IVd), formula (We), formula (IVf), formula (IVg), and formula
(IVh), and where at least one Y is formula (IVa), formula (IVb),
formula (IVc), formula (IVd), formula (IVe), formula (IVf), formula
(IVg), and formula (IVh);
[0059] R' is selected from the group consisting of hydrogen, alkyl,
reporter ligands, heteroatom substituted acyl, carboxylates,
esters, alcohols, alkoxy, hydroxy alkyl, heteroatom substituted
alkyl, heteroatom substituted acyls, carbamides, aldehydes, amines,
sulfur oxides, nitrogen oxides, and halides;
[0060] R.sub.3 is selected from the group consisting of H,
CH.sub.3, and cationic polymers; and
[0061] Z is selected from the group consisting of CO.sub.2H,
NH.sub.2, and SH.
[0062] In particular embodiments, the present invention provides an
oxocarbonamide peptide nucleic acid having the formula (IIIa):
##STR00007##
[0063] wherein W is O or S;
[0064] each L is independently selected from the group consisting
of heteroatom substituted aryl, naturally occurring nucleobases,
non-naturally occurring nucleobases, nucleobase binding groups,
hydrogen, hydroxy, (C.sub.1-C.sub.4)alkanoyl, aromatic moieties,
and reporter ligands, wherein at least one of L.sup.1-L.sup.n is a
naturally occurring nucleobase, non-naturally occurring nucleobase,
nucleobase binding group, or DNA intercalator;
[0065] R' is selected from the group consisting of hydrogen, alkyl,
amino protecting groups, reporter ligands, heteroatom substituted
acyl, carboxylates, esters, alcohols, alkoxy, hydroxy alkyl,
heteroatom substituted alkyl, carbamides, aldehydes, amines, sulfur
oxides, nitrogen oxides, halides, reporter ligands, intercalators,
chelators, peptides, proteins, carbohydrates, lipids, steroids,
oligonucleotides, and soluble and non-soluble polymers;
[0066] R.sub.3 is selected from the group consisting of H,
CH.sub.3, and cationic polymers;
[0067] Z is selected from the group consisting of CO.sub.2H,
CONR'R'', SO.sub.3H, NH.sub.2, SH, SO.sub.2NR'R'', NHR''R''', or
NR'''COR'''', where R', R'', R''', and R'''' are independently
selected from the group consisting of hydrogen, alkyl, amino
protecting groups, reporter ligands, heteroatom substituted acyl,
carboxylates, esters, alcohols, alkoxy, hydroxy alkyl, heteroatom
substituted alkyl, carbamides, aldehydes, amines, amides, sulfur
oxides, nitrogen oxides, halides, reporter ligands, intercalators,
chelators, peptides, proteins, carbohydrates, lipids, steroids,
oligonucleotides, and soluble and non-soluble polymers; and
[0068] n is an integer from 1 to 100.
[0069] In certain aspects of the invention, n may be an integer
from 8 to 60, 10 to 50, 15 to 30, or 18 to 25. In some embodiments,
n is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30, or any range derivable therein.
[0070] In certain aspects of the invention, no Y is CH.sub.2CO. In
some aspects of the invention all Ys are formula (IVa), formula
(IVe), or a combination of formula (IVa) and formula (IVe).
[0071] The oligonucleotide analogs of the present invention may be
employed in a wide range of applications, particularly in
applications involving hybridization. In one embodiment, the
present invention provides a method for detecting a target nucleic
acid molecule, comprising: (a) providing an oxocarbonamide peptide
nucleic acid comprising a sequence complementary to a sequence of a
target nucleic acid molecule; (b) contacting the oxocarbonamide
peptide nucleic acid with the target nucleic molecule under
conditions that allow the oxocarbonamide peptide nucleic acid to
hybridize with the target molecule; and (c) detecting the
hybridization. The target nucleic acid molecule may be, for
example, a DNA or an RNA molecule. The RNA molecule may be, for
example, an mRNA, rRNA, tRNA, miRNA, or siRNA.
[0072] To facilitate the detection of a target molecule, one or
both of the oxocarbonamide peptide nucleic acid probe or the target
molecule may be labeled. A number of different labels may be used
in the present invention such as fluorophores, chromophores,
radiophores, enzymatic tags, antibodies, chemiluminescence,
electroluminescence, metal nanoparticles, quantum dots, magnetic
particles, and affinity labels. One of skill in the art will
recognize that these and other labels not mentioned herein can be
used with success in this invention.
[0073] Examples of affinity labels include, but are not limited to
the following: an antibody, an antibody fragment, a receptor
protein, a hormone, biotin, DNP, a molecular imprint, or any
polypeptide/protein molecule that binds to an affinity label.
[0074] Examples of enzyme tags include enzymes such as urease,
alkaline phosphatase or peroxidase to mention a few. Colorimetric
indicator substrates can be employed to provide a detection means
visible to the human eye or spectrophotometrically, to identify
specific hybridization with complementary nucleic acid-containing
samples. All of these examples are generally known in the art and
the skilled artisan will recognize that the invention is not
limited to the examples described above.
[0075] Examples of fluorophores include, but are not limited to the
following: all of the Alexa Fluor.RTM. dyes, AMCA, BODIPY.RTM.
630/650, BODIPY.RTM. 650/665, BODIPY.RTM.-FL, BODIPY.RTM.-R6G,
BODIPY.RTM.-TMR, BODIPY.RTM.-TRX, Cascade Blue.RTM., CyDyes.TM.,
including but not limited to Cy2.TM., Cy3.TM., and Cy5.TM., DNA
intercalating dyes, 6-FAM.TM., Fluorescein, HEX.TM., 6-JOE, Oregon
Green.RTM. 488, Oregon Green.RTM. 500, Oregon Green.RTM. 514,
Pacific Blue.TM., REG, phycobilliproteins including, but not
limited to, phycoerythrin and allophycocyanin, Rhodamine Green.TM.,
Rhodamine Red.TM., ROX.TM., TAMRA.TM., TET.TM.,
Tetramethylrhodamine, and Texas Red.RTM..
[0076] In certain aspects of the invention, the oxocarbonamide
peptide nucleic acid probe or the target molecule is immobilized on
a solid support. Non-limiting examples of solid supports include:
nitrocellulose, nylon membrane, glass, activated quartz, activated
glass, polyvinylidene difluoride (PVDF) membrane, polystyrene
substrates, polyacrylamide-based substrate, other polymers,
copolymers, or crosslinked polymers such as poly(vinyl chloride),
poly(methyl methacrylate), poly(dimethyl siloxane), photopolymers
(which contain photoreactive species such as nitrenes, carbenes and
ketyl radicals capable of forming covalent links with target
molecules). A solid support may be in the form of, for example, a
bead, a column, or a chip.
[0077] In one embodiment, the present invention provides an array
comprising a plurality of oxocarbonamide peptide nucleic acid
probes immobilized on a solid support. In particular embodiments,
the solid support is a chip or a bead. In certain aspects of the
invention, the array comprises at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000,
10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or
100000, or any range derivable therein, different oxocarbonamide
peptide oligonucleotide probes.
[0078] In certain embodiments, the present invention provides a
method for detecting one or more target nucleic acid molecules in a
multiplexed assay, comprising: (a) providing a plurality of
different oxocarbonamide peptide nucleic acids, wherein each
different oxocarbonamide peptide nucleic acid is covalently
attached to a defined location on an array; (b) contacting a sample
comprising the one or more target nucleic acid molecules with the
array under conditions that allow the one or more target nucleic
acid molecules to hybridize to complementary oxocarbonamide peptide
nucleic acids on the array; and (c) detecting the hybridization. In
particular embodiments, the plurality of nucleic acid molecules is
a plurality of miRNA molecules. In certain embodiments, the array
comprises a plurality of fluorescently encoded microspheres
("beads").
[0079] In another aspect, the invention provides a method for
amplifying a target nucleic acid molecule. The method involves (a)
incubating a first oxocarbonamide peptide nucleic acid of the
invention with a target molecule under conditions that allow the
first oxocarbonamide peptide nucleic acid to bind the target
molecule; and (b) extending the first nucleic acid with the target
molecule as a template. The method may further comprise contacting
the target molecule with a second oxocarbonamide peptide nucleic
acid that binds to a different region of the target molecule than
the first oxocarbonamide peptide nucleic acid. In various
embodiments, the sequence of the target molecule is known or
unknown.
[0080] In certain aspects, the association constant (K.sub.a) of
the oxocarbonamide peptide nucleic acid toward a complementary
target molecule is higher than the association constant of the
complementary strands of the double stranded target molecule. In
some embodiments, the melting temperature of a duplex between the
oxocarbonamide peptide nucleic acid and a complementary target
molecule is higher than the melting temperature of the
complementary strands of the double stranded target molecule.
[0081] In one aspect, the present invention provides pharmaceutical
composition comprising an oxocarbonamide peptide nucleic acid for
treatment of a disease curable by an antisense technology. In one
embodiment, the invention provides a method for inhibiting the
expression of a target nucleic acid in a cell. The method comprises
introducing into the cell a oxocarbonamide peptide nucleic acid of
the invention in an amount sufficient to specifically attenuate
expression of the target nucleic acid. The introduced
oxocarbonamide peptide nucleic acid has a nucleotide sequence that
is complementary to a region of the target nucleic acid sequence.
In another aspect, the invention provides a method for preventing,
stabilizing, or treating a disease, disorder, or condition
associated with a target nucleic acid in a mammal. This method
comprises introducing into the mammal oxocarbonamide peptide
nucleic acid of the invention in an amount sufficient to
specifically attenuate expression of the target nucleic acid,
wherein the introduced oxocarbonamide peptide nucleic acid has a
nucleotide sequence that is complementary to a region of the target
nucleic acid. In particular embodiments, the oxocarbonamide peptide
nucleic acid has a nucleotide sequence that is complementary to a
region of between about 10 to about 100, about 10 to about 50,
about 10 to about 30, about 15 to about 30, or about 17 to about
25, nucleotides of the target nucleic acid sequence. In some
embodiments, the introduced oxocarbonamide peptide nucleic acid is
single stranded or double stranded. Where the oxocarbonamide
peptide nucleic acid is double stranded, both strands may be
oxocarbonamide peptide nucleic acids or one strand may be an
oxocarbonamide peptide nucleic acid and the other strand may be a
DNA, RNA, or an oligonucleotide analog such as a PNA or LNA.
[0082] Exemplary mammals that can be treated using the methods of
the invention include humans, primates, animals of veterinary
interest (e.g., cows, sheep, goats, buffalos, and horses), and
domestic pets (e.g., dogs and cats). Exemplary cells in which one
or more target genes can be silenced using the methods of the
invention include invertebrate, plant, bacteria, yeast, and
vertebrate (e.g., mammalian) cells.
[0083] It is contemplated that any method or composition described
herein can be implemented with respect to any other method or
composition described herein.
[0084] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0085] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0086] Following long-standing patent law, the words "a" and "an,"
when used in conjunction with the word "comprising" in the claims
or specification, denotes one or more, unless specifically
noted.
[0087] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0089] FIG. 1 shows the oxocarbon acids: deltic acid, thio-deltic
acid, squaric acid, thio-squaric acid, croconic acid, thio-croconic
acid, rhodizonic acid, and thio-rhodizonic acid.
[0090] FIG. 2 shows abasic monomers of squaric acid amides and
reactive derivatives of squaric acid.
[0091] FIG. 3 shows squaric acid amide peptide nucleic acid
(SquarPNA) monomers.
[0092] FIG. 4 shows one scheme for the solid phase synthesis of
OxoPNAs.
[0093] FIG. 5 shows one scheme for the synthesis of morpholino
nucleosides for incorporation in OxoPNAs. Morpholino nucleotide
synthesis may be performed according to the method of Girault et
al. (1996) (incorporated by reference).
[0094] FIGS. 6A, 6B, and 6C. FIG. 6A shows one scheme for the
synthesis of peptide nucleic acid monomers according to the method
of Howarth et al. (1997). FIG. 6B shows examples of additional
peptide nucleic acid monomers that may be prepared according to the
scheme shown in FIG. 6A. FIG. 6C shows two examples of peptide
nucleic acid monomers modified with squaric acid.
[0095] FIGS. 7A and 7B. FIG. 7A shows a scheme for the synthesis of
a .beta.-aminoalainine monomer modified with a nucleobase according
to the protocol of Fujii et al. (1998). FIG. 7B shows the
.beta.-aminoalainine nucleoside monomer modified with squaric
acid.
[0096] FIG. 8 shows one scheme for the solid phase synthesis of
OxoPNAs using reactive derivatives of squaric acid. Although
difluoro squaric acid is used in this example, other reactive
halogen derivatives, such as dichloro squaric acid, could be used.
Dilkyl ester derivatives of squaric acid could also be used.
[0097] FIG. 9 shows abasic dimers of squaric acid amides.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
A. The Present Invention
[0098] The present invention provides novel oxocarbon acid
incorporated peptide nucleic acids (OxoPNAs) that provide increased
stability, sensitivity, and specificity as compared to their
natural DNA and RNA counterparts. The OxoPNAs of the invention
generally comprise nucleobases linked to an oxocarbon acid amide
incorporated peptide backbone. The nucleobases may include any of
the four main naturally occurring DNA bases (i.e., thymine,
cytosine, adenine, or guanine) or other naturally occurring
nucleobases (e.g., inosine, uracil, 5-methylcytosine, or
thiouracil) or artificial bases (e.g., bromothymine, azaadenines,
or azaguanines, etc.) attached to a peptide backbone through a
suitable linker. The oxocarbon acid may be squaric acid, deltic
acid, croconic acid, rhodizonic acid, or their corresponding
thioxocarbon acids.
[0099] The present invention also provides a variety of methods
employing the OxoPNAs of the present invention. As described
herein, the oligonucleotide analogs of the present invention may be
employed in a wide range of applications, particularly in
applications involving hybridization. For example, the present
invention provides methods for the detection and functional
analysis of nucleic acid molecules, including miRNAs and other
non-coding RNAs. In addition, the present invention also provides
methods for antisense-based intervention targeted against
disease-associated nucleic acid molecules.
B. Oxocarbon Acid Amide Modified PNAs
[0100] The present invention provides novel oxocarbon acid amide
(i.e., "oxocarbonamide") incorporated peptide oligonucleotides.
Oxocarbon acids are a class of organic compounds that are vinylogs
of carboxylic acids, that is the OH and CO groups are joined
through a vinylic unsaturation forming a cyclic non-aromatic ring.
Furthermore, the carbon atoms not involved in the acidic moiety are
substituted by oxygen and are present as carbonyl or hydroxy
functions. Cyclic oxocarbon compounds have the general formula
C.sub.xO.sub.x, wherein x.gtoreq.3. Examples of oxocarbon acids are
deltic acid, squaric acid, croconic acid, and rhodizonic acid (FIG.
1).
[0101] Squaric acid derivatives have been shown to function as
amino acid-like analogs. For example, Porter et al. demonstrated
that a squaric acid derivative of the thioproline CT5219, a small
molecule VLA-4 antagonist, was also a potent VLA-4 antagonist and
had an improved pharmokinetic profile compared to CT5219 (Porter et
al., 2002).
[0102] The squaryl group has been evaluated as a mimic of the
phosphate group in modified oligodeoxynucleotides (Sato et al.,
2002). Squaric acid is a dibasic acid with two acidic hydroxyl
groups and two carbonyl groups. Nucleophilic substitution of the
squaric acid esters with amines gives the corresponding diamides.
Sato et al. reported the synthesis of oligonucleotide analogues
containing a single squaryldiamide internucleotide linkage between
two thymidines (TsqT). The structure of the TsqT as reported by
Sato et al. is as follows:
##STR00008##
[0103] The present invention employs squaryldiamides, and other
oxocarbonamides, in the design of modified peptide nucleic acids.
In one embodiment, a squaryldiamide modified peptide nucleic acid
of the present invention has the structure:
##STR00009##
[0104] A "peptide nucleic acid," also known as a "PNA,"
"peptide-based nucleic acid analog," or "PENAM," generally has
enhanced sequence specificity, binding properties, and resistance
to enzymatic degradation in comparison to molecules such as DNA and
RNA (Egholm et al., 1993; PCT/EP/01219). A PNA typically comprises
one or more nucleotides or nucleosides that comprise a nucleobase
moiety, a nucleobase linker moiety that is not a 5-carbon sugar,
and/or a backbone moiety that is not a phosphate backbone moiety.
Examples of nucleobase linker moieties described for PNAs include
aza nitrogen atoms, amido and/or ureido tethers (see for example,
U.S. Pat. No. 5,539,082). Examples of backbone moieties described
for PNAs include an aminoethylglycine, polyamide, polyethyl,
polythioamide, polysulfinamide or polysulfonamide backbone moiety.
Various PNAs have been described in U.S. Pat. Nos. 5,786,461,
5,891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331,
5,539,082, and WO 92/20702, each of which is incorporated herein by
reference.
[0105] Other nucleotide analogs include, for example, a locked
nucleic acid or "LNA." An LNA monomer is a bi-cyclic compound that
is structurally similar to RNA nucleosides. LNAs have a furanose
conformation that is restricted by a methylene linker that connects
the 2'-O position to the 4'-C position, as described in Koshkin et
al., 1998a and 1998b and Wahlestedt et al., 2000.
[0106] Typical structures of a deoxyribonucleic acid (DNA), locked
nucleic acid (LNA), and a peptide nucleic acid (PNA) are:
##STR00010##
[0107] The preparation of PNA oligomers may be based on standard
solid phase peptide synthesis protocols such as those disclosed in
WO 92/20702; U.S. Pat. No. 5,539,082; and Mc Cairn et al.
(2006).
[0108] Solid phase synthesis is also a convenient strategy for
making the OxoPNAs of the present invention. Examples of reactive
derivatives of squaric acid and OxoPNA monomers, which may be used
in the synthesis of OxoPNAs are illustrated in FIGS. 1 and 2,
respectively. Examples of synthesis schemes for OxoPNAs are
provided in FIGS. 4 to 8. Although the exemplary synthesis schemes
in FIGS. 4 to 8 show squaric acid amides, it will be understood by
those in the art that other oxocarbon acid amides could also be
used to synthesize OxoPNAs. The synthesis of OxoPNAs may be
performed on commercially available synthesizers using commercially
available reagents. In certain embodiments, resins such as
5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) or
5-(9-Fmoc-aminoxanthen-2-oxy)valeric acid (XAL) may be employed.
The resins may be immobilized with functional groups such as
NH.sub.2 and COOH. The amino or carboxy end of the OxoPNA may be
attached to the resin by establishing an amide bond. In addition,
commercially available microsphere (e.g., Luminex beads) are
functionalized with, for example, COOH and can be modified to make
an amide bond for attachment of OxoPNAs.
[0109] The oxocarbonamide peptide nucleic acids of the present
invention have improved stability, sensitivity, and specificity for
their target sequences, which make them well suited to a variety of
applications including, for example, the detection, analysis, and
capture of miRNAs, and other small nucleic acids; and as antisense
molecules for the selective gene knock-down.
C. Chemical Group Definitions
[0110] As used herein, the term "amino" means --NH.sub.2; the term
"nitro" means --NO.sub.2; the term "halo" designates --F, --Cl,
--Br or --I; the term "mercapto" means --SH; the term "cyano" means
--CN; the term "azido" means --N.sub.3; the term "silyl" means
--SiH.sub.3, and the term "hydroxy" means --OH.
[0111] The term "alkyl" includes straight-chain alkyl,
branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl,
heteroatom-unsubstituted alkyl, heteroatom-substituted alkyl,
heteroatom-unsubstituted alkyl.sub.(Cn), and heteroatom-substituted
alkyl.sub.(Cn). The term "heteroatom-unsubstituted alkyl.sub.(Cn)"
refers to a radical, having a linear or branched, cyclic or acyclic
structure, further having no carbon-carbon double or triple bonds,
further having a total of n carbon atoms, all of which are
nonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For
example, a heteroatom-unsubstituted alkyl.sub.(C1-C10) has 1 to 10
carbon atoms. The groups, --CH.sub.3 (Me), --CH.sub.2CH.sub.3 (Et),
--CH.sub.2CH.sub.2CH.sub.3 (n-Pr), --CH(CH.sub.3).sub.2 (iso-Pr),
--CH(CH.sub.2).sub.2 (cyclopropyl),
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 (n-Bu),
--CH(CH.sub.3)CH.sub.2CH.sub.3 (sec-butyl),
--CH.sub.2CH(CH.sub.3).sub.2 (iso-butyl), --C(CH.sub.3).sub.3
(tert-butyl), --CH.sub.2C(CH.sub.3).sub.3 (neo-pentyl), cyclobutyl,
cyclopentyl, and cyclohexyl, are all non-limiting examples of
heteroatom-unsubstituted alkyl groups. The term
"heteroatom-substituted alkyl.sub.(Cn)" refers to a radical, having
a single saturated carbon atom as the point of attachment, no
carbon-carbon double or triple bonds, further having a linear or
branched, cyclic or acyclic structure, further having a total of n
carbon atoms, all of which are nonaromatic, 0, 1, or more than one
hydrogen atom, at least one heteroatom, wherein each heteroatom is
independently selected from the group consisting of N, O, F, Cl,
Br, I, Si, P, and S. For example, a heteroatom-substituted
alkyl.sub.(C1-C10) has 1 to 10 carbon atoms. The following groups
are all non-limiting examples of heteroatom-substituted alkyl
groups: trifluoromethyl, --CH.sub.2F, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2OH, --CH.sub.2OCH.sub.3, --CH.sub.2OCH.sub.2CF.sub.3,
--CH.sub.2OC(O)CH.sub.3, --CH.sub.2NH.sub.2, --CH.sub.2NHCH.sub.3,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2CH.sub.2Cl,
--CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2OC(O)CH.sub.3,
--CH.sub.2CH.sub.2NHCO.sub.2C(CH.sub.3).sub.3, and
--CH.sub.2Si(CH.sub.3).sub.3.
[0112] The term "aryl" includes heteroatom-unsubstituted aryl,
heteroatom-substituted aryl, heteroatom-unsubstituted
aryl.sub.(Cn), heteroatom-substituted aryl.sub.(Cn), heteroaryl,
heterocyclic aryl groups, carbocyclic aryl groups, biaryl groups,
and single-valent radicals derived from polycyclic fused
hydrocarbons (PAHs). The term "heteroatom-unsubstituted
aryl.sub.(Cn)" refers to a radical, having a single carbon atom as
a point of attachment, wherein the carbon atom is part of an
aromatic ring structure containing only carbon atoms, further
having a total of n carbon atoms, 5 or more hydrogen atoms, and no
heteroatoms. For example, a heteroatom-unsubstituted
aryl.sub.(C6-C10) has 6 to 10 carbon atoms. Non-limiting examples
of heteroatom-unsubstituted aryl groups include phenyl (Ph),
methylphenyl, (dimethyl)phenyl, --C.sub.6H.sub.4CH.sub.2CH.sub.3,
--C.sub.6H.sub.4CH.sub.2CH.sub.2CH.sub.3,
--C.sub.6H.sub.4CH(CH.sub.3).sub.2,
--C.sub.6H.sub.4CH(CH.sub.2).sub.2,
--C.sub.6H.sub.3(CH.sub.3)CH.sub.2CH.sub.3,
--C.sub.6H.sub.4CH--CH.sub.2, --C.sub.6H.sub.4CH.dbd.CHCH.sub.3,
--C.sub.6H.sub.4C.ident.CH, --C.sub.6H.sub.4C.ident.CCH.sub.3,
naphthyl, and the radical derived from biphenyl. The term
"heteroatom-substituted aryl.sub.(Cn)" refers to a radical, having
either a single aromatic carbon atom or a single aromatic
heteroatom as the point of attachment, further having a total of n
carbon atoms, at least one hydrogen atom, and at least one
heteroatom, further wherein each heteroatom is independently
selected from the group consisting of N, O, F, Cl, Br, I, Si, P,
and S. For example, a heteroatom-unsubstituted
heteroaryl.sub.(C1-C10) has 1 to 10 carbon atoms. Non-limiting
examples of heteroatom-substituted aryl groups include the groups:
--C.sub.6H.sub.4F, --C.sub.6H.sub.4Cl, --C.sub.6H.sub.4Br,
--C.sub.6H.sub.4OH, --C.sub.6H.sub.4OCH.sub.3,
--C.sub.6H.sub.4OCH.sub.2CH.sub.3, --C.sub.6H.sub.4OC(O)CH.sub.3,
--C.sub.6H.sub.4NH.sub.2, --C.sub.6H.sub.4NHCH.sub.3,
--C.sub.6H.sub.4N(CH.sub.3).sub.2, --C.sub.6H.sub.4CH.sub.2OH,
--C.sub.6H.sub.4CH.sub.2OC(O)CH.sub.3,
--C.sub.6H.sub.4CH.sub.2NH.sub.2, --C.sub.6H.sub.4CF.sub.3,
--C.sub.6H.sub.4CN, --C.sub.6H.sub.4CHO, --C.sub.6H.sub.4CHO,
--C.sub.6H.sub.4C(O)CH.sub.3, --C.sub.6H.sub.4C(O)C.sub.6H.sub.5,
--C.sub.6H.sub.4CO.sub.2H, --C.sub.6H.sub.4CO.sub.2CH.sub.3,
--C.sub.6H.sub.4CONH.sub.2, --C.sub.6H.sub.4CONHCH.sub.3,
--C.sub.6H.sub.4CON(CH.sub.3).sub.2, furanyl, thienyl, pyridyl,
pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, indolyl, and
imidazoyl.
[0113] The term "aralkyl" includes heteroatom-unsubstituted
aralkyl, heteroatom-substituted aralkyl, heteroatom-unsubstituted
aralkyl.sub.(Cn), heteroatom-substituted aralkyl.sub.(Cn),
heteroaralkyl, and heterocyclic aralkyl groups. The term
"heteroatom-unsubstituted aralkyl.sub.(Cn)" refers to a radical,
having a single saturated carbon atom as the point of attachment,
further having a total of n carbon atoms, wherein at least 6 of the
carbon atoms form an aromatic ring structure containing only carbon
atoms, 7 or more hydrogen atoms, and no heteroatoms. For example, a
heteroatom-unsubstituted aralkyl.sub.(C7-C10) has 7 to 10 carbon
atoms. Non-limiting examples of heteroatom-unsubstituted aralkyls
are: phenylmethyl (benzyl, Bn) and phenylethyl. The term
"heteroatom-substituted aralkyl.sub.(Cn)" refers to a radical,
having a single saturated carbon atom as the point of attachment,
further having a total of n carbon atoms, 0, 1, or more than one
hydrogen atom, and at least one heteroatom, wherein at least one of
the carbon atoms is incorporated an aromatic ring structures,
further wherein each heteroatom is independently selected from the
group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example,
a heteroatom-substituted heteroaralkyl.sub.(C2-C10) has 2 to 10
carbon atoms.
[0114] The term "acyl" includes straight-chain acyl, branched-chain
acyl, cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl,
heteroatom-substituted acyl, heteroatom-unsubstituted
acyl.sub.(Cn), heteroatom-substituted acyl.sub.(Cn), alkylcarbonyl,
alkoxycarbonyl and aminocarbonyl groups. The term
"heteroatom-unsubstituted acyl.sub.(Cn)" refers to a radical,
having a single carbon atom of a carbonyl group as the point of
attachment, further having a linear or branched, cyclic or acyclic
structure, further having a total of n carbon atoms, 1 or more
hydrogen atoms, a total of one oxygen atom, and no additional
heteroatoms. For example, a heteroatom-unsubstituted
acyl.sub.(C1-C10) has 1 to 10 carbon atoms. The groups, --CHO,
--C(O)CH.sub.3, --C(O)CH.sub.2CH.sub.3,
--C(O)CH.sub.2CH.sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2,
--C(O)CH(CH.sub.2).sub.2, --C(O)C.sub.6H.sub.5,
--C(O)C.sub.6H.sub.4CH.sub.3, --C(O)C.sub.6H.sub.4CH.sub.2CH.sub.3,
and --COC.sub.6H.sub.3(CH.sub.3).sub.2, are non-limiting examples
of heteroatom-unsubstituted acyl groups. The term
"heteroatom-substituted acyl.sub.(Cn)" refers to a radical, having
a single carbon atom as the point of attachment, the carbon atom
being part of a carbonyl group, further having a linear or
branched, cyclic or acyclic structure, further having a total of n
carbon atoms, 0, 1, or more than one hydrogen atom, at least one
additional heteroatom, in addition to the oxygen of the carbonyl
group, wherein each additional heteroatom is independently selected
from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For
example, a heteroatom-substituted acyl.sub.(C1-C10) has 1 to 10
carbon atoms. The groups, --C(O)CH.sub.2CF.sub.3, --CO.sub.2H,
--CO.sub.2CH.sub.3, --CO.sub.2CH.sub.2CH.sub.3,
--CO.sub.2CH.sub.2CH.sub.2CH.sub.3, --CO.sub.2CH(CH.sub.3).sub.2,
--CO.sub.2CH(CH.sub.2).sub.2, --C(O)NH.sub.2 (carbamoyl),
--C(O)NHCH.sub.3, --C(O)NHCH.sub.2CH.sub.3,
--CONHCH(CH.sub.3).sub.2, --CONHCH(CH.sub.2).sub.2,
--CON(CH.sub.3).sub.2, and --CONHCH.sub.2CF.sub.3, are non-limiting
examples of heteroatom-substituted acyl groups.
[0115] The term "alkoxy" includes straight-chain alkoxy,
branched-chain alkoxy, cycloalkoxy, cyclic alkoxy,
heteroatom-unsubstituted alkoxy, heteroatom-substituted alkoxy,
heteroatom-unsubstituted alkoxy.sub.(Cn), and
heteroatom-substituted alkoxy.sub.(Cn). The term
"heteroatom-unsubstituted alkoxy.sub.(Cn)" refers to a group,
having the structure --OR, in which R is a heteroatom-unsubstituted
alkyl.sub.(Cn), as that term is defined above.
Heteroatom-unsubstituted alkoxy groups include: --OCH.sub.3,
--OCH.sub.2CH.sub.3, --OCH.sub.2CH.sub.2CH.sub.3,
--OCH(CH.sub.3).sub.2, and --OCH(CH.sub.2).sub.2. The term
"heteroatom-substituted alkoxy.sub.(Cn)" refers to a group, having
the structure --OR, in which R is a heteroatom-substituted
alkyl.sub.(Cn), as that term is defined above. For example,
--OCH.sub.2CF.sub.3 is a heteroatom-substituted alkoxy group.
[0116] The term "alkenyloxy" includes straight-chain alkenyloxy,
branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy,
heteroatom-unsubstituted alkenyloxy, heteroatom-substituted
alkenyloxy, heteroatom-unsubstituted alkenyloxy.sub.(Cn), and
heteroatom-substituted alkenyloxy.sub.(Cn). The term
"heteroatom-unsubstituted alkenyloxy.sub.(Cn)" refers to a group,
having the structure --OR, in which R is a heteroatom-unsubstituted
alkenyl.sub.(Cn), as that term is defined above. The term
"heteroatom-substituted alkenyloxy.sub.(Cn)" refers to a group,
having the structure --OR, in which R is a heteroatom-substituted
alkenyl.sub.(Cn), as that term is defined above.
[0117] The term "alkynyloxy" includes straight-chain alkynyloxy,
branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy,
heteroatom-unsubstituted alkynyloxy, heteroatom-substituted
alkynyloxy, heteroatom-unsubstituted alkynyloxy.sub.(Cn), and
heteroatom-substituted alkynyloxy.sub.(Cn). The term
"heteroatom-unsubstituted alkynyloxy.sub.(Cn)" refers to a group,
having the structure --OR, in which R is a heteroatom-unsubstituted
alkynyl.sub.(Cn), as that term is defined above. The term
"heteroatom-substituted alkynyloxy.sub.(Cn)" refers to a group,
having the structure --OR, in which R is a heteroatom-substituted
alkynyl.sub.(Cn), as that term is defined above.
[0118] The term "aryloxy" includes heteroatom-unsubstituted
aryloxy, heteroatom-substituted aryloxy, heteroatom-unsubstituted
aryloxy.sub.(Cn), heteroatom-substituted aryloxy.sub.(Cn),
heteroaryloxy, and heterocyclic aryloxy groups. The term
"heteroatom-unsubstituted aryloxy.sub.(Cn)" refers to a group,
having the structure --OAr, in which Ar is a
heteroatom-unsubstituted aryl.sub.(Cn), as that term is defined
above. A non-limiting example of a heteroatom-unsubstituted aryloxy
group is --OC.sub.6H.sub.5. The term "heteroatom-substituted
aryloxy.sub.(Cn)" refers to a group, having the structure --OAr, in
which Ar is a heteroatom-substituted aryl.sub.(Cn), as that term is
defined above.
[0119] The term "aralkyloxy" includes heteroatom-unsubstituted
aralkyloxy, heteroatom-substituted aralkyloxy,
heteroatom-unsubstituted aralkyloxy.sub.(Cn),
heteroatom-substituted aralkyloxy.sub.(Cn), heteroaralkyloxy, and
heterocyclic aralkyloxy groups. The term "heteroatom-unsubstituted
aralkyloxy.sub.(Cn)" refers to a group, having the structure --OAr,
in which Ar is a heteroatom-unsubstituted aralkyl.sub.(Cn), as that
term is defined above. The term "heteroatom-substituted
aralkyloxy.sub.(Cn)" refers to a group, having the structure --OAr,
in which Ar is a heteroatom-substituted aralkyl.sub.(Cn), as that
term is defined above.
[0120] The term "acyloxy" includes straight-chain acyloxy,
branched-chain acyloxy, cycloacyloxy, cyclic acyloxy,
heteroatom-unsubstituted acyloxy, heteroatom-substituted acyloxy,
heteroatom-unsubstituted acyloxy.sub.(Cn), heteroatom-substituted
acyloxy.sub.(Cn), alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The
term "heteroatom-unsubstituted acyloxy.sub.(Cn)" refers to a group,
having the structure --OAc, in which Ac is a
heteroatom-unsubstituted acyl.sub.(Cn), as that term is defined
above. For example, --OC(O)CH.sub.3 is a non-limiting example of a
heteroatom-unsubstituted acyloxy group. The term
"heteroatom-substituted acyloxy.sub.(Cn)" refers to a group, having
the structure --OAc, in which Ac is a heteroatom-substituted
acyl.sub.(Cn), as that term is defined above. For example,
--OC(O)OCH.sub.3 and --OC(O)NHCH.sub.3 are non-limiting examples of
heteroatom-unsubstituted acyloxy groups.
[0121] A DNA intercalating agent is a ligand of an appropriate size
and chemical nature to fit in between base pairs of DNA. These
ligands are mostly polycyclic, aromatic, and planar. Non-limiting
examples of DNA intercalators include ethidium bromide, proflavine,
daunomycin, doxorubicin, and thalidomide.
D. Nucleic Acid Detection
[0122] The stability, sensitivity, and specificity of the
oxocarbonamide peptide nucleic acids of the present invention, make
them a useful tool in diagnostics and molecular biology. While the
compounds of the present invention are useful in the detection and
analysis of any nucleic acids, it is contemplated that they will be
particularly useful in the detection of small RNA molecules such as
miRNA molecules, which often require the use of sensitive analysis
tools due to their size and low level of expression.
[0123] In certain embodiments, the oxocarbonamide peptide nucleic
acids of the present invention may be used as hybridization probes
for the detection of complementary nucleic acid sequences.
Sequence-specific nucleic acid hybridization assays (e.g., Northern
blotting, Southern blotting, and microarray analysis) are commonly
used for the detection of specific genetic sequences as indicators
of genetic anomalies, mutations, and disease propensity. In
addition, they are used for the detection of various biological
agents and infectious pathogens.
[0124] As used herein, "hybridization," "hybridizes" or "capable of
hybridizing" is understood to mean the forming of a double or
triple stranded molecule or a molecule with partial double or
triple stranded nature. The term "anneal" as used herein is
synonymous with "hybridize." The term "hybridization," "hybridizes"
or "capable of hybridizing" encompasses the terms "stringent
conditions" or "high stringency" and the terms "low stringency" or
"low stringency conditions." As used herein "stringent conditions"
or "high stringency" are those conditions that allow hybridization
between or within one or more nucleic acid strands containing
complementary sequences, but preclude hybridization of random
sequences. Stringent conditions tolerate little, if any, mismatch
between a nucleic acid and a target strand. Such conditions are
well known to those of ordinary skill in the art, and are preferred
for applications requiring high selectivity. Non-limiting
applications include isolating a nucleic acid, such as a gene or a
nucleic acid segment thereof, or detecting at least one specific
mRNA, miRNA, or siRNA transcript or a nucleic acid segment thereof,
and the like.
[0125] Stringent conditions may comprise low salt and/or high
temperature conditions, such as provided by about 0.02 M to about
0.15 M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. It is understood that the temperature and ionic
strength of a desired stringency are determined in part by the
length of the particular nucleic acids, the length and nucleobase
content of the target sequences, the charge composition of the
nucleic acids, the presence of nucleic acid analogues in the
nucleic acid molecules, and to the presence or concentration of
formamide, tetramethylammonium chloride or other solvents in a
hybridization mixture.
[0126] It is also understood that these ranges, compositions and
conditions for hybridization are mentioned by way of non-limiting
examples only, and that the desired stringency for a particular
hybridization reaction is often determined empirically by
comparison to one or more positive or negative controls. Depending
on the application envisioned it is preferred to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of a nucleic acid towards a target sequence. In a
non-limiting example, identification or isolation of a related
target nucleic acid that does not hybridize to a nucleic acid under
stringent conditions may be achieved by hybridization at low
temperature and/or high ionic strength. Such conditions are termed
"low stringency" or "low stringency conditions," and non-limiting
examples of low stringency include hybridization performed at about
0.15 M to about 0.9 M NaCl at a temperature range of about
20.degree. C. to about 50.degree. C. Of course, it is within the
skill of one in the art to further modify the low or high
stringency conditions to suite a particular application.
[0127] The present invention may be employed in solution
hybridization as well as in solid phase hybridization. The
hybridization conditions selected will depend on the particular
circumstances (depending, for example, on the G+C content, type of
target nucleic acid, source of nucleic acid, size of hybridization
probe, etc.). Optimization of hybridization conditions for the
particular application of interest is well known to those of skill
in the art. Representative solid phase hybridization methods are
disclosed in U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626.
Other methods of hybridization that may be used in the practice of
the present invention are disclosed in U.S. Pat. Nos. 5,849,486 and
5,851,772.
[0128] To detect hybridization, it will be advantageous to employ
an appropriate detection moiety. Recognition moieties incorporated
into primers, incorporated into the amplified product during
amplification, or attached to probes are useful in the
identification of nucleic acid molecules. A number of different
labels may be used for this purpose such as fluorophores,
chromophores, radiophores, enzymatic tags, antibodies,
chemiluminescence, electroluminescence, affinity labels, noble
metal nanoparticles, quantum dots, magnetic particles, etc. One of
skill in the art will recognize that these and other labels not
mentioned herein can be used with success in this invention.
[0129] Examples of affinity labels include, but are not limited to
the following: an antibody, an antibody fragment, a receptor
protein, a hormone, biotin, DNP, or any polypeptide/protein
molecule that binds to an affinity label.
[0130] Examples of enzyme tags include enzymes such as urease,
alkaline phosphatase, or peroxidase to mention a few. Colorimetric
indicator substrates can be employed to provide a detection means
visible to the human eye or spectrophotometrically, to identify
specific hybridization with complementary nucleic acid-containing
samples. All of these examples are generally known in the art and
the skilled artisan will recognize that the invention is not
limited to the examples described above.
[0131] Examples of fluorophores include, but are not limited to the
following: all of the Alexa Fluor.RTM. dyes, AMCA, BODIPY.RTM.
630/650, BODIPY.RTM. 650/665, BODIPY.RTM.-FL, BODIPY.RTM.-R6G,
BODIPY.RTM.-TMR, BODIPY.RTM.-TRX, Cascade Blue.RTM., CyDyes.TM.,
including but not limited to Cy2.TM., Cy3.TM., and Cy5.TM., DNA
intercalating dyes, 6-FAM.TM., Fluorescein, HEX.TM., 6-JOE, Oregon
Green.RTM. 488, Oregon Green.RTM. 500, Oregon Green.RTM. 514,
Pacific Blue.TM., REG, phycobilliproteins including, but not
limited to, phycoerythrin and allophycocyanin, Rhodamine Green.TM.,
Rhodamine Red.TM., ROX.TM., TAMRA.TM., TET.TM.,
Tetramethylrhodamine, and Texas Red.RTM..
[0132] Detection can result in qualitative identification,
semi-quantitative identification, or quantitative identification of
the target molecule. Qualitative detection includes detection of
the presence of the molecule, without any correlation to an amount
of the molecule in the sample that was tested. Semi-quantitative
detection permits not only detection of the target molecule, but
correlation of the signal to a basal level of target molecule in
the sample that was tested. For example, it may indicate a minimum
threshold amount of the target molecule was present in the sample.
Quantitative detection permits the practitioner to determine the
amount of target molecule present in the original sample over a
wide range of amounts. In general, quantitative detection compares
the amount detected to a reference or standard that is either
previously generated (e.g., a standard curve) or generated at the
time of the assay for the target molecule using internal controls.
Numerous techniques for performing quantitative and
semi-quantitative analyses are known to those of skill in the
art.
[0133] Arrays and gene chip technology provide a means of rapidly
screening a large number of nucleic acid samples for their ability
to hybridize to oxocarbonamide peptide nucleic acid molecules
immobilized on a solid substrate. These techniques involve
quantitative methods for analyzing large numbers of miRNA
molecules, or other nucleic acid sequences, rapidly and accurately.
Basically, an array or gene chip consists of a solid substrate upon
which an array of single stranded oxocarbonamide peptide nucleic
acid molecules have been attached. For screening, the chip or array
is contacted with a single stranded DNA or RNA sample, which is
allowed to hybridize under stringent conditions. The chip or array
is then scanned to determine which probes have hybridized.
[0134] The ability to directly synthesize on or attach
polynucleotide probes to solid substrates is well known in the art.
See U.S. Pat. Nos. 5,837,832 and 5,837,860, both of which are
expressly incorporated by reference. A variety of methods have been
utilized to either permanently or removably attach the probes to
the substrate. Exemplary methods include: the immobilization of
biotinylated nucleic acid molecules to avidin/streptavidin coated
supports (Holmstrom, 1993), the direct covalent attachment of
short, 5'-phosphorylated primers to chemically modified polystyrene
plates (Rasmussen et al., 1991), or the precoating of the
polystyrene or glass solid phases with poly-L-Lys or poly L-Lys,
Phe, followed by the covalent attachment of either amino- or
sulfhydryl-modified oligonucleotides using bi-functional
crosslinking reagents (Running et al., 1990; Newton et al., 1993).
When immobilized onto a substrate, the probes are stabilized and
therefore may be used repeatedly. In general terms, hybridization
is performed on an immobilized nucleic acid target or a probe
molecule that is attached to a solid surface such as
nitrocellulose, nylon membrane, or glass. Numerous other matrix
materials may be used, including reinforced nitrocellulose
membrane, activated quartz, activated glass, polyvinylidene
difluoride (PVDF) membrane, polystyrene substrates,
polyacrylamide-based substrate, other polymers such as poly(vinyl
chloride), poly(methyl methacrylate), poly(dimethyl siloxane),
photopolymers (which contain photoreactive species such as
nitrenes, carbenes and ketyl radicals capable of forming covalent
links with target molecules.
[0135] In certain embodiments, the present invention is used in
conjunction with Luminex.RTM. xMAP.RTM. technology. The Luminex
technology allows the quantitation of nucleic acid products
immobilized on fluorescently encoded microspheres. By dyeing
microspheres with 10 different intensities each of two spectrally
distinct fluorochromes, 100 fluorescently distinct populations of
microspheres are produced. By using three or more spectrally
distinct fluorochromes at different intensity levels, even greater
numbers of fluorescently distinct populations can be created. These
individual populations (sets) can represent individual detection
sequences and the magnitude of hybridization on each set can be
detected individually. The magnitude of the hybridization reaction
is measured using a third spectrally distinct fluorochrome called a
reporter. The reporter molecule signals the extent of the reaction
by attaching to the molecules on the microspheres. As both the
microspheres and the reporter molecules are labeled, digital signal
processing allows the translation of signals into real-time,
quantitative data for each reaction. The Luminex technology is
described, for example, in U.S. Pat. Nos. 5,736,330, 5,981,180, and
6,057,107, all of which are specifically incorporated by
reference.
[0136] The present invention may also be used in conjunction with a
competitive binding assay format. In general, this format involves
a detection sequence coupled to a solid surface, and a labeled
sequence complementary to the detection sequence in solution. With
this format, the target sequence in the sample being assayed does
not need to be labeled. Rather, the target sequence's presence in
the sample is detected because it competes with the labeled
complement for hybridization with the immobilized detection
sequence. Thus, if the target sequence is present in the sample,
the signal decreases as compared to a sample lacking the target
sequence.
[0137] The Luminex xMAP technology described above can be used in a
competitive binding assay format. In general, this format would
comprise an oxocarbonamide peptide nucleic acid detection molecule
immobilized on a labeled bead, a labeled sequence complementary to
the detection sequence, exposing the immobilized detection sequence
and the labeled complement to a nucleic acid sample under
hybridizing conditions, and detecting the presence or absence of
the target sequence in the sample. The use of the Luminex
technology in a competitive binding assay format is described in
U.S. Pat. Nos. 5,736,330 and 6,057,107, incorporated herein by
reference.
[0138] Flow cytometry is a useful tool in the analysis of
biomolecules. In the context of the present invention, flow
cytometry is particularly useful in the analysis of microsphere
based assays, such as the Luminex xMAP system. Flow cytometry
involves the separation of cells or other particles, such as
microspheres, in a liquid sample. Generally, the purpose of flow
cytometry is to analyze the separated particles for one or more
characteristics. The basic steps of flow cytometry involve the
direction of a fluid sample through an apparatus such that a liquid
stream passes through a sensing region. The particles should pass
one at a time by the sensor and are categorized based on size,
refraction, light scattering, opacity, roughness, shape,
fluorescence, etc.
[0139] In the context of the Luminex xMAP system, flow cytometry
can be used for simultaneous sequence identification and
hybridization quantification. Internal dyes in the microspheres are
detected by flow cytometry and used to identify the specific
nucleic acid sequence to which a microsphere is coupled. The label
on the target nucleic acid molecule is also detected by flow
cytometry and used to quantify target hybridization to the
microsphere.
[0140] Methods of flow cytometry are well know in the art and are
described, for example, in U.S. Pat. Nos. 5,981,180, 4,284,412;
4,989,977; 4,498,766; 5,478,722; 4,857,451; 4,774,189; 4,767,206;
4,714,682; 5,160,974; and 4,661,913, all of which are specifically
incorporated by reference.
E. miRNA Isolation
[0141] As discussed above, the oxocarbonamide peptide nucleic acids
of the present invention are particularly useful in the detection
of small RNA molecules, such as miRNA, in a sample. miRNAs are
18-25 nucleotide (nt) RNAs that are processed from longer
endogenous hairpin transcripts. The increased sensitivity and
selectivity of OxoPNAs of the present invention overcome the
limitations of their DNA-probe counterparts for the detection of
short nucleotide targets.
[0142] Small RNA molecules may be isolated from a sample, such as a
cell sample, by a variety of methods known in the art. The most
commonly used method is to co-purify the miRNA with total RNA using
a combination of acidified phenol and guanidine isothiocyanate
using care not to remove the highly-soluble short RNA (see, e.g.,
Pfeffer et al., 2003). This method isolates total RNA, which
comprises transfer RNA (tRNA), ribosomal RNA (rRNA), polyA
messenger RNA (mRNA), short interfering RNA (siRNA), small nuclear
RNA (snRNA), and microRNA (miRNA). If desired, the miRNA can be
enriched from the total RNA by size selection using gel
purification (Pfeffer, Id.).
[0143] Other methods for isolating miRNA include the mirVana.TM.
miRNA Isolation Kit (Ambion). The resulting RNA preparation (less
than about 200 nucleotides) is enriched for miRNAs, siRNAs, and/or
snRNAs. In addition, the Absolutely RNA.RTM. Miniprep Kit
(Stratagene) may be used to isolate total RNA comprising miRNA.
Removal of genomic DNA is desirable as its presence in the total
RNA could lead to false or misleading results.
F. Therapeutic Applications
[0144] The oxocarbonamide peptide nucleic acids of the present
invention may be used as double-stranded siRNA molecules,
single-stranded antisense molecules, or as decoy molecules for
nucleic acid binding proteins. These uses may be for therapeutic or
research purposes. Naturally occurring DNA molecules are generally
not well suited to these applications due to the instability of
unmodified DNA in vivo. However, chemically modified
oligonucleotides have been shown to be effective inhibitors of
coding and non-coding RNAs (see Weiler et al., 2006).
[0145] Oxocarbonamide peptide siRNA, antisense, or decoy molecules
may be prepared by solid phase synthesis as described above. For
therapeutic application it may be desirable to incorporate
hydrophilic groups, such as polyethylene glycol (PEG), in to the
OxoPNA in order to increase its hydrophilicity. Cationic polymers
such as polyamidoamine (PMAM) dendrimers or a polyethyleneimine
(PEI) may be combined with OxoPNAs for DNA delivery.
[0146] The nucleotide sequence of the siRNA or antisense molecule
is defined by the nucleotide sequence of its target gene. The siRNA
or antisense molecule contains a nucleotide sequence that is
essentially identical to at least a portion of the target gene.
Preferably, the siRNA or antisense contains a nucleotide sequence
that is completely identical to at least a portion of the target
gene. Of course, when comparing an RNA sequence to a DNA sequence,
an "identical" RNA sequence will contain ribonucleotides where the
DNA sequence contains deoxyribonucleotides, and further that the
RNA sequence will typically contain a uracil at positions where the
DNA sequence contains thymidine. The nucleotide sequence of the
decoy molecule is defined by the recognition sequence of the target
protein.
[0147] The cell containing the target gene or protein may be
derived from or contained in any organism (e.g., plant, animal,
protozoan, virus, bacterium, or fungus). The plant may be a
monocot, dicot or gynmosperm; the animal may be a vertebrate or
invertebrate. Preferred microbes are those used in agriculture or
by industry, and those that a pathogenic for plants or animals.
Fungi include organisms in both the mold and yeast morphologies.
Examples of vertebrates include fish and mammals, including cattle,
goat, pig, sheep, hamster, mouse, rat, and human; invertebrate
animals include nematodes, insects, arachnids, and other
arthropods. Preferably, the cell is a vertebrate cell. More
preferably, the cell is a mammalian cell.
[0148] The cell having the target gene or protein may be from the
germ line or somatic, totipotent or pluripotent, dividing or
non-dividing, parenchyma or epithelium, immortalized or
transformed, or the like. The cell can be a gamete or an embryo; if
an embryo, it can be a single cell embryo or a constituent cell or
cells from a multicellular embryo. The term "embryo" thus
encompasses fetal tissue. The cell having the target gene or
protein may be an undifferentiated cell, such as a stem cell, or a
differentiated cell, such as from a cell of an organ or tissue,
including fetal tissue, or any other cell present in an organism.
Cell types that are differentiated include adipocytes, fibroblasts,
myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells,
megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils,
basophils, mast cells, leukocytes, granulocytes, keratinocytes,
chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells, of
the endocrine or exocrine glands.
[0149] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these terms also
include their progeny, which is any and all subsequent generations
formed by cell division. It is understood that all progeny may not
be identical due to deliberate or inadvertent mutations. A host
cell may be "transfected" or "transformed," which refers to a
process by which exogenous nucleic acid is transferred or
introduced into the host cell. A transformed cell includes the
primary subject cell and its progeny. As used herein, the terms
"engineered" and "recombinant" cells or host cells are intended to
refer to a cell into which an exogenous nucleic acid sequence has
been introduced. Therefore, recombinant cells are distinguishable
from naturally occurring cells which do not contain a recombinantly
introduced nucleic acid.
[0150] A tissue may comprise a host cell or cells to be transformed
or contacted with a nucleic acid delivery composition and/or an
additional agent. The tissue may be part or separated from an
organism. In certain embodiments, a tissue and its constituent
cells may comprise, but is not limited to, blood (e.g.,
hematopoietic cells (such as human hematopoietic progenitor cells,
human hematopoietic stem cells, CD34.sup.+ cells CD4.sup.+ cells),
lymphocytes and other blood lineage cells), bone marrow, brain,
stem cells, blood vessel, liver, lung, bone, breast, cartilage,
cervix, colon, cornea, embryonic, endometrium, endothelial,
epithelial, esophagus, facia, fibroblast, follicular, ganglion
cells, glial cells, goblet cells, kidney, lymph node, muscle,
neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin,
small intestine, spleen, stomach, or testes.
G. Anchor Molecules
[0151] The oxocarbonamide peptide nucleic acids of the present
invention may be used as anchor molecules for the attachment of
different functional molecules to DNA via specific Watson-Crick
base pairing. The coupling of molecules with different biological
function to plasmids or other nucleic acid molecules of interest
can improve the targeting of genetic material in non-viral gene
delivery systems. Coupling methods based on chemical linkage of
peptides to plasmid DNA can interfere with gene expression. Thus,
coupling via specific Watson-Crick base pairing provides an
attractive alternative to chemical linkage. Those in the art are
familiar with methods of coupling peptides and other molecules to
plasmid DNA using nucleic acid analogs, such as those described for
PNA, bisPNA, and LNA (see e.g., Lundin et al., 2005; Branden et
al., 1999; Rebuff et al., 2002; Hertoghs et al., 2003; Branden et
al., 2002).
H. Nucleic Acid Analogues for Programmable Assembly
[0152] The oxocarbonamide peptide nucleic acids of the present
invention may also be used in the programmed assembly of nanoscale
devices. Nucleic acid guided assembly has been used to organize
gold nanoparticles (Mirkin et al., 1996; Alivisatos et al., 1996;
Mucic et al., 1998), nanowires (Mbindyo et al., 2003), quantum dots
(Parak et al., 2002); Mitchell et al., 1999), carbon nanotubes
(Dwyer et al., 2004), dendrimers (DeMattei et al., 2004),
micron-size polystyrene beads (Milam et al., 2003), virus particles
(Strable et al., 2004), and to attach nano- and microparticles to
substrates (Kannan et al., 2004; Hartmann et al., 2002); Niemeyer
et al., 2001). The oxocarbonamide peptide nucleic acids of the
present invention provide advantages in programmed assembly over
natural DNA due to increased stability and greater affinity between
complementary oligomers.
I. Kits
[0153] Any of the compositions described herein may be comprised in
a kit. In one embodiment, the present invention provides a kit
comprising one or more oxocarbonamide peptide nucleic acid
molecules. In certain aspects of the invention, the kit comprises a
plurality of oxocarbonamide peptide nucleic acid molecules having
at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,
400, 500, 600, 700, 800, 900, 1000, 10000, 20000, 30000, 40000,
50000, 60000, 70000, 80000, 90000, or 100000 different nucleic acid
sequences. In another embodiment of the invention, the kit may
include components for making a nucleic acid array, and thus, may
include, for example, a solid support. In some aspects of the
invention, the kits will comprise pre-fabricated arrays, such as,
for example, microspheres coupled to oxocarbonamide peptide nucleic
acid probes. It may also include one or more buffers, such as
hybridization buffer or a wash buffer.
[0154] The kits may comprise suitably aliquoted nucleic acid
compositions of the present invention, whether labeled or
unlabeled, as may be used to isolate, separate, detect, or amplify
a targeted nucleic acid. The components of the kits may be packaged
either in aqueous media or in lyophilized form. The container means
of the kits will generally include at least one vial, test tube,
flask, bottle, syringe or other container means, into which a
component may be placed, and preferably, suitably aliquoted. Where
there is more than one component in the kit, the kit also will
generally contain a second, third or other additional containers
into which the additional components may be separately placed.
However, various combinations of components may be comprised in a
vial. The kits of the present invention also will typically include
a means for containing the oxocarbonamide peptide nucleic acids,
and any other reagent containers in close confinement for
commercial sale. Such containers may include cardboard or injection
or blow-molded plastic containers into which the desired vials,
bottles, etc. are retained.
[0155] When the components of the kit are provided in one or more
liquid solutions, the liquid solution may be an aqueous solution,
with a sterile aqueous solution being particularly preferred.
[0156] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0157] A kit may also include instructions for employing the kit
components. Instructions may include variations that can be
implemented.
[0158] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of certain
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the methods described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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