U.S. patent application number 10/407543 was filed with the patent office on 2003-09-18 for method of detection of nucleic acids with a specific sequence composition.
Invention is credited to Weininger, Arthur M., Weininger, Susan.
Application Number | 20030175789 10/407543 |
Document ID | / |
Family ID | 25334160 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175789 |
Kind Code |
A1 |
Weininger, Susan ; et
al. |
September 18, 2003 |
Method of detection of nucleic acids with a specific sequence
composition
Abstract
This invention is a novel method for detecting and localizing
specific nucleic acid sequences in a sample with a high degree of
sensitivity and specificity. The method and novel compositions used
in the method involve the use of Probe Nucleic Acids, the
production of nucleic acid binding regions and the use of nucleic
acid Target Binding Assemblies to detect and localize specific
Target Nucleic Acids. The detection and localization of the Target
Nucleic Acid is accomplished even in the presence of nucleic acids
which have similar sequences. The method provides for a high degree
of amplification of the signal produced by each specific binding
event. In particular, methods and compositions are presented for
the detection of HIV and HPV nucleic acid in samples. These methods
and compositions find use in diagnosis of disease, genetic
monitoring, forensics, and analysis of nucleic acid mixtures. Some
of the novel compositions used in the detection method are useful
in preventing or treating pathogenic conditions.
Inventors: |
Weininger, Susan; (Seattle,
WA) ; Weininger, Arthur M.; (Seattle, WA) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
2421 N.W. 41ST STREET
SUITE A-1
GAINESVILLE
FL
326066669
|
Family ID: |
25334160 |
Appl. No.: |
10/407543 |
Filed: |
April 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10407543 |
Apr 3, 2003 |
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08860844 |
Sep 29, 1997 |
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08860844 |
Sep 29, 1997 |
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PCT/US95/15944 |
Dec 7, 1995 |
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PCT/US95/15944 |
Dec 7, 1995 |
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08353476 |
Dec 9, 1994 |
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5871902 |
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Current U.S.
Class: |
435/6.15 |
Current CPC
Class: |
C12Q 1/708 20130101;
C12Q 1/6811 20130101; C12Q 1/6811 20130101; C12Q 1/6804 20130101;
C12Q 1/6813 20130101; A61P 31/18 20180101; C12Q 1/703 20130101;
C12Q 1/682 20130101; C12Q 1/6853 20130101; C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 2537/143 20130101; C12Q 2537/149
20130101; C12Q 2525/301 20130101; A61P 31/14 20180101; C12Q
2537/149 20130101; C12Q 2537/161 20130101; C12Q 2537/161 20130101;
C12Q 2525/301 20130101; C12Q 2537/143 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of using a target binding assembly (TBA) wherein said
TBA comprises a plurality of nucleic acid recognition units
assembled by assembly sequences that are not ligated together,
wherein each of said nucleic acid recognition units binds to a
specific nucleic acid sequence on a target double stranded nucleic
acid molecule; and wherein the combined affinity of said plurality
of nucleic acid recognition units is such that said TBA
specifically binds to the target double stranded nucleic acid
molecule but does not bind to non-target molecules; and wherein
said method comprises administering to a patient a therapeutically
or prophylactically effective amount of said TBA, or nucleic acid
which codes for and produces said TBA, such that the TBA binds a
target double stranded nucleic acid molecule to achieve a desired
prophylactic or therapeutic result.
Description
CROSS-REFERENCE TO A RELATED APPLICATIONS
[0001] This application is a continuation of co-pending application
Ser. No. 08/860,844; filed Jun. 9, 1997; which is a 371 application
of PCT/US95/15944; which is a continuation-in-part application of
Ser. No. 08/353,476, filed Dec. 9, 1994, now U.S. Pat. No.
5,871,902.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention provides a method and compositions for use in
binding, detecting, and amplifying the detection of specific Target
Nucleic Acid sequences in a sample with fidelity and accuracy, even
in the presence of closely related but different nucleic acids. The
binding may involve the chaperoning and assembly of specific
molecules into Target Binding Assemblies which specifically bind
Target Binding Regions formed by the hybridization of Probe Nucleic
Acids and Target Nucleic Acid sequences. The amplifying may involve
the chaperoning and/or assembly of specific molecules into Booster
Binding Assemblies which specifically bind Booster Binding Regions
formed by the hybridization of Booster Nucleic Acids with Probe
Nucleic Acids, Target Nucleic Acids, or other Booster Nucleic
Acids. A method, and compositions, involving Hairpin Nucleic Acids
is also provided to enable control of the size of specifically or
non-specifically elongated Booster Nucleic Acids and Booster
Binding Assemblies used in the amplification. The detecting
involves providing one or more detectable labels, including
radioactive, light- or fluorescent-emitting, enzymatic, or other
detectable or signal-generating molecules, in association with the
Probe Nucleic Acid, the Target Binding Assembly, the Booster
Nucleic Acid, the Booster Binding Assembly, or the Hairpin Nucleic
Acid. A method is presented for isolating nucleic acid fragments
from an organism which has TBA component binding sites in order to
create a probe nucleic acid and a TBA which is unique for that
fragment and/or organism. Therapeutic and prophylactic uses of the
Target Binding Assemblies and compositions for such use are also
provided.
[0004] 2. Background and Description of Related Art
[0005] There are an increasing number of cases in which it is
important to be able to detect nucleic acids containing a specific
sequence, hereinafter named Target Nucleic Acids (TNAs), in a
sample. It is desirable to be able to detect the TNAs with the
smallest number of processing steps, with the simplest components
and to the exclusion of other similar but different nucleic acids,
hereinafter named Cousin Nucleic Acids (CNAs). It is desirable to
be able to detect specific TNAs to the exclusion of any and all
CNAs in the detection sample without the necessity of amplification
or other post-detection processing.
[0006] There are numerous methods which use immobilized or tagged
nucleic acids as probes for TNAs. However, using known methods, it
is difficult to discriminate between a TNA bound to the Probe
Nucleic Acid (PNA) as opposed to a CNA bound to the PNA. For
example, one or more base mismatches between the PNA and a CNA can
still result in a CNA-PNA hybridization which is almost
indistinguishable from a TNA-PNA hybridization. Thus, hybridization
alone is not an optimal indicator that a PNA has hybridized to a
unique TNA.
[0007] There are many situations in which a PNA would be used to
try to determine whether a TNA was present in a sample which may
contain CNAs. Hybridization of the PNA to any CNA in this situation
would limit the diagnostic value that the PNA might have for the
detection of a TNA, absent additional verification. Furthermore, it
is desirable to be able to detect and localize TNAs with low copy
numbers in samples which may contain many copies of CNAs, without
the necessity of creating additional copies of the TNA. It would
also be desirable to be able to confirm the presence of CNAs,
independent of the TNAs, without the necessity of separating the
CNAs and TNAs in the sample.
[0008] Furthermore, it would be desirable to be able to amplify the
signal of even a low frequency hybridization of a particular
TNA-PNA. For this purpose, a method of polymerizing multiple copies
of a label, hereinafter referred to as a Booster Nucleic Acid (BNA)
onto the TNA-PNA would be desirable.
[0009] The instant invention provides methods and compositions for
achieving the foregoing desired objectives. As revealed by the
following review, the instant compositions and methods have not
been reported or suggested in the art. A general and comprehensive
review of the state of art of nucleic acid detection is provided in
Keller, H., M. M. Manak (1989) DNA Probes, Stockton Press.
[0010] A method has been reported for detecting base pair
mismatches by chemical means in order to determine whether a PNA
has hybridized to a CNA rather than to a TNA. In U.S. Pat. No.
4,794,075 to Ford et al., a method for distinguishing fragments of
DNA which contain single base mismatches from their perfectly
paired homologs is discussed. Single stranded regions within a
duplex fragment are modified with carbodiimide, which reacts with
unpaired guanine (G) and thymine (T) residues in DNA. Linear duplex
DNA molecules do not react, while DNA molecules with single base
mismatches react quantitatively. Following reaction with
carbodiimide, the DNA molecules are fractionated on high percentage
polyacrylamide gels such that modified and unmodified fragments can
be distinguished. Ford et al. applied this technique in order to
locate and purify DNA sequence differences responsible for
phenotype variation and inherited disease. Although this method is
useful for following variations in genetic material, it has a large
number of steps, it requires costly components, and it does not
offer a direct means of determining whether a PNA has hybridized to
the TNA exclusive of CNAs in the sample.
[0011] There have been some attempts to assure that at least a
portion of the hybridization between the PNA and another nucleic
acid is complementary. One method involves the monitoring of
transcription products which are produced if the PNA hybridizes to
a nucleic acid sufficiently to be transcribed from a promoter site
contained in the probe. U.S. Pat. No. 5,215,899 to Dattagupta
discloses how specific nucleic acid sequences are amplified through
the use of a hairpin probe which, upon hybridization with and
ligation to a target sequence, is capable of being transcribed. The
probe comprises a single stranded self-complementary sequence
which, under hybridizing conditions, forms a hairpin structure
having a functional promoter region, and further comprises a single
stranded probe sequence extending from the 3' end of the hairpin
sequence. Upon hybridization with a target sequence complementary
to the probe sequence and ligation of the 3' end of the hybridized
target sequence to the 5' end of the hairpin probe, the target
sequence is rendered transcribable in the presence of a suitable
RNA polymerase and appropriate ribonucleoside triphosphates
(rNTPs). Amplification is accomplished by hybridizing the desired
TNA sequence with the probe, ligating the TNA to the PNA, adding
the RNA polymerase and the rNTPs to the separated hybrids, and
allowing transcription to proceed until a desired amount of RNA
transcription product has accumulated. That method generally and
specifically involves the use of hairpin DNA formed with a single
stranded unpaired end to anneal a target sequence. When the target
sequence is bound, the production of RNA transcription products is
enabled. Thus, the method involves the detection of secondary
transcription products rather than the use of a nucleic acid
binding assembly to directly immobilize and/or localize a target
sequence. A CNA could easily bind to the probe, and the lack of
complementarity would not necessarily interfere with the formation
of a CNA-PNA hybrid which could then support the production of
unwanted transcription products.
[0012] A CNA bound to the PNA might be detected if the lack of
complementarity interferes with the susceptibility of the hybrid
CNA-PNA pair to be cut by a restriction endonuclease. In U.S. Pat.
No. 5,118,605 to Urdea and U.S. Pat. No. 4,775,619 to Urdea, novel
methods for assaying a nucleic acid analyte were provided, which
employ polynucleotides having oligonucleotide sequences
substantially homologous to a sequence of interest in the analyte,
where the presence or absence of hybridization at a predetermined
stringency provides for the release of a label from a support.
Various techniques are employed for binding a label to a support,
whereupon cleavage of either a single or double strand, a label may
be released from a support, and the release of the label can be
detected as indicative of the presence of a particular
polynucleotide sequence in a sample. However, this technique has
the shortcoming that a CNA-PNA pair could be cut by the restriction
endonuclease, even if there is a mismatch, so long as the mismatch
was outside of the endonuclease recognition region. This would lead
to failure of the assay to identify a CNA-PNA hybrid.
[0013] Another method uses a branched DNA probe to detect nucleic
acids. U.S. Pat. No. 5,124,246 to Urdea et al. discloses linear or
branched oligonucleotide multimers useful as amplifiers in
biochemical assays which comprise (1) at least one first
single-stranded oligonucleotide unit (PNA) that is complementary to
a single-stranded oligonucleotide sequence of interest (TNA), and
(2) a multiplicity of second single-stranded, oligonucleotide units
that are complementary to a single-stranded labeled
oligonucleotide. Although amplified sandwich nucleic acid
hybridizations and immunoassays using the multimers are described,
the method has the limitation that PNA-CNA hybridization could
occur and would result in production of unwanted signal.
[0014] In addition to methods for identification of TNAs, methods
have been disclosed for the amplification of this DNA. In U.S. Pat.
No. 5,200,314 to Urdea, an analyte polynucleotide strand having an
analyte sequence (TNA) is detected within a sample containing
polynucleotides by contacting the analyte polynucleotide with a
capture probe (PNA) under hybridizing conditions, where the capture
probe has a first binding partner specific for the TNA, and a
second binding sequence specific for a solid phase third binding
partner. The resulting duplex is then immobilized by specific
binding between the binding partners, and non-bound polynucleotides
are separated from the bound species. The analyte polynucleotide is
optionally displaced from the solid phase, then amplified by PCR.
The PCR primers each have a polynucleotide region capable of
hybridizing to a region of the analyte polynucleotide, and at least
one of the primers further has an additional binding partner
capable of binding a solid-phase binding partner. The amplified
product is then separated from the reaction mixture by specific
binding between the binding partners, and the amplified product is
detected. Although it is possible to confirm (by PCR) that a
particular nucleic acid has hybridized with the PNA, confirmation
is expensive and involves multiple steps.
[0015] As for reports that involve the interaction of a double
stranded nucleic acid and a DNA-binding protein, a method has been
described whereby a sequence of immobilized DNA which contains
binding sites for a single protein is used to purify that protein.
U.S. Pat. No. 5,122,600 to Kawaguchi et al. discloses a
DNA-immobilized microsphere comprising DNA chains having base
sequences which specifically bind a particular protein, and a
carrier having a particle size of not more than 50 .mu.m and not
less than 0.01 .mu.m which does not adsorb any protein, said
carrier and said DNA chains being bound to each other by a chemical
bond, and a process for purifying a protein using said microsphere.
As this is a purification method for a protein, it does not
disclose a method of detection of a TNA nor a method whereby more
than one protein is bound to a double stranded nucleic acid for the
purposes of detection and localization of specific TNA
sequences.
[0016] In EP 0 453 301, a method for detecting a polynucleotide
target sequence in a sample was described wherein sequences in a
TNA are detected by hybridizing a first and a second PNA to the
TNA. Each of said first and second PNAs contained a pre-formed
duplex sequence, or a duplex that is formed through chain
extension, capable of binding a nucleotide sequence specific
binding protein. A method for binding a nucleotide specific binding
protein to a duplex formed between a TNA and a PNA only upon
formation of a duplex between the PNA and TNA is neither disclosed
nor suggested.
[0017] In U.S. Pat. No. 4,556,643, a method was disclosed for the
non-radioactive detection of specific nucleotide sequences in a
sample which involved hybridization of a probe containing DNA
binding protein specific sequences. However, this disclosure
neither taught nor suggested a method for binding a nucleotide
specific binding protein to a duplex formed between a TNA and a PNA
only upon formation of a duplex between sequences present in the
PNA and sequences present in the TNA.
BRIEF SUMMARY OF THE INVENTION
[0018] Disclosed are methods by which specific Target Nucleic Acid
(TNA) sequences are detected through the use of Probe Nucleic Acids
(PNAs) which, upon hybridization with TNAs, are capable of binding
Target Binding Assemblies (TBAs). Each TBA binds at least one
specific region of the PNA-TNA hybrid pair, the Target Binding
Region (TBR). The TBA is comprised of one or more molecules, one or
more of which can bind to TBR sequences in a specific and sequence
or conformation dependent manner. The TBA may comprise one or more
piloting sequences, called "PILOTS" or "Asymmetry Sequences," which
assemble and constrain the nucleotide binding components of the TBA
to specific geometries. The PILOTS act to assemble specific nucleic
acid recognition units or other pilots to which specific nucleic
acid recognition units are attached into the TBAs in a
predetermined fashion. The TBA may also contain one or more
molecules which anchor or localize the TBA. Novel TBAs having
unique discriminating characteristics which surprisingly render the
TBAs useful not only as diagnostic tools but also as prophylactic
or therapeutic compounds, are also disclosed. Disclosed are methods
and compositions for utilization of the PNAs, TBRs, TBAs, and TBA
PILOTS, including their utilization as components of diagnostic and
forensic test kits and the utilization of the novel TBAs as
prophylactic or therapeutic agents.
[0019] The PNAs, in addition to TNA-specific sequences, may also
contain one or more sequences, 1/2 BBRs, capable of hybridizing
with complementary 1/2 BBRs in Booster Nucleic Acids (BNAs).
Through hybridization of added BNAs to the starter 1/2 BBRs present
in the PNAs, extensions of the PNAs are made in the form of PNA-BNA
and then BNA-BNA hybrids. These extensions can contain one or more
Booster Binding Regions (BBRs). Each BBR is capable of binding a
Booster Binding Assembly (BBA). The BBA is comprised of molecules,
one or more of which can bind to a BBR in a specific and sequence
or conformation dependent manner. The BBA may comprise one or more
piloting sequences, called "PILOTS" or "Asymmetry Sequences," which
assemble and constrain the nucleotide binding components of the TBA
to specific geometries. The PILOTS act to assemble specific nucleic
acid recognition units or other pilots to which specific nucleic
acid recognition units are attached into the BBAs in a
predetermined fashion. The BBA may contain molecules which anchor
or localize the BBA or which allow for detection of the bound BBAs
and thereby of the TBA-TNA-PNA complexes to which they, in turn,
are bound. Disclosed are methods and compositions for utilization
of the 1/2 BBRs, BNAs, BBRs, BBAs, and BBA PILOTS, including their
utilization as components of diagnostic and forensic test kits.
[0020] Methods and compositions are disclosed for the use of
Hairpin Nucleic Acids (HNAs) as capping structures. The HNAs
contain a self-hybridizing region and a single stranded 1/2 BBR
which, under hybridizing conditions, can hybridize directly to the
1/2 BBRs in the PNAs or the 1/2 BBRs in BNAs already bound to the
PNAs, to terminate the extension of BNAs onto the PNA or onto other
BNAs.
[0021] Methods and compositions are disclosed for test procedures
and the production of a test kit containing PNAs, TBAs, TBRs, BNAs,
BBRs, BBAs and HNAs for the detection, localization and
differentiation of specific nucleic acid sequences, including
nucleic acid sequences which are found in human cells, in the Human
Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), and in
other nucleic acid containing systems including viruses and
bacteria.
[0022] Accordingly, it is an object of this invention to provide
methods and compositions for use in binding, detecting, and
amplifying the detection of specific Target Nucelic Acid sequences
in a sample with fidelity and accuracy, even in the presence of
closely related but different nucleic acid sequences. Accordingly,
it is an object of this invention to provide methods and
compositions for the creation of Target Binding Assemblies which
specifically bind Target Binding Regions formed by the
hybridization of Probe Nucleic Acids and Target Nucleic Acid
sequences.
[0023] Another object of this invention is to provide a method and
compositions for the creation of Booster Binding Assemblies which
specifically bind Booster Binding Regions formed by the
hybridization of Booster Nucleic Acid sequences with Probe Nucleic
Acids, Booster Nucleic Acids and Hairpin Nucleic Acids.
[0024] Another object of this invention is to provide a method and
compositions containing Hairpin Nucleic Acids which enable the
control of the size of specifically or non-specifically elongated
Booster Nucleic Acids and Booster Binding Assemblies used in
amplification of PNA-TNA hybridization events.
[0025] Another object of this invention is to provide a method and
compositions for use in the selection, assembly and or chaperoning
of specific molecules, each with nucleic acid binding
discriminating capabilities, into Target and Booster Binding
Assemblies.
[0026] Another object of this invention is to provide a method and
compositions for use in amplifying the detection of Target Binding
assemblies bound to Target Binding Regions using Booster Binding
Assemblies and Booster Nucleic Acids.
[0027] Another object of this invention is to provide a method and
compositions which allow the use of one or more detectable labels,
including but limited to radioactive labels, light emitting,
fluorescent, enzymatic or other signal generating molecules. These
labels are used in association with Probe Nucleic Acids, Target
Binding Assemblies, Booster Binding Assemblies, Booster Nucleic
Acids or Hairpin Nucleic Acids.
[0028] Another object of this invention is to provide a method for
isolating nucleic acid fragments form an organism which has TBA
component binding sites in order to create Probe Nucleic Acids and
TBAs which are unique for that fragment or organism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The following illustrations are contained in FIG. 1: FIG.
1-I is a PNA containing a 1/2 TBR, which is a single-stranded
sequence which is complementary to a TNA and a 1/2 BBR sequence.
FIG 1-IIa is a TNA to which is added the components of FIG. 1-I,
and, under hybridizing conditions, binds the PNA to form the
components of FIG. 1-IIIa, a PNA-TNA hybrid containing at least one
TBR. FIG. 1-IVa is a BNA which is added to the components of FIG.
1-IIIa and, under hybridizing conditions, binds the 1/2 BBR of FIG.
1-IIIa to form a PNA-BNA hybrid containing a BBR shown in FIG.
1-Va.
[0030] FIG. 1-IIb is a BNA which is added the components of FIG.
1-I, and which, under hybridizing conditions, binds the PNA to form
the components of FIG. 1-IIIb, a PNA-TNA hybrid containing a BBR.
FIG. 1-IVb is a TNA to which is added the components of FIG. 1-IIIb
and which, under hybridizing conditions, binds the 1/2 TBR of FIG.
1-IIIb to form a PNA-BNA hybrid containing a TBR shown in FIG.
1-Vb.
[0031] FIG. 1-IIc is a HNA which is added to the components of FIG.
1-I and which, under hybridizing conditions, binds the PNA to form
the components of FIG. 1-IIIc, a PNA-HNA hybrid containing a BBR.
FIG. 1-IVc is a TNA which is added to the components of FIG. 1-IIIc
and which, under hybridizing conditions, binds the 1/2 TBR of FIG.
1-IIIc to form a PNA-BNA hybrid containing a BBR shown in FIG.
1-Vc.
[0032] The hybrids which form the TBRs and BBRs are useful in the
present invention. The PNAs and BNAs, as indicated in FIG. 1, may
contain no attached support and/or indicator (OSA), or an attached
support or other means of localization, including, but not limited
to, attachment to beads, polymers, and surfaces, and/or
indicators.
[0033] FIG. 2a is a diagram of strategies for polymerization of
BNAs onto PNAs and capping by HNAs.
[0034] FIG. 2b is a diagram of additional strategies for amplifying
PNA-TNA signals via polymerization of BNAs and capping by HNAs.
[0035] FIG. 3 is a diagram showing the use of BNAs containing
multiple 1/2 BBRs per BNA.
[0036] FIG. 4a is a diagram showing the binding of TBAs and BBAs to
TBRs and BBRs, and the ability of the TBA to discriminate between
TNAs and CNAs. According to this embodiment, if the TBA is
immobilized, either on a bead, microtiter plate surface, or any
other such surface, only complexes such as complex X would be
retained and detected, while complexes such as complex XI would
not.
[0037] FIG. 4b is a diagram exemplifying events similar to those
shown in FIG. 4a but in a slightly different order of
occurrence.
[0038] FIG. 5 is a diagram exemplifying PNAs containing between one
1/2 TBR and no 1/2 BBR to PNAs containing up to five 1/2 TBRs and
one 1/2 BBR. The (a) and (b) members of each numeral (I, II, III,
IV, V) form a set which, upon hybridization to a TNA, provide TBRs
either with ((a) members) or without ((b) members) an available 1/2
BBR for amplification via hybridization to BNAs having
complementary 1/2 BBRs.
[0039] FIG. 6a is a diagram exemplifying a particular TNA having
two 1/2 TBRs which, upon binding an appropriate PNA, forms two
closely associated TBRs capable of binding two TBAs. A 1/2 BBR is
also provided for amplification.
[0040] FIG. 6b is a diagram showing the same events as in FIG. 6a
except here, a double TBA is used so that discrimination between
single TBRs that occur in normal cellular samples may be
discriminated from abnormal, double TBRs.
[0041] FIG. 6c is a diagram showing the same scenario as in FIG. 6a
except that here, five TBRs are identified in the TNA. Each TBR may
be bound to a TBA same or different, and each TBA may be
differentially labeled, allowing for confirmation that all five
sites are present in the TNA.
[0042] FIG. 6d is a diagram of the same events as in FIG. 6c except
here, a double TBA is shown, extending what is shown in FIG. 6b to
the use of the double TBA. An example of the TNA shown in item II
in FIGS. 6a, 6b, 6c and 6d is HIV single stranded DNA or RNA.
[0043] FIG. 7 shows the HIV LTR as a TNA, and two PNAs, and a
strategy for detection of the TNA using the PNAs.
[0044] FIG. 8 is a schematic of one embodiment of the invention
wherein a target binding assembly is used to bind a hybrid TNA-PNA,
and booster binding assemblies are used to bind polymerized
BNAs.
[0045] FIG. 9 is a schematic of a modular TBA in which assembly
sequences, linker sequences, and asymmetry sequences are used to
chaperone desired nucleic acid recognition units together to form a
TBA.
[0046] FIG. 10 shows modular TBAs useful in detection of
HIV-specific sequences.
[0047] FIG. 11 shows modular TBAs useful in the detection of human
papillomavirus sequences. Each unit of E2 is actually a dimer of
the DNA binding portion of E2.
[0048] FIG. 12a is a schematic of TNA fractionation and shift in
mobility due to binding of a TBA.
[0049] FIG. 12b is a schematic of TNA fractionation and enhanced
shift in mobility due to binding of BBAs in addition to TBAs.
[0050] FIG. 13 shows a detection strategy for deletion sequences;
an example of use of this strategy is for a human papillomavirus
integration assay.
[0051] FIG. 14 shows assembly of higher order TBAs through use of
nucleic acid recognition units, linker, assembly, and asymmetry
sequences such that various Target Binding Assemblies specific to
binding sites in the HIV LTR are formed.
[0052] FIG. 15 shows assembly of higher order TBAs through use of
DNA recognition units, linker, assembly, and asymmetry sequences
such that various Target Binding Assemblies specific to binding
sites in the HPV genome are formed.
[0053] FIG. 16 shows the discrimination achieved by using a complex
TBA and the ability of endogenous competitor target binding
molecules to eliminate binding of the TBA to a cousin nucleic acid
but not from the TNA which contains the appropriate orientation of
more than one site recognized by the TBA.
[0054] FIG. 17 shows the ability of a TBA to specifically be
targeted to bind to sites of sequence mismatch and to
preferentially bind those sites over cousin sites which do not
contain all of the targeted mismatches.
BRIEF DESCRIPTION OF THE SEQUENCES
[0055] SEQ ID NO. 1 corresponds to FIG. 5-Ia-1 and shows the class
I MHC NF-kB binding site.
[0056] SEQ ID NO. 2 corresponds to FIG. 5(Ia) and shows the
B2-microglobulin NF-kB binding site.
[0057] SEQ ID NO. 3 corresponds to FIG. 5(Ia) and shows the kappa
immunoglobulin NF-kB binding site.
[0058] SEQ ID NO. 4 corresponds to FIG. 5(Ia) and shows one of the
HIV NF-kB binding sites.
[0059] SEQ ID NO. 5 corresponds to FIG. 5(Ia) and shows one of the
HIV NF-kB binding sites.
[0060] SEQ ID NO. 6 corresponds to FIG. 5(Ia) and shows the c-myc
NF-kB binding site.
[0061] SEQ ID NO. 7 corresponds to FIG. 5(IIa) and shows a double
HIV NF-kB binding site.
[0062] SEQ ID NO. 8 corresponds to FIG. 5(IIa) and shows a double
HIV NF-kB binding site.
[0063] SEQ ID NOS. 9-16 correspond to FIG. 5(IIa) and show a double
binding site with one site being an HIV NF-kB binding site, and the
other site being an HIV SP1 binding site.
[0064] SEQ ID NOS. 17-18 correspond to FIG. 5(IIa) and show a
double HIV SP1 binding, site.
[0065] SEQ ID NOS. 19-31 correspond to FIG. 5(IIIa) and show a
double HIV NF-kB binding site and an HIV SP1 binding site.
[0066] SEQ ID NOS. 32-33 correspond to FIG. 5(IVa) and show a
quadruple binding site where two sites are HWV NF-kB binding sites
and two sites are HIV SP1 binding sites.
[0067] SEQ ID NO. 34 corresponds to FIG. 5 VIa) and shows a
quintuple binding site where two sites are HIV NF-kB binding sites
and three sites are HIV SP1 binding sites.
[0068] SEQ ID NO. 35 is an example of a 1/2 BBR, in this case the
OL1, OL2 and OL3 elements of the bacteriophage lambda left
operator, including intervening sequences.
[0069] SEQ ID NO. 36 is an example of a 1/2 BBR, in this case the
OR3, OR2 and OR1 elements of the bacteriophage lambda right
operator, including intervening sequences.
[0070] SEQ ID NO. 37 is the HIV LTR.
[0071] SEQ ID NO. 38 is a PNA complementary to PNA of the HIV
LTR.
[0072] SEQ ID NO. 39 is a PNA complementary to a different PNA of
the HIV LTR than SEQ ID NO. 38.
[0073] SEQ ID NO. 40 is a PNA complementary to part of the HIV LTR
and it also contains a 1/2 BBR and an overhang sequence for
polymerizing BNAs onto the PNA.
[0074] SEQ ID NO. 41 is a BNA complementary to the SEQ ID NO. 40
1/2 BBR.
[0075] SEQ ID NO. 42 is a BNA that will polymerize onto the SEQ ID
NO. 41 BNA and which, with SEQ ID NOS. 40 and 41, creates a PstI
recognition site.
[0076] SEQ ID NO. 43 is a BNA that is complementary to the SEQ ID
NO. 42 BNA and which completes a BamHI recognition site.
[0077] SEQ ID NO. 44 is an HNA which has a BamHI recognition site
that will hybridize with the BamHI recognition site created by SEQ
ID NOS. 42 and 43 to the growing polymer.
[0078] SEQ ID NO.45 is a second PNA which, like SEQ ID NO. 40, is
complementary to part of the HIV LTR, but not to the same sequence
as SEQ ID NO. 40. SEQ ID NO. 45 also encodes a 1/2 BBR and an
overhang which will allow polymerization of BNAs starting with a
Sph1 recognition site.
[0079] SEQ ID NOS. 46-62 are human papillomavirus (HPV) specific
PNAs which, upon hybridization with HPV sequences, form TBRs which
bind HPV DNA binding proteins.
[0080] SEQ ID NOS. 63-71 are NF-kB DNA recognition units for
incorporation into TBAs.
[0081] SEQ ID NO. 72 is a nuclear localization sequence.
[0082] SEQ ID NO. 73 is a SP1 sequence recognition unit.
[0083] SEQ ID NO. 74 is a TATA binding protein recognition
unit.
[0084] SEQ ID NOS. 75-84 are papillomavirus E2 DNA recognition
units.
[0085] SEQ ID NOS. 85-92 are asymmetry sequences.
[0086] SEQ ID NO. 93 is an arabidopsis TATA binding protein
recognition unit.
[0087] SEQ ID NO. 94 is an HPV-16-E2-1 DNA binding protein
recognition unit.
[0088] SEQ ID NO. 95 is an HPV-16-E2-2 DNA binding protein
recognition unit.
[0089] SEQ ID NO. 96 is an HPV-18-E2 DNA binding protein
recognition unit.
[0090] SEQ ID NO. 97 is an HPV-33-E2 DNA binding protein
recognition unit.
[0091] SEQ ID NO. 98 is a bovine papillomavirus E2 DNA binding
protein recognition unit.
[0092] SEQ ID NOS. 99-102 are exemplary linker sequences.
[0093] SEQ ID NO. 103 is an exemplary nuclear localization signal
sequence (NLS).
[0094] SEQ ID NOS. 104-108 are exemplary chaperone sequences.
[0095] SEQ ID NOS. 109-116 are exemplary assembled TBA
sequences.
[0096] SEQ ID NO. 117 is a consensus NF-kB binding site.
[0097] SEQ ID NO. 118 an HIV Tat amino acid sequence.
1 Abbreviations 1 single stranded nucleic acid 2 double-stranded
nucleic acid 3 binding region on nucleic acid 4 no support or
indicators, or solid support, or other means of localization,
including, but not limited to, attachment to beads, polymers, and
surfaces, or indicators = GSA BBA booster binding assembly BBR
booster binding region BNA booster nucleic acid CNA cousin nucleic
acid 1/2 BBR single-stranded region which, when hybridized to the
complementary sequence from an HNA or a BNA, can bind a BBA 1/2 TBR
single-stranded region of the PNA which, when hybridized to the
complementary sequence from a TNA, can bind a TBA OSA optional
support or attachment, circle with box PNA probe nucleic acid TBA
target binding assembly TBR target binding region TNA target
nucleic acid HNA Hairpin Nucleic Acid
Definitions
[0098] It should also be understood from the disclosure which
follows that when mention is made of such terms as target binding
assemblies (TBAs), booster binding assemblies (BBAs), DNA binding
proteins, nucleic acid binding proteins or RNA binding proteins,
what is intended are compositions comprised of molecules which bind
to DNA or RNA target nucleic acid sequences (TNAs) irrespective of
the specificity of the category of binding molecules from which
they are derived. Thus, for example, a TBA adapted to bind to human
immunodeficiency virus sequences may be most similar to an NF-kB
transcriptional factor which typically binds DNA sequences.
However, as used herein, it will be understood that the TBA may be
adapted for optimal use to bind to RNA sequences of a particular
sequence composition or conformation.
[0099] The fidelity of the detection method disclosed herein
depends in large measure on the selective binding of TBAs and BBAs
to particular nucleic acid motifs. It should be understood
throughout this disclosure that the basis of TBA and BBA
discrimination of TNAs from related sequences (cousin nucleic acids
or CNAs) may be the formation of precise probe nucleic acid
(PNA)-target nucleic acid (TNA) hybrid segments (PNA-TNA hybrids).
However, the basis of discrimination may just as well be the
formation of a particular conformation, and may not require the
complete absence of mismatched-base pairing in the TNA-PNA hybrid.
Accordingly, the basis of TBA or BBA operation should be understood
throughout to depend on discrimination of any property unique to
the TNA-PNA hybrid as opposed to any properties displayed by any
PNA-CNA hybrids that may be formed in a test sample contacted with
a given PNA.
DETAILED DISCLOSURE OF THE INVENTION
[0100] The present invention provides a method for specifically
identifying a target nucleic acid (TNA) in a sample through the use
of target binding assemblies (TBAs) which incorporate specific
nucleic acid binding proteins. By using probe nucleic acids (PNAs)
specific to a given TNA sequence, and a TBA which is specific to
the duplex target binding region (TBR) formed upon formation of
hybrid TNA-PNA sequences, a stable TBA-TNA-PNA complex is formed.
By additionally providing specific amplifiable sequences in the
PNA, in addition to sequences which specifically contribute to the
formation of the TBR recognized by the TBA, the binding of the PNA
to the TNA is detected and the detection amplified. For this
purpose, any of a number of nucleic acid amplification systems,
including polymerase chain reaction, or the use of branched DNA,
each branch of which contains a detectable label, may be used. In
particular, a novel method of amplification is described herein
where the amplifiable portion of the PNA contains sequences onto
which booster nucleic acids (BNAs) may be polymerized. Upon
formation of each BNA-PNA hybrid, a booster binding region (BBR) is
formed to which a booster binding assembly (BBA) binds
specifically. If detectably labeled, the BBAs or BNAs provide
essentially unlimited amplification of the original TNA-PNA binding
event.
[0101] According to this invention, the TNA will be understood to
include specific nucleic acid sequences. The TBA will be understood
to be any molecular assembly which can specifically and tightly
bind to a formed TNA-PNA hybrid. The TBA will contain one or more
molecules whose sequences are sufficient to bind to the TBR.
Nucleic acid binding domains which are known can either be used
directly as components of the TBA or modified according to the
teachings provided herein. The most readily available molecules
with such sequences are the DNA-binding domains of DNA-binding
proteins. Specifically, many DNA or RNA binding proteins are known
which can either be used directly as the known, unmodified protein,
or the TBA may be a nucleic acid binding protein, modified
according to the specific teachings provided herein. In the latter
case, specific modifications that are desirable would include
optimization of binding affinities, removal of unwanted activities
(such as nuclease activity and reorganization of the TBA in the
presence of other molecules with an affinity for components of the
TBA), optimization of selectivity of a target sequence over closely
related sequences, and optimization of stability.
[0102] Examples of DNA binding proteins which could be used
according to this invention are the DNA-binding portions of the
transcription factor NF-kB (p50 and p65), NF-WL6, NF-AT, rel, TBP,
the papilloma virus' E2 protein, sp1, the repressors cro and CI
from bacteriophage lambda, and like proteins are well known
proteins whose DNA binding portion has been isolated, cloned,
sequenced, and characterized. In addition, any other DNA-binding
protein or portion of a protein that is necessary and sufficient to
bind to a TBR hybrid or a BBR is included. This includes proteins
or portions of wild-type proteins with altered DNA binding activity
as well as protein created with altered DNA-binding specificity,
such as the exchange of a DNA-binding recognition helix from one
protein to another. In addition, proteins which exhibit nucleic
acid binding and other nucleic acid functions, such as restriction
endonucleases, could be used as the nucleic acid binding function.
Proteins which bind to target regions in DNA-RNA hybrids as well as
RNA-RNA hybrids are included. (See, for example, Shi 1995,
DeStefano 1993, Zhu 1995, Gonzales 1994, Salazar 1993, Jaishree
1993, Wang 1992, Roberts 1992, Kainz 1992, Salazar 1993(b)). The
binding assemblies may be constructed with the use of a molecule
which chaperones portions of the binding assembly so that specific
component combinations and geometries can be achieved. This
molecule is designated here as a PILOT. Pilots can be comprised of
proteins or any combination of organic and inorganic materials
which achieve the combinatorial selection and/or to induce specific
geometries between members of the TBA or BBAs. A chaperone is a
stable scaffold upon which a TBA or BBA may be constructed such
that the correct conformation of the TBA or BBA is provided while
at the same time eliminating undesirable properties of a naturally
occurring nucleic acid binding protein. As a specific example of
this embodiment, a modified version of the pleiotropic
transcription factor, NF-kB, is provided using a modified
bacteriophage lambda cro protein as the chaperone. Each NF-kB
binding dimer retains the picomolar binding affinity for the NF-kB
binding site while at the same time the binding assembly presents
several advantageous manufacturing, stability, and specificity
characteristics.
[0103] In view of the foregoing, the various aspects and
embodiments of this invention are described below in detail.
[0104] 1. The Probe Nucleic Acids (PNAs) and their preparation. The
PNAs of the present invention comprise at least three principal
parts joined together. With reference to FIG. 1(I) of the drawings,
the first part of the PNA is one or more sequences of bases,
designated "1/2 TBR." With reference to FIG. 1(I and IIa) of the
drawings, the 1/2 TBR-in the PNA is complementary to a sequence of
interest in a sample, the TNA containing a 1/2 TBR. With reference
to FIG. 1(IIIa) of the drawings, the TNA, when added to the PNA
under hybridizing conditions, forms a PNA-TNA hybrid containing a
TBR. With reference to FIG. 1(I) of the drawings, the second part
of the PNA is a sequence of bases, designated "1/2 BBR." With
reference to FIG. 1(I, IIb, IIc, and IVa) of the drawings, the 1/2
BBR in the PNA is complementary to a 1/2 BBR contained in a BNA or
a HNA. With reference to FIG. 1(IIIb, IIIc, and Va) of the
drawings, the BNA or HNA, when added to the PNA under hybridizing
conditions, forms a PNA-BNA hybrid or PNA-HNA hybrid, respectively,
containing a BBR. With reference to FIG. 1(I) of the drawings, the
third part of the PNA is the OSA, designated by a circle with a box
around it. The OSA is no support and/or an indicator, or solid
support, or other means of localization, including but not limited
to, attachment to beads, polymers, and surfaces and/or indicators
which is/are covalently attached to, or non-covalently, but
specifically, associated with the PNA. The OSA may be an atom or
molecule which aids in the separation and/or localization such as a
solid support binding group or label which can be detected by
various physical means including, but not limited to, adsorption or
imaging of emitted particles or light. Methods for attaching
indicators to oligonucleotides or for immobilizing oligonucleotides
to solid supports are well known in the art (see Keller and Manak,
supra, herein incorporated by reference).
[0105] The PNA of the present invention can be prepared by any
suitable method. Such methods, in general, will include
oligonucleotide synthesis and cloning in a replicable vector.
Methods for nucleic acid synthesis are well-known in the art. When
cloned or synthesized, strand purification and separation may be
necessary to use the product as a pure PNA. Methods of preparing
RNA probes are well known (see for example Blais 1993, Blais 1994,
which uses in vitro transcription from a PCR reaction incorporating
a T7 RNA polymerase promoter).
[0106] The length and specific sequence of the PNA will be
understood by those skilled in the art to depend on the length and
sequence to be detected in a TNA, and the strictures for achieving
tight and specific binding of the particular TBA to be used (see
discussion on TBAs below). In general, PNAs of sequence lengths
between about 10 and about 300 nucleotides in length are adequate,
with lengths of about 15-100 nucleotides being desirable for many
of the embodiments specifically exemplified herein.
[0107] It should also be understood that the PNA may be constructed
so as to contain more than one 1/2 TBR and to produce more than one
TBR for one or more TBAs, same or different, as well as complex
TBRs recognized by novel duplex and multiplex TBAs (see description
below regarding these novel TBAs) upon hybridization of the PNAs
and TNAs. FIG. 5 illustrates specific PNAs which contain one or
more 1/2 TBRs. Specific sequences which correspond to the 1/2 TBR
sequences illustrated in FIG. 5(Ia, IIa, IIIa, IVa, and Va) are SEQ
ID NOS. 1-34 (see Description of Sequences above).
[0108] As shown in FIGS. 2a and 2b, the PNA, containing a 1/2 TBR,
may be hybridized with one or more BNAs (see description below) and
the chain of BNAs polymerized to any desired potential length for
amplification of the TNA-PNA hybridization event. Preferably,
between about 0 and about 10 1/2 BBRs will be present in the
PNA.
[0109] As shown in FIGS. 6a and 6b, the PNA may contain several 1/2
TBRs, same or different, which can hybridize with several 1/2 TBRs
in a TNA. Each time a 1/2 TBR in the PNA matches a 1/2 TBR in a
TNA, a Target Binding Region, TBR, is formed which can bind a TBA.
Furthermore, it is not essential that all of the TBRs be on a
single, contiguous PNA. Thus, in one embodiment of the invention,
two different PNAs are used to detect sequences on a particular
TNA. As an illustration of this aspect of the invention, FIG. 7
shows one representation of the human immunodeficiency virus (HIV)
long terminal repeat (LTR). As is known in the art, the HIV LTR
comprises two NF-kB binding sites and three SP1 binding sites, in
close proximity, wherein NF-kB and SP1 are known DNA binding
proteins. FIG. 7 provides two PNAs, PNA1 (SEQ ID NO. 38) and PNA2
(SEQ ID NO. 39), each of which is complementary to the opposite
strand shown as a TNA (SEQ ID NO. 37), which shows the two NF-kB
binding sites and the three SP1 binding sites of the HIV LTR.
According to this aspect of the invention, PNA1 specifically
hybridizes with that section of the TNA shown in FIG. 7 with bases
underscored with a "+" symbol, while PNA2 specifically hybridizes
with that section of the TNA shown in FIG. 7 with bases underscored
with an "=" symbol. Each of PNA1 or PNA2 may also contain sequences
(indicated by the symbols "#" or "*") which will hybridize with a
BNA's 1/2 BBR sequences (see below). In addition, each of PNA1 and
PNA2 may be differentially tagged with an OSA, such as a
fluorophore such as a fluorescein or a rhodamine label, which would
allow confirmation that both probes have become bound to the TNA.
If only one label or neither label is detected, it is concluded
that the TNA is not present in the sample being tested.
[0110] In a further aspect of the embodiment shown in FIG. 7, a
method for altering the specificity of the instant assay method is
shown. By changing the length of the gap between PNA1 and PNA2,
such that the region of TNA remaining unhybridized is altered, one
practicing this invention is able to alter the discrimination of
the assay.
[0111] In order to more clearly exemplify this aspect of the
invention, it is necessary to emphasize that the TBR may have a
helical structure. Thus, while PNA1 creates TBRs on one "face" of
the helix, PNA2 creates a TBR on either the same or a different
face of the helix, depending on the distance between the middle of
each TBR (underlined in FIG. 7). If the middle of each binding site
is an integral product of 10.5 bases apart, the TBRs will be on the
same side of the helix, while non-integer products of 10.5 bases
apart would place the TBRs on opposite sides of the helix. In this
fashion, any cooperativity in binding by the TBA recognizing the
PNA1 TBR and the TBA recognizing the PNA2 TBR can be manipulated
(see Hochschild, A., M. Ptashne [1986] Cell 44:681-687, showing
this effect for the binding of bacteriophage lambda repressor to
two different operator sites located at different distances from
each other in a DNA helix). As described by Perkins et al. ([1993]
EMBO J. 12:3551-3558), cooperativity between NF-kB and the SP1
sites is required to achieve activation of the HIV LTR. However,
for the purpose of the instant invention, the double NF-kB-triple
SP1 binding site motif in the HIV LTR may be taken advantage of by
providing a single, novel binding protein capable of binding both
sites simultaneously, but only if the spacing between the sites is
geometrically feasible. This is controlled both by the structure of
the selected TBA and by the PNAs used. Thus, in the embodiment
exemplified in FIG. 7, the two probes may be used with a large
enough interprobe region of single-stranded DNA remaining such
that, even if the NF-kB and SP1 binding sites are on opposite sides
of the helix, the single-stranded region between the probes
provides a sufficiently flexible "hinge" so that the DNA can both
bend and twist to accommodate the geometry of the TBA.
Alternatively, a more stringent assay may be designed by narrowing
the interprobe distance such that the DNA may only bend, but not
twist. Finally, the probes may be so closely spaced, or a single
PNA used, such that the DNA can only bend but not twist. Thus, this
figure exemplifies and enables the production of detection systems
with any given desired degree of discrimination between target
nucleic acids having similar sequences, but different
juxtapositions of these sequences.
[0112] In terms of a diagnostic or forensic kit for HIV, those
skilled in the art would understand that the aforementioned aspects
of this invention allow for the tailoring of the components of the
diagnostic or forensic kit to match what is known at any given time
about the prevalent strains of HIV or another pathogen or disease
condition. It will also be appreciated by those skilled in the art
that, while detection of HIV infection is not the only utility of
the instant invention, due to the mutability of the HIV genome, it
is probably one of the most complex test environments for such a
diagnostic. It is precisely in such a mutable environment, however,
where the flexibility of the instant method, coupled with its
ability to discriminate between very closely related sequences, may
be most clearly appreciated. In less mutable environments, some of
the sophistication to which this invention is amenable need not be
utilized. Thus, in a diagnostic kit for papillomavirus infection,
all of the discrimination characteristics of the TBA-TBR
interaction are available, along with the ability to amplify the
signal using the BNAs and BBAs, but a single, simple PNA, such as
any one of SEQ ID NOS. 46-62, may be used which identifies unique
papillomavirus sequences, which also are known to bind to a TBA
such as the papillomavirus E2 protein or truncated DNA binding
portions thereof (see Hegde et al. [1992] Nature 359:505-512;
Monini et al. [1991] J. Virol. 65:2124-2130).
[0113] In applying the instant method to the detection of a
particular TNA for the purposes of assessing whether certain
nucleic acids are present which are associated with the progression
of melanoma, hepatoma, breast, cervical, lung, colon, prostate,
pancreatic or ovarian cancers, the TNA may be obtained from biopsy
materials taken from organs and fluids suspected of containing the
cancerous cells. For the detection of genetic deficiencies, the TNA
may be obtained from patient samples containing the affected cells.
For detection of fermentation contaminants and products in the
manufacture of food, chemical or biotechnology products or in the
bioremediation of wastes, the TNA may be obtained from samples
taken at various stages in the fermentation or treatment process.
For detection of food or drug pathogens or contaminants, the TNA
sample may be obtained from the food or drug, swabs of food or
surfaces in contact with the food, fluids in contact with the food,
processing materials, fluids and the like associated with the
manufacture of or in contact with the food, drug, or biological
samples taken from those in contact with the food or drug or the
like.
[0114] 2. The Booster Nucleic Acids (BNAs), Booster Binding Regions
(BBRs) and their preparation. The BNAs of the present invention are
comprised of at least one or more 1/2 BBRs coupled to an OSA. The
1/2 BBRs can hybridize to complementary 1/2 BBRs contained in the
PNA, other BNAs or an HNA.
[0115] With reference to FIG. 1(I, IIb and IIIb) of the drawings,
the simplest BNA is comprised of two parts. With reference to FIG.
1(IIb) of the drawings, the first part of the simplest BNA is a
sequence of bases which is complementary to the sequence in the PNA
which is designated "1/2 BBR." With reference to FIG. 1(IIb) of the
drawings, the second part of the simplest BNA is the OSA,
designated by a circle with a box around it. The OSA is no support
and/or indicator, or solid support, or other means of localization,
including but not limited to, attachment to beads, polymers, and
surfaces and/or indicators which are covalently attached to, or
non-covalently, but specifically, associated with the BNA.
[0116] With reference to FIG. 2a(II and III) of the drawings, the
BNA may contain more than one 1/2 BBR sequence. The BNA illustrated
in FIG. 3(II) contains a sequence which is complementary to the PNA
illustrated in FIG. 3(I) and two other 1/2 BBR sequences. The BNA
illustrated in FIG. 3(III) contains two 1/2 BBR sequences which are
complementary to two of the 1/2 BBR sequences in the BNA
illustrated in FIG. 3(II), plus up to "n" additional 1/2 BBRs for
polymerization of additional BNAs.
[0117] Under hybridizing conditions, the BNA illustrated in FIG.
3(II), when combined with the PNA illustrated in FIG. 3(I), creates
the PNA-BNA hybrid illustrated in FIG. 3(IVa) containing a BBR and
an unhybridized extension with two additional 1/2 BBR sequences or
"booster" sequences. The BBRs created by said hybridization can be
identical, similar or dissimilar in sequence. The BBRs created by
said hybridization can bind identical, similar or dissimilar BBAs
(see below). The BNAs may have prepared analogously to the
PNAs.
[0118] Under hybridizing conditions, the BNA-BNA hybrid illustrated
in FIG. 3(IVb), when combined with the PNA illustrated in FIG.
3(Vb), creates the PNA-BNA hybrid illustrated in FIG. 3(VI)
containing a BBR, two additional BNA-BNA hybrids containing BBRs,
and an unhybridized extension with an additional 1/2 BBR sequence,
a "booster" sequence. The BBRs created by said hybridization can be
identical, similar or dissimilar in sequence. The BBRs created by
said hybridization can bind identical, similar or dissimilar BBAs
(see below). The BNAs may be prepared in a fashion analogous to
preparation of the PNAs.
[0119] 3. The Target Nucleic Acids (TNAs) and their preparation.
The first step in detecting and amplifying signals produced through
detection of a particular TNA according to the present method is
the hybridization of such target with the PNA in a suitable
mixture. Such hybridization is achieved under suitable conditions
well known in the art.
[0120] The sample suspected or known to contain the intended TNA
may be obtained from a variety of sources. It can be a biological
sample, a food or agricultural sample, an environmental sample and
so forth. In applying the instant method to the detection of a
particular TNA for the purposes of medical diagnostics or
forensics, the TNA may be obtained from a biopsy sample, a body
fluid or exudate such as urine, blood, milk, cerebrospinal fluid,
sputum, saliva, stool, lung aspirates, throat or genital swabs and
the like. In addition, detection may be in situ (see for example
Embretson 1993; Patterson 1993; Adams 1994).
[0121] Accordingly, PNAs specific to vertebrates (including mammals
and including humans) or to any or all of the following
microorganisms of interest may be envisioned and used according to
the instant method:
2 Corynebacteria Corynebacterium diphtheria Bacillus Bacillus
thuringiensis Pneumococci Diplococcus pneumoniae Streptococci
Streptococcus pyogenes Streptococcus salivarius Staphylococcus
Staphylococcus aureus Staphylococcus albus Pseudomonas Pseudomonas
stutzen Neisseria Neisseria meningitidis Neisseria gonorrhea
Enterobacteriaceae Escherichia coli Aerobacteria aerogenes
Klebsiella pneumoniae The coliform bacteria Salmonella typhosa
Salmonella choleraesuis The Salmonellae Salmonella typhimurium
Shigellae dysenteriae Shigellae schmitzii Shigellae arabinotarda
Shigellae flexneri The Shigellae Shigellae boydii Shigellae sonnei
Other enteric bacilli Proteus vulgaris Proteus mirabilis Proteus
species Proteus morgani Pseudomonas aeruginosa Alcaligenes faecalis
Vibrio cholerae Hemophilus-Bordetella group Hemophilus influenza,
H. ducryi Hemophilus hemophilus Hemophilus aegypticus Hemophilus
parainfluenzae Bordetella pertussis Pasteurellae Pasteurella pestis
Pasteurella tulareusis Brucellac Brucella melitensis Brucella
abortus Brucella suis Aerobic Spore-Forming Bacilli Bacillus
anthracis Bacillus subtilis Bacillus megaterium Bacillus cereus
Anaerobic Spore-Forming Bacilli Clostridium botulinum Clostridium
tetani Clostridium perfringens Clostridium novyi Clostridium
septicum Clostridium histolyticum Clostridium tertium Clostridium
bifermentans Clostridium sporogenes Mycobacteria Mycobacterium
tuberculosis hominis Mycobacterium bovis Mycobacterium avium
Mycobacterium leprae Mycobacterium paratuberculosis Actinomycetes
(fungus-like bacteria) Actinomyces isaeli Actinomyces bovis
Actinomyces naeslundii Nocardia asteroides Nocardia brasiliensis
The Spirochetes Treponema pallidum Treponema pertenue Treponema
carateum Spirillum minus Streptobacillus monilformis Borrelia
recurrens Leptospira icterohemorrhagiae Leptospira canicola
Trypanasomes Mycoplasmas Mycoplasma pneumoniae Other pathogens
Listeria monocytogenes Erysipelothrix rhusiopathiae Streptobacillus
moniliformis Donvania granulomatis Bartonella bacillformis
Rickettsiae (bacteria-like parasites) Rickettsia prowazekii
Rickettsia mooseri Rickettsia rickettsii Rickettsia conori
Rickettsia australis Rickettsia sibiricus Rickettsia akari
Rickettsia tsutsugamushi Rickettsia burnetti Rickettsia quintana
Chlamydia (unclassifiable parasites bacterial/viral) Chlamydia
agents (naming uncertain) Fungi Cryptococcus neoformans Blastomyces
dermatidis Histoplasma capsulatum Coccidoides immitis
Paracoccidioides brasiliensis Candida albicans Aspergillus
fumigatus Mucor corymbifera (Absidia corymbifera) Rhizopus oryzae
Rhizopus arrhizus Phycomycetes Rhizopus nigricans Sporotrichum
schenkii Flonsecaea pedrosoi Fonsecaea compact Fonsecacae
dermatidis Cladosporium carrioni Phialophora verrucosa Aspergillus
nidulans Madurella mycetomi Madurella grisea Allescheria boydii
Phialophora jeanselmei Microsporum gypsum Trichophyton
mentagrophytes Keratinomyces ajelloi Microsporum canis Trichophyton
rubrum Microsporum adouini Viruses Adenoviruses Herpes Viruses
Herpes simplex Varicella (Chicken pox) Herpes zoster (Shingles)
Virus B Cytomegalovirus Pox Viruses Variola (smallpox) Vaccinia
Poxvirus bovis Paravaccinia Molluscum contagiosurn Picornaviruses
Poliovirus Coxsackievirus Echoviruses Rhinoviruses Myxoviruses
Influenza (A, B, and C) Parainfluenza (1-4) Mumps virus Newcastle
disease virus Measles virus Rinderpest virus Canine distemper virus
Respiratory syncytial virus Rubella virus Arboviruses Eastern
equine encephalitis virus Western equine encephalitis virus Sindbis
virus Chikugunya virus Semliki forest virus Mayora virus St. Louis
encephalitis virus California encephalitis virus Colorado tick
fever virus Yellow fever virus Dengue virus Reoviruses Reovirus
types 1-3 Retroviruses Human immunodeficiency viruses (HIV) Human
T-cell lymphotrophic virus I & II (HTLV) Hepatitis Hepatitis A
virus Hepatitis B virus Hepatitis nonA-nonB virus Hepatitis, C, D,
E Tumor viruses Rauscher leukemia virus Gross virus Maloney
leukemia virus Human papilloma viruses
[0122] It would be understood by one of skill in the art that it is
generally required to treat samples suspected of containing a
particular TNA in such a fashion as to produce fragments that can
easily hybridize with the PNA. It may be necessary to treat the
test sample to effect release of or to extract the TNA for
hybridization, such as by exposing blood or other cells to a
hypotonic environment, or otherwise disrupting the sample using
more vigorous means. When the TNA is thought to be present in
double stranded form, it would naturally be desirable to separate
the strands to render the TNA hybridizable in single stranded form
by methods well known in the art, including but not limited to
heating or limited exposure to alkaline conditions which may be
neutralized upon addition of the single stranded PNA to allow
hybridization to occur. Methods for preparing RNA targets are well
known (see Waterhouse 1993, Mitchell 1992).
[0123] Fragmentation of nucleic acid samples containing TNAs is
usually required to decrease the sample viscosity and to increase
the accessibility of the TNAs to the PNAs. Such fragmentation is
accomplished by random or specific means known in the art. Thus,
for example, specific nucleases known to cut with a particular
frequency in the particular genome being analyzed, may be used to
produce fragments of a known average molecular size. In addition,
other nucleases, phosphodiesterases, exonucleases and
endonucleases, physical shear and sonication are all methods
amenable for this purpose. These processes are well known in the
art. The use of restriction enzymes for the purpose of DNA
fragmentation is generally preferred. However, DNA can also be
fragmented by a variety of chemical means such as the use of the
following types of reagents: EDTA-Fe(II) (according to Stroebel et
al. [1988] J. Am. Chem. Soc. 110:7927; Dervan [1986] Science
232:464); Cu(II)-phenanthroline (according to Chen and Sigman
[1987] Science 237:1197); class IIS restriction enzyme (according
to Kim et al. [1988] Science 240:504); hybrid DNAse (according to
Corey et al. [1989] Biochem. 28:8277); bleomycin (according to
Umezawa et al. [1986] J. Antibiot. (Tokyo) Ser. A, 19:200);
neocarzinostatin (Goldberg et al. [1981] Second Annual
Bristol-Myers Symposium in Cancer Research, Academic Press, New
York, p.163); and methidiumpropyl-EDTA-Fe(II) (according to
Hertzberg et al. [1982] J. Am. Chem. Soc. 104:313). Removal of
proteins, as by treatment with a protease, is also generally
desirable and methods for effecting protein removal from nucleic
acid samples, without appreciable loss of nucleic acid, are well
known in the art.
[0124] The TNAs of the present invention should be long enough so
that there is a sufficient amount of double-stranded hybrid
flanking the TBR so that a TBA can bind unperturbed by the
unligated fragment ends. Typically, fragments in the range of about
10 nucleotides to about 100,000 nucleotides, and preferably in the
range of about 20 nucleotides to about 1,000 nucleotides are used
as the average size for TNA fragments. Examples of specific TNA
sequences that could be detected are sequences complementary to the
PNA sequences described herein for detection of normal cellular,
abnormal cellular (as in activated oncogenes, integrated foreign
genes, genetically defective genes), and pathogen-specific nucleic
acid sequences, for which specific nucleic acid binding proteins
are known, or which can be produced according to methods described
in this disclosure. With reference to FIG. 7, a specific
HIV-related TNA is shown as SEQ ID NO. 37.
[0125] 4. Extensions to the PNA using BNAs, their preparation, and
signal amplification. Under hybridizing conditions, BNAs can be
added that hybridize to the PNAs, PNA-BNA hybrids, BNAs and/or
BNA-BNA hybrids. The aforementioned additions can be made in a
non-vectorial polymeric fashion or in a vectorial fashion, with a
known order of BNAs.
[0126] With reference to FIG. 2a, a simple booster is presented. A
booster polymer is produced by adding two BNAs, illustrated in FIG.
2a(Ib and Ic), which when combined under hybridizing conditions
with the PNA, form PNA-BNA-BNA hybrids, comprised of the PNA and
"booster" extensions", illustrated in FIG. 2a(IIa,IIb,IIc and IId)
leaving at least one unpaired 1/2 BBR sequence. Each unpaired 1/2
BBR sequence, illustrated in FIG. 2a(IIa, IIb, IIe, IId) can
hybridize with additional BNAs to form additional "booster"
extensions. Each unpaired 1/2 BBR sequence, illustrated in FIG.
2a(IIa,IIb,IIc and IId) can hybridize with added HNAs, illustrated
in FIG. 2a(IIIa and IIIb). The hybridization of the ITNAs, which
cannot hybridize additional BNAs, acts to "cap" the addition of the
BNAs onto the PNA, as illustrated in FIG. 2a(IVa, IVb, IVc and
IVd).
[0127] With reference to FIG. 2b, it is possible to control and
specify the order and components of extensions to the PNA. If a
single BBR is required, a HNA containing the complementary sequence
to the 1/2 BBR in the PNA is added to the PNA to produce a single
BBR and to "cap" any "booster" extensions to the PNA. If additional
BBRs are to be added to the PNA, a controlled extension of the PNA
can be accomplished.
[0128] With reference to FIG. 2b, a simple booster is presented.
Vectorial polymer extension is accomplished by adding a BNA which
is specific for the PNA, as illustrated in FIG. 2b(Ia and IIa),
which when combined under hybridizing conditions with the PNA, form
PNA-BNA-BNA hybrids, comprised of the PNA and "booster" extensions.
These extensions, if labeled with an OSA, provide a method for
greatly amplifying any signal produced upon binding of a PNA to a
TNA in the sample. Furthermore, by binding labeled BBAs to the BBRs
in the polymer, additional amplification is achieved.
[0129] Any of a number of methods may be used to prepare the BNAs,
including, e.g., synthesis via known chemistry or via recombinant
DNA production methods. In the latter method, an essentially
unlimited number of BNAs may be produced simply and inexpensively,
for example, by production in prokaryotes (E. coli for example) of
a plasmid DNA having multiple repeats of the specific BNA sequences
flanked by restriction sites having overhanging ends. In this
fashion, for example, the bacteriophage lambda left or right
operator sites, or any other DNA or other nucleic acid sequence
known to specifically and tightly bind a particular BBA, such as a
DNA or RNA binding protein, may be produced in an essentially
unlimited number of copies, with each copy flanked by an EcoRI,
PstI, BamHI or any of a number of other common restriction nuclease
sites. Alternatively, a polymer at repeated sites may be excised by
unique restriction sites not present within the polymer. Large
quantities of pBR322, pUC plasmid or other plasmid having multiple
copies of these sequences are produced by methods well known in the
art, the plasmid cut with the restriction enzyme flanking the
polymerized site, and the liberated multiple copies of the
operators isolated either by chromatography or any other convenient
means known in the art. The BNA, prior to use, is then strand
separated and is then amenable for polymerization onto a PNA
encoding a single stranded complementary copy of the operator as a
1/2 BBR. The BNAs may be polymerized vectorially onto the PNA by
using different restriction enzymes to flank each repeat of the
polymer in the plasmid used to produce multiple copies of the BNA.
Alternatively, the BNA polymer may be hybridized to the PNA via
overhangs at one or both ends of the BNA polymer, without the need
to strand separate and anneal each BNA segment. Examples of
specific BNA sequences are provided above in the section entitled
Description of Sequences, as SEQ ID NOS. 35-36. To stabilize the
BNA polymer, DNA ligase may be used to covalently link the
hybridized BNAs.
[0130] 5. The Hairpin Nucleic Acids (HNAs) and their preparation.
The HNAs of the present invention comprise at least two principal
parts joined together: A single-stranded sequence, which is
complementary to a 1/2 BBR, and a double-stranded nucleic acid
region formed, under hybridizing conditions, by the
self-association of self-complementary sequences within the HNA.
With reference to FIG. 1(IIc) of the drawings, the 1/2 BBR in the
HNA may be constructed so as to be complementary to the 1/2 BBR
sequence in the PNA. With reference to FIG. 1(I, IIc and IIIc) of
the drawings, the aforementioned HNA, when added to the PNA under
hybridizing conditions, forms a PNA-HNA hybrid containing a BBR.
With reference to FIG. 1(IIIc, IVc and Vc) of the drawings, a
PNA-HNA hybrid, under hybridizing conditions, upon addition of the
TNA, can form a TNA-PNA-HNA hybrid containing a TBR and a BBR.
[0131] With reference to FIGS. 2a and 2b, the HNAs can be used to
"cap" or terminate the addition of BNA extensions to the PNA. The
two BNAs in FIG. 2a(Ib and Ie) can associate to form the hybrid
shown in FIG. 3(IVb) or can hybridize directly and individually to
the PNA as illustrated in FIG. 2a(Ia-c, IIa-d). The two HNAs (shown
in FIG. 2a(IIIa and IIIb)) can terminate the hybridization of the
BNA to other BNAs which extend from the PNA, as illustrated in FIG.
2a(IVa-d). The HNA in FIG. 2a(IIIa) can terminate the PNA-BNA
hybrids shown in FIG. 2a(IIb and IId) and any PNA-BNA hybrid with a
single stranded 1/2 BBR which is complementary to the 1/2 BBR in
the HNA illustrated in FIG. 2a(IIIa). Similarly, the HNA in FIG.
2a(IIIb) can terminate the PNA-BNA hybrids shown in FIG. 2a(IIa and
IIe) and any PNA-BNA hybrid with two single stranded 1/2 BBRs which
are complementary to the 1/2 BBRs in the HNA illustrated in FIG.
2a(IIIb).
[0132] HNAs are constructed that will terminate PNA-BNA hybrids
which are constructed from the sequential addition of BNAs to the
PNA as illustrated in FIG.(2b). The single stranded 1/2 BBR
sequences illustrated in FIG. 2b(Ia, IIIa, Va, and VIIa) are
specifically complementary to the single stranded 1/2 BBR sequences
illustrated in FIG. 2b(Ib,IIIb,Vb and VIIb) and produce the unique
capped PNA-BNA-HNA hybrids illustrated in FIG. 2b(Ic,IIIc,Vc and
VIIe).
[0133] The self-complementary sequences in the HNA and the loop
sequence which links the self-complementary hairpin sequences can
be of any composition and length, as long as they do not
substantially impede or inhibit the presentation of the
single-stranded 1/2 BBR that comprises part of the HNA by the HNA
or selectively bind the BBA or the TBA. The loop sequences should
be selected so that formation of the loop does not impede formation
of the hairpin. An examples of an HNA useful in this application is
provided as SEQ ID NO. 44 (see Description of Sequences above).
[0134] 6. The Target Binding Assemblies (TBAs) and their
preparation. A TBA may be any substance which binds a particular
TBR formed by hybridization of particular TNAs and PNAs, provided
that the TBA must have at least the following attributes:
[0135] (a) The TBA must bind the TBR(s) in a fashion that is highly
specific to the TBR(s) of interest. That is, the TBA must
discriminate between TBRs present in the TNA-PNA hybrid and similar
duplex sequences formed by PNA-CNA hybrids. The TBA must bind the
PNA-CNA hybrid with a sufficiently low avidity that upon washing
the TBA-TNA-PNA complex, the PNA-CNA hybrid is displaced and the
PNA-TNA hybrid is not displaced;
[0136] (b) The TBA must avidly bind the TBR(s) created by the
hybridization of the TNA with the PNA. Binding affinities in the
range of 10.sup.-5 to about 10.sup.-12 or higher are generally
considered sufficient. As noted below, in some instances, it might
be desirable to utilize a particular TBA which has a very low
avidity for a particular TBR, but which has a greatly increased
affinity when a particular configuration of multiple TBRs is
provided so that the square of the affinity of the TBA for each TBR
becomes the affinity of relevance to that particular TBA.
[0137] Examples of the DNA binding components useful in the
formation of TBAs include, but are not limited to NF-kB,
papillomavirus E2 protein, transcription factor SP1, inactive
restriction enzymes, antibodies, etc. Each of these proteins has
been recognized in the art to contain sequences which bind to
particular nucleic acid sequences and the affinities of these
interactions are known. Naturally, the method of the instant
invention is not limited to the use of these known DNA binding
proteins or fragments thereof. From the instant disclosure, it
would be apparent to one of ordinary skill that the instant method
could easily be applied to the use of novel TBAs exhibiting at
least the required attributes noted above. Thus, for example, in WO
92/20698, a sequence specific DNA binding molecule comprising an
oligonucleotide conjugate formed by the covalent attachment of a
DNA binding drug to a triplex forming oligonucleotide was
described. The method of that disclosure could be used to produce
novel TBAs for use according to the instant disclosure, provided
that the TBAs thus formed meet the criteria described above. In
addition, the methods of U.S. Pat. Nos. 5,096,815, 5,198,346, and
WO88/06601, herein incorporated by reference, may be used to
generate novel TBAs for use according to the method of this
invention. Specific antibodies or portions thereof could be used
(see for example Blais 1994).
[0138] Where the TBA is a protein, or a complex of proteins, it
will be recognized that any of a number of methods routine in the
art may be used to produce the TBA. The TBA may be isolated from
its naturally occurring environment in nature, or if this is
impractical, produced by the standard techniques of molecular
biology. Thus, using NF-kB as an example, using the DNA binding
portions of p50 or p65 subunits, this binding assembly could be
produced according to recombinant methods known in the art (see for
example Ghosh [1990] Cell 62:1019-1029, describing the cloning of
the p50 DNA binding subunit of NF-kB and the homology of that
protein to rel and dorsal).
[0139] Many DNA and other nucleic acid binding proteins are known
which can be used as or in TBAs according to this invention. Once
the amino acid sequence of any DNA, RNA:DNA, RNA or other nucleic
acid binding protein is known, an appropriate DNA sequence encoding
the protein can either be prepared by synthetic means, or a cDNA
copy of the mRNA encoding the protein from an appropriate tissue
source can be used. Furthermore, genomic copies encoding the
protein may be obtained and introns spliced out according to
methods known in the art. Furthermore, the TBAs may be chemically
synthesized.
[0140] Once an appropriate coding sequence has been obtained,
site-directed mutagenesis may be used to alter the amino acid
sequence encoded to produce mutant nucleic acid binding proteins
exhibiting more desirable binding characteristics than those of the
original nucleic acid binding protein. As an example of this
process, the amino acid sequence of the DNA binding portions of
NF-kB can be altered so as to produce an NF-kB' molecule which more
tightly binds the NF-kB binding site (see examples
below--HIV-Detect and HIV-Lock).
[0141] To provide further insight into this aspect of the
invention, the following considerations are to be noted. Using
NF-kB as an example, a TBA may be prepared using the naturally
occurring NF-kB molecule. However, because this molecule is present
in vanishingly small quantities in cells, and because the subunits
of this DNA binding protein have been cloned, it would be more
reasonable to prepare large quantities of the complex via
recombinant DNA means as has already been accomplished for this
protein (see for example Ghosh [1990] Cell 62:1019-1029). NF-kB is
a pleiotropic inducer of genes involved in immune, inflammatory and
growth regulatory responses to primary pathogenic (viral, bacterial
or stress) challenges or secondary pathogenic (inflammatory
cytokine) challenges. NF-kB is a dimeric DNA binding protein
comprising a p50 and a p65 subunit, both of which contact and bind
to specific DNA sequences. In an inactivated state, NF-kB resides
in the cellular cytoplasm, complexed with a specific inhibitor,
I-kB, to form a cytoplasmic heterotrimer. Upon activation, the
inhibitor is decomplexed, and the p50-p65 dimer relocates via a
specific nuclear localization signal (NLS) to the cell's nucleus
where it can bind DNA and effect its role as a transcriptional
activator of numerous genes (see Grimm and Baeuerle [1993] Biochem.
J. 290:297-308, for a review of the state of the art regarding
NF-kB).
[0142] The p50-p65 dimer binds with picomolar affinity to sequences
matching the consensus GGGAMTNYCC (SEQ ID NO. 117), with slightly
different affinities depending on the exact sequence. It is worth
noting that homodimers of p50 and p65 have also been observed to
occur. These homodimers display different-biochemical properties as
well as slightly different affinities of binding sequences within
and similar to the above consensus. Thus, depending on the desired
binding characteristics of the TBA, a p50-p65 heterodimer, a
p50-p50 homodimer, or a p65-p65 homodimer or fragments of the
aforementioned dimers may be used.
[0143] One way in which various novel TBAs may be produced for use
according to this invention is shown schematically in FIG. 9. The
nucleic acid recognition units of the TBA may be assembled and
associated with similar or dissimilar TBA nucleic acid recognition
units via a "chaperone." The chaperone is a structure on which the
various TBA recognition elements are built and which confers
desirable properties on the nucleic acid recognition units. The
chaperone is comprised of any sequence which provides assembly
sequences such that same or different nucleic acid recognition
units are brought into close and stable association with each
other. Thus, for example, in the case of a TBA designed to tightly
bind NF-kB TBRs, a TBA is assembled by providing lambda cro
sequences as assembly sequences, linked to the nucleic acid binding
sequences for either NF-kB p50 or p65. The p50 or p65 nucleic acid
binding sequences are linked to the cro sequences at either the
carboxy or amino terminus of cro and either the carboxy or amino
terminus of the nucleic acid recognition unit of the p50 or p65.
Linking sequences are optionally provided to allow appropriate
spacing of the nucleic acid recognition units for optimal TBR
binding.
[0144] The assembly sequences, exemplified above by cro and CO
sequences (SEQ ID NOS. 104-108), comprise any stable oligopeptides
which naturally and strongly bond to like sequences. Thus, in the
case of cro, it is well known that a dimer of cro binds to the
bacteriophage lambda operator sites (Anderson et al. [1981] Nature
290:754-758; Harrison and Aggarwal [1990] Ann. Rev. Biochem.
59:933-969). The monomer units of cro tightly and specifically
associate with each other. Thus, by linking DNA recognition unit
sequences to the cro sequences, close and tight association is
achieved.
[0145] The optional linker sequences comprise any amino acid
sequence which does not interfere with TBA assembly or nucleic acid
binding, and which is not labile so as to liberate the nucleic acid
recognition unit from the complete TBA. It is desirable but not
necessary that the linker sequences be covalently linked to other
binding assembly components. The association should be specific so
as to aid in the assembly and manufacture of the binding
assemblies. Examples of such sequences include, but are not limited
to, such well known sequences as are found linking various domains
in structural proteins. Thus, for example, in the lambda repressor
protein, there is a linking sequence between the DNA binding domain
and the dimerization domain which is useful for this purpose. Many
other such sequences are known and the precise sequence thereof is
not critical to this invention, provided that routine
experimentation is conducted to ensure stability and
non-interference with target nucleic acid binding. Examples of such
sequences are provided herein as Met Ser and SEQ IN NOS. 99-102.
Insertion of specific, known proteolysis sites into these linkers
is also an integral part of this invention. The presence of such
sites in the linker sequences would provide manufacturing
advantages, allowing different molecules to be assembled on the
chaperone scaffold.
[0146] In addition to the nucleic acid recognition units, optional
linking sequences, and assembly sequences, the novel TBAs of this
invention optionally have asymmetry or PILOT TNA sequences and one
or more OSA units. The asymmetry sequences are provided to
encourage or prevent certain desirable or undesirable associations.
For example, in the event that a TBA having homodimeric p50 DNA
recognition units is desired, the asymmetry sequences are provided
to disrupt the naturally stronger association of NF-kB p50 subunits
and p65 subunits, while not disrupting the assembly sequences from
bringing together p50 subunits. Examples of such sequences are
provided herein as SEQ ID NOS. 85-92 and SEQ ID NOS. 105 and
106.
[0147] In a different configuration, NF-kB p50 subunit sequences
are brought into close association with transcription factor SP1
DNA recognition unit sequences. This is desirable in the event that
an NF-kB/SP1 binding motif is of significance, as in the HIV LTR
where a motif of at least six DNA binding protein recognition
sites, two NF-kB, three SP1, and a TATA site are known to exist.
Since it is also known that the second NF-kB and first SP1 site are
significant to regulation of HIV transcription (Perkins et al.
[1993] Embo J. 12:3551-3558), this particular configuration of TBA
is useful not only in the detection of HIV, but as a therapeutic or
prophylactic against HIV infection (see below). In a similar
fashion, the long control region (LCR) of human papillomavirus may
be used as a key control region for probing according to this
method.
[0148] In view of the different elements that can be associated,
cassette fashion, according to this method of TBA formation, an
essentially unlimited variety of TBAs are produced. In FIG. 10, a
series of different molecules, referred to as "HIV-detect I-IV" are
exemplified wherein "CHAP" denotes the chaperone, "nfkb" denotes
NF-kB subunits, "sp1" denotes the nucleic acid recognition unit of
the SP1 transcription factor, and "TATA" denotes a dimer of the DNA
recognition unit of a TATA sequence DNA binding protein (TBP), also
known as a TATA binding protein, or TBP. These configurations are
further exemplified below and are all integral parts of the instant
invention.
[0149] In yet another configuration, the modular structure shown in
FIG. 9 is adapted to detection and or treatment or prophylaxis of a
completely different pathogen. In FIG. 11, in a similar fashion to
the above described "HIV-detect I-IV" molecules, a series of
"HPV-Detect I-IV" molecules is produced. In this embodiment,
advantage is taken of the DNA binding properties of the E2 protein
of human papillomavirus (HPV). In addition, the roles of SP1 and
TBP are taken advantage of by providing specific DNA recognition
units adapted to bind to these sequences in the HPV genome. In the
formation of the E2-specific TBAs for use in detecting HPV
infection, it may be desirable to use any of SEQ ID NOS. 75-84 or
93-98 as the E2 DNA recognition units. A TBA containing a bovine E2
dimer and a human E2 dimer DNA binding domain may be particularly
useful.
[0150] The various sequences described above may either be
chemically linked using pure oligopeptide starting materials, or
they may be linked through provision of recombinant nucleic acids
encoding, via the well known genetic code, the various subelements.
In the event of recombinant production, linking cro coding
sequences to sequences of nucleic acid recognition units to form
TBAs is advantageous because not only does cro act as assembly
sequences in the chaperone, it also acts to direct the proper
folding of the nucleic acid recognition elements. Exemplary
sequences for chaperones are provided herein as SEQ ID NOS.
104-108. Furthermore, in the event that higher order structures
comprising multiple binding sites is desired, as in a pentameric
NF-kB/NF-kB/SP1/SP1/SP1 TBA, proper design of the asymmetry
sequences allows such structures to be made.
[0151] In the foregoing fashion, TBAs are prepared which bind to
their cognate binding sites with high affinity. For example, the
NF-kB DNA binding components of the TBAs of FIG. 10 are expected to
bind to the HIV-LTR with an affinity of between about 10.sup.-8 and
10.sup.-12 molar. Sequences useful as the DNA recognition units are
provided as SEQ ID NOS. 63-71, 73-84, 93-98, and 104-108 and
exemplified further below.
[0152] In view of the foregoing description of directed assembly of
nucleic acid binding proteins using assembly and asymmetry (or
piloting) sequences, those skilled in the art will recognize that a
generally applicable method for assembling protein structures is
provided by this invention. The generality of this method is
demonstrated further by consideration, by way of further example,
of the use of an antibody-epitope interaction in the assembly of
desired structures. By way of specificity, a DNA binding protein
structure may be assembled by linking an NF-kB p50 subunit to an
antigen, such as a circularized (through disulfide bonds)
melanocyte stimulating hormone (MSH). This pro-MSH molecule may
then be bound by an anti-MSH antibody to provide a novel nucleic
acid binding assembly, with the antigen and antibody acting as
assembly sequences.
[0153] The modular structure provided by FIG. 9 reveals that a
great variety of TBAs may be assembled using different combinations
of components. Thus, representative embodiments of this general
structure are provided as SEQ ID NOS. 109-116.
[0154] 7. The Booster Binding Assemblies (BBAs) and their
preparation. A BBA may be any substance which binds a particular
BBR formed by hybridization of particular PNAs and BNAs, including
when multiple BNAs (up to and including "n" BNAs, i.e., BNA.sub.n,
wherein "n" is theoretically 0-.infin., but practically is between
about 0 and 100) are polymerized onto the PNA for signal
amplification, provided that the BBA must have at least the
following attributes:
[0155] (a) The BBA must bind the BBRs in a fashion that is highly
specific to the BBR of interest. That is, the BBA must discriminate
between BBRs present in the PNA-BNA hybrid and similar duplex
sequences in BNA-CNA hybrids or other CNAs. Thus, where even a
single base mismatch or conformational differences with or without
base mismatches occur in the production of the PNA-BNA.sub.n or
PNA-BNA.sub.n-HNA hybrid, the BBA must bind the hybrid with a
sufficiently low avidity that upon washing the
TBA-TNA-PNA-BNA.sub.n complex, the BBA is displaced from the CNA
sequences but not the BBR sequences.
[0156] (b) The BBA must avidly bind the BBR(s). Binding affinities
in the range of 10.sup.-5 to about 10.sup.-9 or higher are
generally considered sufficient.
[0157] Examples of BBAs include, but are not limited to cro, and
the bacteriophage lambda repressor protein, CI. In addition, see
U.S. Pat. No. 4,556,643, herein incorporated by reference, which
suggests other DNA sequences and specific binding proteins such as
repressors, histones, DNA modifying enzymes, and catabolite gene
activator protein. See also EP 0 453 301, herein incorporated by
reference, which suggests a multitude of nucleotide sequence
specific binding proteins (NSSBPs) such as the tetracycline
repressor, the lac repressor, and the tryptophan repressor. Each of
these BBAs has been recognized in the art to bind to particular,
known nucleic acid sequences and the affinities of these
interactions are known. Naturally, the method of the instant
invention is not limited to the use of these known BBAs. From the
instant disclosure, one of ordinary skill could easily apply the
use of novel BBAs exhibiting at least the required attributes noted
above to the instant method.
[0158] Examples of novel BBAs useful according to this aspect of
the invention include novel proteins based on the motif of a known
DNA or RNA or DNA:RNA binding protein such as cro or the .lambda.
CI repressor protein. Preferably, such modifications are made to
improve the handling of these components of the invention. Thus, it
may be desirable to add a high concentration of cro to an assay.
One of the negative qualities of cro is that at high
concentrations, the binding of cro to its DNA target comes into
competition with cro-cro interactions. Thus, for example, a
chaperoned or mutated cro may be produced which does not have this
shortcoming. Examples of such altered chaperones are SEQ ID NOS.
105-106 and 108. Methods known in the art, such as production of
novel target binding proteins using variegated populations of
nucleic acids and selection of bacteriophage binding to particular,
pre-selected targets (i.e., so-called phage-display technology, see
discussion above for production of novel TBAs) may be used to
produce such novel BBAs as well as the aforementioned novel
TBAs.
[0159] Where the BBA is a protein, or a complex of proteins, it
will be recognized that any of a number of methods routine in the
art may be used to produce the BBA. The BBA may be isolated from
its naturally occurring environment in nature, or if this is
impractical, produced by the standard techniques of molecular
biology. Thus, for example, the sequence of the cro protein is
known and any molecular clone of bacteriophage lambda may be used
to obtain appropriate nucleic acids encoding cro for recombinant
production thereof. In addition, the TBAs described herein may be
used as BBAs, provided that different TBAs are used to bind TBRs
and BBRs.
[0160] 8. The use of BBAs and BBRs to localize and amplify the
localization of the PNA-TNA-TBA complexes (see FIG. 8). In one
embodiment of this invention, the highly specific and extremely
tight binding of TBAs comprised of nucleic acid binding components
is used to produce an amplifiable nucleic acid sandwich assay.
According to one aspect of this embodiment, a solid support is
coated with a first TBA creating an immobilized TBA. In solution, a
PNA and TNA are contacted under hybridizing conditions and then
contacted with the immobilized TBA. Only those PNA-TNA interactions
which form the specific TBR recognized by the immobilized TBA are
retained upon wash-out of the solid surface which binds the TBA-TBR
complex.
[0161] Detection of the bound TBR is accomplished through binding
of Booster Nucleic Acids, BNAs, to the 1/2 BBRs present on the PNAs
under hybridizing conditions. In this manner, even if only a single
TBA-TBR complex is bound to the immobilized TBA, a large, amplified
signal may be produced by polymerizing multiple BNAs to the
immobilized TNA. Each BNA which binds to the TNA forms a BBR which
can be bound by BBAs which, like the TBAs immobilized on the solid
surface, may be chosen for their very tight and specific binding to
particular nucleic acid structures. Thus, according to this
embodiment, the immobilized TBA may contain the DNA binding portion
of NF-kB, which very specifically and tightly binds to NF-kB
binding sites formed upon hybridization of the TNA and PNA to form
such a site.
[0162] Because it is well known that there are NF-kB binding sites
both in the normal human genome and in the long terminal repeats of
human immunodeficiency virus (HIV), this invention provides a
method of discriminating between the "normal" human sites and the
sites present in cells due to HIV infection. Therefore, in a test
designed to determine the presence or absence of HIV DNA in a
sample of human DNA, the HIV NF-kB binding sites may be viewed as
the TNA, and the normal human NF-kB binding sites may be viewed as
CNAs. According to the method of this invention, discrimination
between these TNAs and CNAs is accomplished by taking advantage of
the fact that in the HIV LTR, there are two NF-kB binding sites,
followed by three SP1 sites (see, for example, Koken et al. [1992]
Virology 191:968-972), while cellular NF-kB binding sites with the
same sequences are not found in tandem.
[0163] In cases where the TNA contains more than one 1/2 TBR and it
is desirable to pursue the therapeutic and prophylactic
applications of the TBAs, it may be desirable to use more than one
TBA, each with the capacity to bind a TBR in the TNA-PNA complex.
In this case, it may be advantageous to select, as components of
the TBAs, DNA-binding or RNA-binding domains with lesser affinity
for its TBR than the wild-type DNA-binding or RNA-binding domain.
Given that the TBAs which are involved in the binding to the
multiple TBRs can either assemble together before binding to their
TBRs or assemble together after binding to their TBRs, the
individual TBAs will not block the corresponding TBRs in the other
genomes than the target genome unless the TBRs are spatially
capable of binding the assembled TBA complex. One feature of the
multimeric assembly of TBAs which is specifically claimed here as
part of this invention is that such a multimeric assembly is
expected to have a much reduced affinity for a single site within
the TNA. However, since the binding is dramatically increased
relative to any one TBA, the TBA complex would be expected to not
compete for the binding of any single TBR with the corresponding
native proteins in situ but bind tightly to sequences in the
PNA-TNA hybrid containing the TBRs for each of the nucleic
acid-binding components assembled in the TBA. The TBA complex
should be assembled and linkers adjusted in the individual TBAs so
as to allow the nucleic acid-binding regions contained in the TBA
complex to simultaneously reach and bind to these targets.
[0164] Once the TNA-PNA hybrids have formed and been contacted with
the immobilized TBA, unbound nucleic acid is washed from the
immobilized surface and the immobilized hybrids detected. This is
accomplished in any one of several ways. In one aspect of this
invention, the PNA is labeled with an OSA, such as a radionuclide,
colored beads, or an enzyme capable of forming a colored reaction
product. Furthermore, in addition to having one or more 1/2 TBRs,
the PNA also may contain at least one 1/2 BBR. The 1/2 BBR
sequences are chosen so as to be complementary to unique 1/2 BBR
sequences in BNAs. In the embodiment described above, for example,
where the TBA is NF-kB and the TBR formed upon TNA-PNA
hybridization is one or more NF-kB binding sites, the 1/2 BBRs may
provide hybridizable (that is, single-stranded, complementary)
sequences of the left or right bacteriophage lambda operators (see,
for example, Ptashne [1982] Scientific American 247:128-140, and
references cited therein for sequences of these operators). These
may be polymerized onto the PNA 1/2 BBRs in a vectorial fashion
(see FIGS. 2 and 3) providing up to "n" BBRs, and each BBR forms a
cro binding site.
[0165] Enzymatically, radioactively, or otherwise labeled cro, is
contacted with the TBA-TNA-PNA-(BNA).sub.n complex. In this
fashion, a highly selective and amplified signal is produced.
Signal produced using a PNA having a single 1/2 TBR indicates
success of the assay in achieving TBA-TBR binding and
polymerization of the BNAs to produce signal from cellular sites
(i.e. from CNAs). Absence of signal when a dimerized TBA is used
indicates that in the TNA, there were no HIV LTRs as no double
NF-kB binding sites were present. On the other hand, presence of
signal using the dimer NF-kB indicates HWV infection. As a specific
example of the foregoing description of this embodiment of the
invention, see Example 6 describing an HWV test kit.
[0166] Naturally, those skilled in the art will recognize that the
foregoing description is subject to several modifications in the
choice of PNAs, TNAs, TBAs, BNAs, and BBAs. Furthermore, in systems
other than HWV, those skilled in the art will recognize that the
general method described above could be likewise applied. However,
these other applications may be simpler than the above described
method as the TBAs used may not recognize any normal cellular sites
and therefore resort to dimerization or other methods of
discriminating between TNAs and CNAs may be less critical. In
designing probes and binding assemblies for these other systems,
the skilled artisan will be guided by the following principles and
considerations.
[0167] In the above-described embodiment, the appeal of using the
DNA-binding portions of NF-kB protein as the TBA and the NF-IcB
recognition binding elements as the TBRs is that these elements
form an important "control point" for the replication of HIV. That
is, it is known that HIV is required to use NF-kB as a critical
feature in its replicative life cycle. Similar control points for
other pathogens are chosen and used as a basis for detection
according to the methods described herein.
[0168] From the foregoing description of general features of this
invention and the mode of its operation, one skilled in the art
will recognize that there are a multiplicity of specific modes for
practicing this invention. By way of example, the method of this
invention is adaptable to a method and devices using
chromatographic test kits described in U.S. Pat. Nos. 4,690,691 and
5,310,650 (the '691 and '650 patents). In those patents, a porous
medium was used to immobilize either a TNA or a capture probe, and
a solvent was used to transport a mobile phase containing either a
labeled PNA, if the TNA was immobilized, or the TNA, if a capture
probe was immobilized, into the "capture zone." Once the TNA was
bound in the capture zone, either by directly immobilizing it or
through capture, a labeled PNA was chromatographed through the
capture zone and any bound label was detected.
[0169] Adapting the instant invention to such a system provides the
improvement of using a Target Binding Assembly in the capture zone
and therefore, the capture of only perfectly matched TBR sequences
or other TBRs representing nucleic acid confirmations specifically
bound by the TBA within the TNA-PNA duplexes by virtue of the
previously described sensitive discrimination by the TBA between
TNAs and CNAs.
[0170] Once the TNA-PNA hybrids become bound to the immobilized
TBA, the signal is amplified by adding BNAs or chromatographing
BNAs through the capture zone. Finally, the signal may be further
amplified by adding BBAs or chromatographing labeled BBAs through
the capture zone. In this fashion, the ease of performing the
analysis steps described in the '691 and '650 patents is improved
upon herein by providing the additional ability to increase the
specificity and, through amplification, the sensitivity of the
method described in those patents. The disclosure of the '691 and
'650 patents is herein incorporated by reference for the purpose of
showing the details of that method and for the teachings provided
therein of specific operating conditions to which the compositions
and methods of the instant invention are adaptable.
[0171] Those skilled in the art will also recognize that the method
of the instant invention is amenable to being run in microtiter
plates or to automation. The use of machines incorporating the
method of this invention therefore naturally falls within the scope
of the instant disclosure and the claims appended hereto. Thus, for
example, this invention is adaptable for use in such instruments as
Abbott Laboratories' (Abbott Park, Ill.) IMx tabletop analyzer. The
IMx is currently designed to run both fluorescent polarization
immunoassay (FPZA, see Kier [1983] KCLA 3:13-15) and microparticle
enzyme immunoassay (MEZA, see Laboratory Medicine, Vol. 20, No. 1,
January 1989, pp. 47-49). The MEZA method is easily transformed
into a nucleic acid detection method using the instant invention by
using a TBA as a capture molecule coated onto a submicron (<0.5
.mu.m on average) sized microparticle suspended in solution. The
microparticles coated with TBA are pipetted into a reaction cell.
The IMx then pipettes sample (hybridized PNA-TNA) into the reaction
cell, forming a complex with the TBA. After an appropriate
incubation period, the solution is transferred to an inert glass
fiber matrix for which the particles have a strong affinity and to
which the microparticles adhere. Either prior to or after filtering
the reaction mixture through the glass fiber matrix, BNAs and BBAs
are added, or another signal amplification and detection means is
used which depends on specific formation of TNA-PNA hybrids. The
immobilized complex is washed and the unbound material flows
through the glass fiber matrix.
[0172] The bound complexes are detected by means of alkaline
phosphatase labeled BBAs or otherwise (radioactively,
enzymatically, fluorescently) labeled BBAs. In the case of alkaline
phosphatase labeled BBAs, the fluorescent substrate 4-methyl
umbelliferyl phosphate or like reagent may be added. Alternatively,
the enzyme may be bypassed by directly labeling BBAs with this or a
like reagent. In any event, fluorescence or other signal is
proportional to the amount of PNA-TNA hybrids present.
[0173] The fluorescence is detected on the surface of the matrix by
means of a front surface fluorometer as described by the
manufacturer of the IMx. With minor adjustments that can be made
through routine experimentation to optimize an instrument such as
the IMx for nucleic acid hybridization and nucleic acid-TBA
interactions, the instant invention is completely adaptable to
automated analyses of TNA samples.
[0174] 9. Other diagnostic applications of this invention. While
the foregoing description enables the use of the instant invention
in a number of different modes, many additional utilities of this
invention are readily appreciated, for example, in a mobility
retardation system.
[0175] In this embodiment of the invention, an improvement of the
well known electrophoretic mobility shift assay (EMSA) is conducted
as follows (See FIGS. 12a and 12b):
[0176] A sample of DNA is fragmented, either through random
cleavage or through specific restriction endonuclease treatment.
The DNA in the sample is then split into two equal aliquots and a
specific TNA is added to the first aliquot but not to the second.
The first and second aliquot are then electrophoresed in an
acrylamide or agarose gel, and the pattern of DNA bands (either
visualized through ethidium bromide binding or through being
radioactively labeled prior to electrophoresis is then compared for
the two aliquots. Fragments of DNA having binding sites to which
the TBA is specific are retarded in their migration through the
electrophoretic medium. By using an appropriate TBA, any number of
DNA or other nucleic acid sequences may be tracked in this
fashion.
[0177] In a modification of the EMSA described above, fragmented
TNA is hybridized with a PNA and fractionated in a first dimension.
The fractionated DNA is then reacted with an appropriate TBA and
the change in mobility of the DNA fragments is noted. Enhancement
of the retardation is possible by adding BBAs as described above.
(See, for example, Vijg and references cited therein for known
techniques of two (2) dimensional nulceic acid electrophoresis, to
which the instant method may be applied).
[0178] 10. Therapeutic applications. Because of the very tight and
selective nucleic acid binding characteristics of the novel TBAs
described herein, therapeutic utilities are contemplated in
addition to the diagnostic utilities of these compounds. Thus, a
TBA comprising tight and specific binding for the HIV-LTR, by
virtue of having an NF-kB p50 and an SP1 DNA recognition unit in
close association (see FIG. 10, HIV-Detect II) is useful to bind up
the HIV-LTR and thereby prevent transcription from this key element
of the HIV genome. The unique features of the assembly sequences of
the TBA allow recombinant vectors to introduce DNA encoding such a
TBA into a cell and the proper folding of the expressed sequences.
Once inside the cell, the nuclear localization signals of the p50
subunit directs the transport of the TBA to the nucleus where it
binds tightly to the LTR of any integrated HIV, effectively
shutting the pathogen down. In a prophylactic mode, one that is
concerned about potential HIV exposure is administered a sufficient
dose of a TBA or a recombinant vector able to express the TBA, so
as to lock up any HIV that might have entered the person. In this
mode, the use of the TBA is analogous to passive protection with a
specific immune globulin. In the therapeutic or prophylactic mode,
NLS sequences are used in place of the OSAs used in the diagnostic
mode. Exemplary NLS sequences are provided as SEQ ID NOS. 72 and
103 (see also Heinzinger 1994 and Bukinsky 1993, describing NLS
sequences of the HIV Vpr and gag proteins respectively). In any
event, the TBA is administered in a pharmaceutically-acceptable
carrier, known in the art such as a sterile salt solution or
associated with a liposome or in the form of a recombinant vector,
preferably one which directs expression of the TBA in a chosen cell
type, or by a protein delivery system.
[0179] II. Embodiments of the Invention
[0180] In view of the foregoing description and the examples which
follow, those skilled in the art will appreciate that this
disclosure describes and enables various embodiments of this
invention, including:
[0181] 1. A probe nucleic acid (PNA) comprising:
[0182] (a) a single-stranded sequence, 1/2 TBR, which is capable of
forming, under hybridizing conditions, a hybrid, TBR, with a 1/2
TBR present in a target nucleic acid (TNA);
[0183] (b) zero, one or more, and preferably one to ten single
stranded sequences, 1/2 BBR, which is capable of forming, under
hybridizing conditions, a hybrid BBR, with a 1/2 BBR present in a
booster nucleic acid (BNA); and
[0184] (c) an OSA, which is no attached support and/or indicator,
or an attached support or other means of localization, including,
but not limited to, attachment to beads, polymers, and surfaces,
and/or indicators;
[0185] wherein said TBR is capable of binding with high affinity to
a TBA, said TBA being a substance capable of discriminating between
a paired TBR and a TBR having unpaired nucleotides, and further,
wherein said BBR is capable of binding with high affinity to a BBA,
said BBA being a substance capable of discriminating between a
paired BBR and a BBR having unpaired nucleotides. This embodiment
includes TBRs which are nucleic acid binding protein recognition
sites, such as the HIV LTR, and other nucleic acid binding protein
recognition sites in other pathogens, some of which are noted
above. The PNA of this embodiment of the invention may produce a
TBR which is a nucleic acid binding protein recognition site
present in the genome of a pathogen or is a binding site associated
with a pathogenic condition in the human genome or a contaminant in
a fermentation process.
[0186] 2. A booster nucleic acid (BNA) comprising:
[0187] (a) a 1/2 BBR which has a sequence which is complementary to
a 1/2 BBR sequence in a PNA or another BNA already hybridized to
the PNA and which is capable of forming, under hybridizing
conditions, a hybrid, BBR, with the PNA;
[0188] (b) an OSA attached support or other means of localization,
including, but not limited to, attachment to beads, polymers, and
surfaces, and/or indicators; and
[0189] (c) additional hybridization sites, 1/2 BBRs, for
hybridization with additional BNAs;
[0190] wherein said BBR is capable of binding with high affinity to
a BBA, said BBA being a substance capable of discriminating between
a paired BBR and a BBR having unpaired nucleotides.
[0191] 3. A Hairpin Nucleic Acid (HNA) comprising a single-stranded
sequence, 1/2 BBR, which under hybridizing conditions is capable of
forming a hairpin while at the same time binding to a BNA to form a
BBR capable of binding a BBA, wherein said BBR is capable of
binding with high affinity to a BBA, said BBA being a substance
capable of discriminating between a paired BBR and a BBR having
unpaired nucleotides.
[0192] 4. A method for detecting a specific TNA sequence,
comprising the steps of:
[0193] (a) hybridizing said TNA with a PNA as described above;
[0194] (b) hybridizing said PNA with a BNA containing a 1/2 BBR
whose sequence is complementary to a 1/2 BBR sequence in the
PNA;
[0195] (c) adding the products of steps (a) and (b) containing a
TBR and a BBR, to a surface, liquid or other medium containing a
TBA;
[0196] (d) adding BBAs to the mixture in step (c) wherein said BBA
comprises:
[0197] (i) a molecule or a portion of a molecule which is capable
of selectively binding to a BBR; and
[0198] (ii) a detectible indicator; and
[0199] (e) detecting signal produced by the indicator attached to
the BBA. This method includes the use of a protein indicator,
including enzymes capable of catalyzing reactions leading to
production of colored reaction products. It also includes
indicators such as a radionuclide or colored beads.
[0200] 5. A method for detecting the presence in a sample of a
specific Target Nucleic Acid, TNA, which comprises:
[0201] (a) contacting said sample with a Probe Nucleic Acid, PNA,
which, upon hybridization with said TNA if present in said sample,
forms a Target Binding Region, TBR, which is capable of binding a
Target Binding Assembly, TBA;
[0202] (b) contacting said sample, already in contact with said
PNA, with a TBA capable of binding to any TBRs formed by the
hybridization of said PNA and said TNA in the sample.
[0203] 6. A method for detecting or localizing specific nucleic
acid sequences with a high degree of sensitivity and specificity
which comprises:
[0204] (a) adding PNAs containing a 1/2 BBR and a 1/2 TBR to a
sample containing or suspected of containing TNAs containing 1/2
TBR sequences, to form a complex having target binding regions,
TBRs, formed by the hybridization of complementary 1/2 TBRs present
in the PNAs and TNAs respectively;
[0205] (b) binding the TBRs formed in step (a) to an immobilized
TBA to form a TBA-TNA-PNA complex;
[0206] (c) adding Booster Nucleic Acids, BNAs, containing booster
binding regions, 1/2 BBRs, to the complex formed in step (b) such
that the 1/2 BBRs in the BNAs hybridize with the 1/2 BBR sequences
present in the PNAs or to 1/2 BBRs present in BNAs already bound to
the PNA, to form BBRs, such that TBA-TNA-PNA-(BNA).sub.n complexes
are formed;
[0207] (d) adding Hairpin Nucleic Acids, HNAs, containing 1/2 BBR
sequences, to the complex formed in step (c) such that the 1/2 BBRs
in the HNAs hybridize with any available 1/2 BBR sequences present
in the BNAs of the complex of step (c), thereby capping the
extension of the BNAs onto the TBA-TNA-PNA-(BNA).sub.n complexes of
step (c) to form TBA-TNA-PNA-(BNA).sub.n-HNA complexes;
[0208] (e) adding Booster Binding Assemblies, BBAs, linked to
indicator moieties, to the TBA-TNA-PNA-(BNA).sub.n-HNA complexes
formed in step (d) to form TBA-TNA-PNA-(BNA-BBA).sub.n-HNA
complexes; and
[0209] (f) detecting the signals produced by the indicator moieties
linked to the TBAs, PNAs, BNAs, BBAs or ITNAs in the
TBA-TNA-PNA-(BNA-BBA).sub.n- -HNA complexes of step (e);
[0210] wherein:
[0211] the TNA comprises:
[0212] (i) one or more specific 1/2 TBR nucleic acid sequences, the
presence or absence of which in a particular sample is to be
confirmed;
[0213] the PNA comprises:
[0214] (i) a single-stranded sequence, 1/2 TBR, which is capable of
forming, under hybridizing conditions, a hybrid, TBR, with a 1/2
TBR present in a target nucleic acid (TNA);
[0215] (ii) a single stranded sequence, 1/2 BBR, which is capable
of forming, under hybridizing conditions, a hybrid BBR with a 1/2
BBR present in a booster nucleic acid (BNA); and
[0216] (iii) an OSA, which is no attached support and/or indicator,
or an attached support or other means of localization, including,
but not limited to, attachment to beads, polymers, and surfaces,
and/or indicators;
[0217] the BNA comprises:
[0218] (i) a 1/2 BBR, as shown in FIG. 1(Ib), which has a sequence
which is complementary to a 1/2 BBR sequence in a PNA and which is
capable of forming, under hybridizing conditions, a hybrid, BBR,
with the PNA;
[0219] (ii) an OSA attached support or other means of localization,
including, but not limited to, attachment to beads, polymers, and
surfaces, and/or indicators;
[0220] (iii) additional hybridization sites, 1/2 BBRs, for other
BNAs; and
[0221] (iv) sequences, 1/2 BBRs, which can hybridize to BNAs
already hybridized to the PNA;
[0222] the BBA comprises:
[0223] (i) a molecule or a portion of a molecule which is capable
of selectively binding to a BBR; and
[0224] (ii) no attached support and/or indicator, or an attached
support or other means of localization, including, but not limited
to, attachment to beads, polymers, and surfaces, and/or
indicators;
[0225] and the TBA comprises:
[0226] (i) a molecule or a portion of a molecule which is capable
of selectively binding to a TBR; and
[0227] (ii) no attached support and/or indicator, or an attached
support or other means of localization, including, but not limited
to, attachment to beads, polymers, and surfaces, and/or
indicators.
[0228] 7. An improvement to a solid phase hybridization method for
detecting the presence of a target polynucleotide involving:
immobilizing a target polynucleotide, if present in a test sample,
directly or via an intermediate capture structure, on a solid phase
at a capture site; before, during or after said immobilization,
attaching a detectable label to said target polynucleotide, if
present; and detecting said label, if any, at said capture site;
the improvement comprising:
[0229] (a) using a Target Binding Assembly, TBA, as the means for
achieving immobilization of said target polynucleotide, wherein
said TBA binds only to a perfect hybrid formed between a specific
Probe Nucleic Acid, PNA, and said target nucleic acid such that a
perfect Target Binding Region, TBR, recognizable by said TBA is
formed; and
[0230] (b) including in the PNA a single stranded sequence, 1/2
BBR, capable of binding a Booster Nucleic Acid, BNA, containing a
single stranded complementary 1/2 BBR which, upon hybridization
with the 1/2 BBR in the PNA, forms a BBR capable of binding labeled
Booster Binding Assemblies, BBAs.
[0231] 8. A target binding assembly, TBA, comprising one or more
nucleic acid recognition units, linker sequence(s), assembly
sequence(s), asymmetry sequence(s), nuclear localization signal
sequence(s) (NLS) and OSA(s). The nucleic acid recognition unit may
be an NF-kB binding unit, an SP1 binding unit, a TATA binding unit,
a human papillomavirus binding unit, an HIV LTR binding unit, or a
binding unit for any other fragment of specific sequence the
detection of which is desirable and which can be achieved through
specific association with the TBA. Such recognition units include,
but are not limited to those exemplified herein as SEQ ID NO. 63,
SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID
NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72,
and SEQ ID NO. 73. Linker sequences such as oligopeptides which do
not interfere with the nucleic acid recognition function of the
nucleic acid recognition unit and which provide stability and
control over the spacing of the nucleic acid recognition unit from
the remainder of the TBA. Examples of such linker sequences are
well known in the art and include, but are not limited to
oligopeptide sequences from the interdomain primary sequence of a
structural protein. Assembly sequences include oligopeptide
sequences which direct the folding and association of nucleic acid
recognition units. A preferred example of such sequences are
oligopeptides derived from the bacteriophage lambda cro protein.
The asymmetry sequence directs the association of nucleic acid
recognition and assembly sequences in a predetermined order. Such
asymmetry sequences are exemplified by sequences derived from
insulin, relaxin, gonadotropic hormone, FSH, HCG, LH, ACTH,
including but not limited to SEQ ID NOS. 85-92. With reference to
FIGS. 14 and 15, SEQ ID NO. 85 is an "A" and SEQ ID NO. 86 is a "B"
sequence; SEQ ID NO. 87 is an "A" and SEQ ID NO. 88 is a "B"
sequence' SEQ ID NO. 89 is a human relaxin "A" and SEQ ID NO. 90 is
a human relaxin "B" sequence; SEQ ID NO. 91 is a skate relaxin "A"
and SEQ ID NO. 92 is a skate relaxin "B" sequence. In addition, the
TBA may contain nuclear localization signal sequences, NLS, which
direct the migration and uptake of a protein or complex associated
with said NLS into the nucleus of a cell. Examples of such NLS
sequences are provided as SEQ ID NOS. 72 and 103. Preferred
embodiments of the TBA include but are not limited to HIV Detect
I-IV or HPV Detect I-IV, and SEQ ID NOS. 109-116.
[0232] 9. Methods of using the novel TBAs of this invention
include, but are not limited to a method of using the TBA to bind a
particular nucleic acid sequence in a target nucleic acid sample
which comprises:
[0233] (a) fragmenting the nucleic acid in the target nucleic acid
sample;
[0234] (b) contacting, under hybridizing conditions, the fragmented
nucleic acid with a probe nucleic acid complementary to the
particular nucleic acid sequence of interest, wherein said probe
nucleic acid, upon hybridization with said particular nucleic acid
sequence of interest forms a target binding region to which said
TBA specifically binds.
[0235] In this method, the probe nucleic acid, in addition to
sequences complementary to said particular nucleic acid sequence of
interest, also may have additional sequences to which a booster
nucleic acid can bind to form a booster binding site to which a
labeled booster binding assembly can bind to provide a signal
showing and amplifying the binding of the probe nucleic acid to the
target nucleic acid sequence of interest.
[0236] An additional aspect of this invention not requiring
fragmentation of Target Nucleic Acid, involves administration of
the TBA to a patient in need of such treatment of a therapeutically
or prophylactically effective amount of said TBA, which comprises
administering the TBA, either in the form of a purified protein
complex or in the form of a recombinant vector which, upon entry
into the patient is able to express the TBA, such that the TBA
binds the particular nucleic acid sequence to achieve the desired
prophylactic or therapeutic result. This may include providing a
dosage which can be determined by routine experimentation to be
sufficient to prevent establishment of an active infection by a
pathogen. Dosages of purified TBAs may be in the range of about
0.001 to 100 mg/kg. When provided as a recombinant expression
vector which will direct the in vivo expression and folding of the
TBA, dosages of the recombinant nucleic acid may be substantially
lower, particularly if provided in the form of non-pathogenic viral
vector. The methods of using the TBAs also include monitoring the
shift in mobility of nucleic acids in target nucleic acid samples
as a function of the size such that binding of the TBA to a
particular fragment in the sample modifies the mobility of the
fragment. This aspect of the method provides a useful method of
analyzing nucleic acid fragments for particular aberrations, such
as might be found associated with metastases.
[0237] 10. Diagnostic or forensic kits useful in determining the
presence of an infection, the susceptibility to a disease, or the
origin of a particular nucleic acid containing sample.
[0238] 11. A method of assembling multimeric TBAs in vivo which
comprises introducing nucleic acids encoding component TBAs into a
cell. The component TBAs should each contain a nucleic acid
recognition unit, assembly sequences, asymmetry sequences, and
nuclear localization signal sequences. Linker sequences, optionally
included if TBA footprinting experiments indicate the need for such
linkers to attain optimal geometry of the multimeric TBA. Upon in
vivo expression of each component TBA and proximal binding, via the
nucleic acid recognition unit of each component TBA to nucleic acid
sequences encountered in the nucleus or elsewhere in the cell,
component expressed TBAs are directed to assemble via the included
assembly and asymmetry sequences into multimeric TBAs. As described
above, such multimeric TBAs will have the advantage of binding
specifically with high affinity to TBRs in a specific target
sequence, but not at all or with very low affinity to cousin
nucleic acids.
[0239] The foregoing description of the invention will be
appreciated by those skilled in the art to enable preferred
embodiments as well as the best mode of this invention. Without
limiting the subject matter to the specifics of the examples
provided hereinafter, the following examples are provided to
further guide those skilled in the art on methods of practicing
this invention. Standard recombinant DNA techniques as disclosed in
Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., and more recent texts are not disclosed
as these are now well within the skill of the ordinary artisan.
EXAMPLE 1
Preparation of PNAs and Labeling of PNAs
[0240] Probe nucleic acids, PNAs, may be prepared by means well
known in the art. Thus, single stranded polynucleotide PNAs of
defined sequence may be prepared via solid phase chemical synthesis
according to Merrifield. PNAs may be prepared by automated
synthesis using commercially available technology, such as resins
and machines produced or marketed by Applied Biosystems, ABI, or
other manufacturers. Alternatively, through known recombinant DNA
methods, particular PNA sequences are synthesized in vivo, for
example by cloning a duplex PNA into a vector which can replicate
in E. coli, large quantities of the duplex PNA may be prepared.
Multimers of the PNA may be cloned into the vector such that for
each mole of vector, several moles of PNA is liberated upon
digestion of the vector with a restriction fragment flanking the
PNA sequence. Subsequent to synthesis or recombinant production,
the PNAs are purified by methods well known in the art such as by
gel electrophoresis or high pressure liquid chromatography (HPLC).
If the PNA is produced as a duplex, prior to use in a hybridization
assay for detection of target nucleic acid sequences, the strands
of the PNA are separated by heating or other methods known in the
art.
[0241] The specific sequence of bases in the PNA is chosen to
reflect the sequence to be detected in a TNA, with the proviso
that, according to this invention, the PNA contains a 1/2 TBR
sequence, which is one that upon hybridization of the PNA and TNA,
a TBR is formed. As there are an essentially unlimited number of
such sequences known in the art, the choice of the PNA sequence is
amenable to selection by the skilled researcher for any given
application. The sequence of the HIV LTR is one such sequence,
which upon hybridization of a PNA encoding portions of the LTR with
TNAs encoding the HIV LTR, TBRs capable of binding the NF-kB or SP1
DNA binding proteins are formed.
[0242] In addition to sequences which will form a TBR upon
hybridization, the PNA also may contain a 1/2 BBR. This sequence is
one which, upon hybridization with a booster nucleic acid, BNA,
forms a BBR which is capable of binding a BBA. The BBA is
preferably a DNA binding protein having high affinity for the BBR
sequence.
[0243] In this particular example, hybridization between a PNA
having as a 1/2 TBR, SEQ ID NO. 4 and, at the 3' end of that
sequence, a 1/2 BBR sequence shown as SEQ ID NO. 35. The PNA
encoding these sequences is either used without labeling or is
labeled with a radioactive isotope such as P.sup.32, S.sup.35, or a
similar isotope, according to methods known in the art.
Alternatively, the PNA is bound to a bead of between 0.01 to 10
.mu.m, which may be colored for easy visual detection. This label
forms the OSA as described in the specification. This probe
hybridizes with HIV LTR sequences to form a TBR that binds NF-kB.
In addition, the PNA hybridizes with BNAs having a complementary
1/2 BBR to form a bacteriophage lambda left operator that binds
either cro or lambda repressor proteins.
[0244] In a manner similar to that described above, PNAs are used
wherein the 1/2 TBR is any one of SEQ ID NO. 5 or SEQ ID NOS. 7-34,
and a 1/2 BBR, such as SEQ ID NO. 35 or SEQ ID NO. 36 is either at
the 3' end or 5' end of the 1/2 TBR.
EXAMPLE 2
Preparation and Labeling of BNAs
[0245] Similar to the methods described in Example 1 for
preparation and labeling of PNAs, BNAs are prepared and labeled
according to methods known in the art. As described in U.S. Pat.
No. 4,556,643, herein incorporated by reference (see particularly
Example 1), nucleic acid sequences encoding particular nucleic acid
binding sequences may be mass produced by cloning into a replicable
vector. Furthermore, similar to that disclosure, the 1/2 TBR and
1/2 BBR sequences may be co-linearly produced in this fashion, with
the distinction, however, that according to the instant invention,
the 1/2 TBR sequence itself forms a nucleic acid binding component
recognition site and the 1/2 BBR, while forming a nucleic acid
binding component recognition site, also provides a means of
amplifying the signal produced upon binding of the 1/2 TBR to
complementary sequences in the TNA by providing for polymerization
of BNAs onto the TNA bound PNA. To enable this, a sequence such as
SEQ ID NO. 35, which encodes the left operator of bacteriophage
lambda, is provided with additional sequences such that an overhang
sequence is created on one or both ends of the BNA upon
hybridization with the PNA.
[0246] As a specific example, vectorial polymerization of BNAs onto
a TNA is provided by SEQ ID NOS. 40-43. In this example, SEQ ID NO.
40 encodes two 1/2 TBRs which will hybridize with two 1/2 TBRs in a
TNA to form two NF-kB binding sites, while at the same time
providing a bacteriophage lambda left operator 1/2 BBR, which
additionally is terminated at the 3' end with the recognition site
for the restriction enzyme PstI. Addition of the BNA, SEQ ID NO.
41, with the 1/2 BBR complementary to the 1/2 BBR on the PNA, SEQ
ID NO. 40, completes the BBR while at the same time completing the
PstI recognition site, leaving a four base overhang for
hybridization with additional BNAs. Accordingly, SEQ ID NO. 42 is
added which has a four base pair sequence at the 3' end which is
complementary to the four-base overhang remaining from the
hybridization of SEQ ID NOS. 40 and 41. In addition, SEQ ID NO. 42
is provided with a five base sequence at its 5' end which forms
part of a BamHI recognition site. The growing polymer of BNAs is
extended further by addition of the BNA SEQ ID NO. 43, which is
complementary to SEQ ID NO. 42, completing the BBR while at the
same time completing the BamHI recognition site and leaving a four
base overhang which may be further hybridized with BNAs having
complementary sequences. In this fashion, the BNAs may be
hybridized extensively so as to greatly amplify the signal of a
single PNA-TNA hybridization event.
[0247] As with the PNAs described in Example 1, the BNAs may be
used in an unlabeled form or may be labeled according to methods
known in the art and described in Example 1. It will also be
appreciated that, rather than produce the BNA polymer by sequential
addition of BNAs to the PNA-TNA complex, the BNA polymer may be
preformed and added directly to the PNA-TNA complex. One simple
method for preforming such a BNA polymer includes the recombinant
production of a vector in which multimers of the BNA are provided
with a unique restriction site at either end of the polymer. This
polymer of BNAs containing multiple BBRs is cut out of the vector
and hybridizes to a single stranded 1/2 BBR remaining in the PNA
upon hybridization of the PNA and the TNA. This is accomplished by
providing a single stranded sequence in the PNA complementary to an
overhang produced in the BNA polymer when it is excised from the
production vector.
EXAMPLE 3
Production of HNAs and Their Use for Capping BNA Polymers
[0248] The HNAs of this invention are produced according to methods
known in the art for polynucleotide production as described in
Examples 1 and 2 for PNAs and BNAs. In the production of the HNAs,
however, the sequence of the HNA is specifically designed so that a
substantial portion of the HNA forms a self-complementary
palindrome to form a hairpin, while at the same time, leaving in
single stranded form enough bases to be able to hybridize with
single stranded sequences in the growing chain of BNAs described in
Example 2.
[0249] In this Example, a HNA of SEQ ID NO. 44 is provided to cap
the extension of BNAs onto the PNA in Example 2 after the addition
of the BNA, SEQ ID NO. 43. This is accomplished because SEQ ID NO.
44, while having a palindromic sequence that forms a stable
hairpin, also has a sequence at the 5' end of the HNA which
completes the BamHI sequence formed by the hybridization of SEQ ID
NO. 42 and SEQ ID NO. 43. Naturally, termination of the polymer
after addition of only 3 BNAs is for the purpose of simplicity in
demonstrating the invention. As described above, this
polymerization may be continued essentially indefinitely to amplify
the signal of the PNA-TNA hybridization event. Once the HNA
hybridizes to the growing chain of BNAs, the polymer is capped and
no further extension of the polymer is possible.
EXAMPLE 4
Preparation of TBAs and BBAs, Labeling, and Immobilization
Thereof
[0250] The TBAs and BBAs which may be used according to the instant
invention include any substance which can specifically bind to the
TBRs and BBRs formed by hybridization of the PNAs, TNAs and BNAs.
Use of DNA binding proteins forms one example of such
substances.
[0251] For this example, the TBA is the dimer of the DNA binding
portion of p50, and the BBA is the lambda cro protein. These
proteins may be produced according to methods known in the art. The
genes for both of these proteins have been cloned. Thus, these
proteins are recombinantly produced and purified according to
methods known in the art. Furthermore, these proteins are labeled,
either with a radioisotope, such as radioactive iodine, or with an
enzyme, such as beta-galactosidase or horseradish peroxidase, or
with a fluorescent dye such as fluorescein or rhodamine, according
to methods well known in the art. In addition, either or both of
the TBA and BBA may be immobilized on a solid surface such as the
surface of a microtiter plate or the surface of a bead, such as a
colored bead of diameter anywhere from 0.01 to 10 .mu.m. The labels
on the TBAs and BBAs may be the same or different.
[0252] In this example, the TBA containing the dimeric p50 DNA
binding domain is labeled with rhodamine, while the BBA, cro, is
labeled with fluorescein. Accordingly, upon hybridization of the
PNAs, TNAs, BNAs and HNAs as described in this patent disclosure
and the foregoing and following examples, the nucleic acid hybrids,
if formed, are contacted with excess labeled TBA and cro. The
fluorescence of these labels is measured according to known methods
and, detection of both signals is indicative of the presence of 1/2
TBR sequences in the TNA. The differential signal produced by the
fluorescence of the NF-kB and cro is a measure of the degree to
which the polymerization of BNAs onto the PNA-TBA hybrid has
resulted in amplification of the signal. Amplification from one to
over a thousand fold is contemplated according to the method of
this invention.
EXAMPLE 5
Hybridization of two PNAs with a TNA and Discrimination Between a
TNA and a CNA
[0253] The PNAs, PNA1, SEQ ID NO. 40 and PNA2, SEQ ID NO. 45, are
used in about ten-fold molar excess over the concentration of TNAs
in a test sample. For this example, an isolated duplex HIV LTR,
wherein one strand of which has the sequence SEQ ID NO. 37, shown
in FIG. 7, and the other strand of which is complementary to the
sequence shown in FIG. 7, is used as the TNA. A duplex isolated CNA
is also used in this example, one strand of which has the same
sequence as SEQ ID NO. 37, except that, in the first NF-kB binding
site shown in FIG. 7, at the center of the binding site, position 1
in FIG. 7, instead of a "T," there is an "A," the complementary
strand of which therefore mismatches with the SEQ ID NO. 40 PNA at
that location.
[0254] SEQ ID NO. 40 and SEQ ID NO. 45 are both added to separate
reactions, the first containing the above described TNA and the
second containing the above described CNA. The samples are
solubilized in an appropriate hybridization buffer, such as 10 mM
Tris (pH 7.5), 1 mM EDTA. The samples are heated to about
90.degree. C. for about five minutes to strand separate the duplex
TNAs and CNAs in the samples, and then the samples are allowed to
cool to allow strands of PNAs, TNAs and CNAs to anneal.
[0255] Once the hybridization has gone to completion, which can be
determined according to known methods such as by calculating the
t1/2 based on base compositions and annealing temperature according
to known methods, the SEQ ID NO. 40 PNA is polymerized by addition
of BNAs as in Example 2 and the SEQ ID NO. 45 PNA2 probe is
polymerized with BNAs starting with Sph1 recognition site overhang.
Following addition of the BNAs and a brief hybridization period,
the separate samples are added to beads coated with covalently
immobilized NF-kB, and the NF-kB is allowed to bind to any TBRs
formed in the TNA and CNA samples. After about 15 minutes of
binding, the samples are washed twice with about three volumes of
an appropriate washing buffer, such as 10 mM Tris, pH 7.5, 100 mM
NaCl, or another buffer pre-determined not to interfere with NF-kB,
or bacteriophage lambda CI repressor protein binding activity.
After each wash, the beads are allowed to settle under gravity or
by brief centrifugation. This removes any nucleic acids which do
not have a perfect NF-kB binding site formed by hybridization of
the PNAI and TNA sequences.
[0256] After the final wash, bacteriophage lambda CI repressor
protein labeled with a radioactive isotope, such as with
radioactive iodine, or labeled with an enzyme, such as horseradish
peroxidase, with colored beads, or with a fluorescent label is
added to each sample. The samples are then washed several times
(about 3) with several volumes (about 2) of an appropriate washing
buffer such as 10 mM Tris, pH 7.5, 100 mM NaCl, or another buffer
pre-determined not to interfere with NF-kB, or bacteriophage lambda
CI repressor protein binding activity. After each wash, the beads
are allowed to settle under gravity or by brief centrifugation.
Following the last settling or centrifugation, the bound label is
quantitated by detecting the bound radioactivity, liberated color
in an enzymatic assay, color of bound beads, or fluorescence
detection. Alternatively, an anti-Cl antibody can be added and a
standard sandwich enzyme linked immunoassay or radioimmunoassay
performed to detect bound repressor. In addition, as a negative
control (background), all of the foregoing manipulations are
carried out in tandem with a sample in which beads are used having
no immobilized NF-kB.
[0257] As a result of the foregoing assay, the control and CNA
containing samples have similarly low signals while the TNA
containing sample has a signal well above background.
EXAMPLE 6
A Test Kit for the Detection of HIV
[0258] A. Kit Contents:
[0259] 1. Microtiter plate.
[0260] 2. 1 mg/mL solution of recombinantly produced NF-kB in
tris-buffered saline.
[0261] 3. Tube containing single stranded HIV PNAs (a mixture of
pre-mixed oligonucleotides encoding two NF-kB 1/2 binding sites,
i.e. a mixture of SEQ. ID. Nos.7 and 8).
[0262] 4. Tube containing single stranded human genomic PNA, SEQ ID
NO. 1.
[0263] 5. Tube of nuclease (PstI).
[0264] 6. Tube of protease.
[0265] 7. Tube containing pre-polymerized BNA's, 100 repeat units
of bacteriophage lambda O.sub.R, capped with an HNA but with free
1/2 BBRs available for binding to PNA-TNA hybrids.
[0266] 8. Tube of horseradish peroxidase (hrp) conjugated cro.
[0267] 9. Tube of hrp colored substrate.
[0268] 10. Tris buffered saline, 100 mL.
[0269] 11. Lancet.
[0270] 12. Reaction tubes A, B, C, each containing 250 .mu.L of
distilled water.
[0271] 13. Medicine dropper.
[0272] B. Assay Method:
[0273] (a) The microtiter plate (item 1) is coated with the
solution of recombinantly produced NF-kB (item 2) at a
concentration of 1 mg/mL in tris buffered saline overnight at
4.degree. C. with rocking.
[0274] (b) Three drops of blood of the test taker is obtained by
pricking a finger with the lancet (reagent 11), and a drop of blood
is dispensed into each of reaction tubes A, B, and C (reagent
12).
[0275] (c) Into each tube is dispensed one drop of protease
solution (reagent 6) with the medicine dropper (item 12) and the
tube agitated and allowed to sit for 5 minutes.
[0276] (d) One drop of nuclease (item 5) is added to each of tubes
A-C using the medicine dropper and the tubes agitated and allowed
to sit for 10 minutes.
[0277] (e) One drop of item 3 is added to tube A (test sample); one
drop of item 4 is added to tube B (positive control); and one drop
of saline (item 12) is added to tube C as a negative control. The
tubes are heated to 50.degree. C. in hot water and allowed to cool
to room temperature over one hour.
[0278] (f) While the hybridization is allowed to occur in step (d),
the excess protein is drained from the surface and the microtiter
plate, from step (a), and the plate is rinsed with tris buffered
saline (tube 10).
[0279] (g) The contents of tubes A-C from step (e) are transferred
to three wells of the microtiter plate and allowed to stand for 1
hour with rocking.
[0280] (h) The microtiter wells containing the contents of tubes
A-C are rinsed with tris buffered saline and emptied.
[0281] (i) One drop of item 7 is added to each well and allowed to
hybridize with any 1/2 BBR sites bound to the plate, over one hour,
followed by three rinses with tris buffered saline.
[0282] (j) One drop of item 8 is added to each well and cro is
allowed to bind to any bound BNA's over 10 minutes, followed by
five, one mL washes with tris-buffered saline.
[0283] (k) One drop of hrp substrate is added to each well and
color allowed to develop.
[0284] C. Results:
[0285] If wells A and B both show color development, and well C
does not, the test is valid and the subject has been infected with
HIV. If only well A shows color development, or if well C shows
color development, the test has been performed incorrectly, and is
invalid. If wells A and C show no color development but well B
does, the test is valid and the individual has not been infected
with HIV.
EXAMPLE 7
Production of Various Novel TBAs
[0286] Novel TBAs for use according to the instant invention are
prepared as follows:
[0287] (a) NFkB/NF-kB (HIV-Detect I). A nucleic acid encoding any
one of SEQ ID NOS. 63-71 or a like NF-kB DNA binding protein, is
fused, in frame, to a nucleotide sequence encoding an assembly
sequence, such as cro, such that the NF-kB DNA recognition sequence
is encoded at amino or carboxy terminus of the cro sequence.
Optionally, a linker sequence is provided between the NF-kB
sequence and the cro sequence. At the other terminus of cro, a
nuclear localization signal sequence, such as SEQ ID NO. 72, is
optionally provided. Further, asymmetry sequences are optionally
provided at the cro terminus unused by the NF-kB recognition
sequence. Examples of complete TBAs are shown below.
[0288] (b) NF-kB/SP1 (HIV-Detect II). In a similar fashion to that
described in (a) above, a recombinant coding sequence encoding an
NF-kB recognition domain is prepared. In a separate construct,
instead of SEQ ID NOS. 63-72, the coding sequence for the DNA
recognition portion of SP1 is included. Such a sequence should
encode all or a functional part of SEQ ID NO. 73, which is that
portion of the SP1 transcription factor exhibiting DNA binding (see
Kadonaga et al. [1987] Cell 51:1079-1090). The NF-kB-encoding
vector and the SP1-encoding vector are then co-transfected into an
appropriate expression system such as is well known in the art. A
monomeric NF-kB recognition unit is added to complete the NF-kB
recognition dimer after the assembly of the SP1 and NF-kB
recognition units by the chaperone. The asymmetry sequences prevent
the formation of NF-kB or SP1 dimers and direct, instead, the
formation of NFkB-SP1 heterodimers (i.e., HIV-Detect II), which are
then isolated from the expression system (mammalian or bacterial
cells) by known methods.
[0289] (c) SP1/SP1 TBAs (HIV-Detect III). As described in (b)
above, an SP1-encoding TBA construct is prepared. However, only
this construct is transfected into the expression system, and
asymmetry sequences allowing the formation of SP1-SP1 dimers are
included.
[0290] (d) SP1-TATA (HIV-Detect IV). As described in (b) above, an
SP1-encoding TBA recombinant is produced. In addition, a
recombinant encoding a TBA having the binding sequence, SEQ ID NO.
74, or like sequence encoding a TATA recognition unit is prepared
with asymmetry sequences complementary to those included in the SP1
TBA-encoding construct. These constructs are co-transfected and the
heterodimers isolated by standard methods, including affinity
purification on a DNA column having the appropriate SP1-TATA target
binding regions.
[0291] (e) SP1-E2 (HPV-Detect I). An SP1-encoding construct is
prepared as in (b) above. An E2 TBA-encoding construct is prepared
by using a sequence encoding any one of SEQ ID NOS. 75-84 and 94-98
which are papillomavirus E2 DNA recognition units (see Hegde et al.
[1992] Nature 359:505-512) or like recognition units, is prepared
and co-transformed or co-transfected with the SP1 TBA-encoding
construct. Monomeric E2 recognition unit is added to the complete
E2 recognition dimer after the assembly of the E2-SP1 recognition
unit by the chaperone. The heterodimer HPV-Detect I is isolated
according to known methods.
[0292] (f) E2-E2 (HPV-Detect II). As described above in (e), an E2
TBA-encoding construct is prepared, except that asymmetry sequences
are included which permit the formation of E2 dimers. The expressed
dimers are then isolated by known methods including affinity for a
dimeric E2 binding site on a DNA affinity column.
[0293] (g) E2-TATA (HPV-Detect III). As described above in (e) and
(d), E2 and TATA binding TBAs are prepared (respectively), except
that asymmetry sequences are included which enhance the formation
of heterodimers rather than homodimers. These constructs are then
co-expressed and the heterodimers are isolated.
[0294] (h) TATA-TATA (HPV-Detect IV). As described above in (a) and
(d), a TATA binding TBA-encoding construct is prepared using
asymmetry sequences that encourage this homodimer formation and the
homodimer is isolated.
[0295] (i) Other TBAs. As described above for HIV and HPV TBAs,
TBAs for any given pathogen or disease state may be produced by
identifying specific DNA binding proteins and forming an expression
construct using appropriate linker, assembly, and asymmetry
sequences.
EXAMPLE 8
[0296] In a similar fashion to the assay described in Example 5, a
more stringent assay is produced by using the duplex NF-kB-SP1
binding protein prepared according to Example 6. Accordingly, the
probes shown in FIG. 7 and used in Example 5 may be lengthened to
reduce the interprobe distance and thereby reduce the flexibility
of the DNA in the DNA.
EXAMPLE 9
Production of "High-Order" TBAs
[0297] By the appropriate use of asymmetry sequences, TBAs are
produced which are dimers, trimers, tetrameres, pentamers, or
hexamers of particular DNA recognition units. In this fashion, a
hexameric TBA is produced by making a first NF-kB p50 dimeric TBA
using asymmetry sequences which enable dimer formation. In
addition, the asymmetry sequences enable the tetramerization of the
p50 dimer with an SP1-SP1 dimer. Finally, additional asymmetry
sequences direct the hexamerization with a dimer exhibiting nuclear
localization sequences. This is accomplished by incorporating, for
example, asymmetry sequences from insulin, which in nature forms
hexamers. This hexamer formation is directed by the sequences, SEQ
ID NOS. 85(A) and 86(B), 87(A) and 88(B), 89(A) and 90(B), and
91(A) and 92(B) (see FIGS. 13 and 14).
[0298] Because of the extremely high affinity for the HIV-LTR that
can be generated using a multimeric TBA, the compounds having this
structure and which can be used for this purpose are referred to
herein as "HIV-Lock."
[0299] An optimal HIV-Lock is defined by footprinting (according to
methods well known in the art) TBAs bound to TBRs in the HIV LTR to
confirm that the binding affinity of each DNA binding protein
contributing to the formation of the multimeric TBA complex is
downshifted relative to the affinity for any natural target
sequence (i.e. CNAs) from which the DNA binding recognition unit of
the TBA is derived. Any concomitant loss in binding affinity for
the HIV TBRs is more than compensated for upon formation of the
multimer as described below.
[0300] There may be competition between the binding of each
component TBA for its TBR and assembly, via asymmetry sequences to
form the multimer. This is obviated by adjusting the linkers
between the chaperone and asymmetry sequences in each TBA component
such that these competing events are uncoupled. The resultant
reduction in the dimensionality of diffusion (effective
concentration increase) for the TBA asymmetry and assembly
components results in efficient formation of the multimeric
complex.
[0301] On the basis of the footprinting, the length and composition
of linkers is adjusted to achieve optimal discrimination between
target HIV sequences and natural sequences. In this fashion,
although each component TBA will have a low affinity for CNA and
TBR sequences, the multimeric complex will have an extremely high
affinity for the now expanded TBR recognized by the multimeric
complex (the square of the affinity of each TBR recognized by each
component TBA of the multimeric TBA), while still having a low
affinity for CNAs. In the same fashion, other multimeric TBA
complexes, aside from HIV-Lock, are prepared.
[0302] TBAs which can be formed in this fashion include the
following sequences, which are assembled by linking either the
protein subunits or nucleic acid sequences encoding these subunits,
as follows:
3 Set Link Sequences from Groups A I + II + III B IV + V + III C IV
+ III
[0303] wherein groups I-V consist of sequences selected from:
4 Group Selected from Sequences I Any of SEQ ID NOS. 85-92 II Met
Ser, linked to any of SEQ ID NOS 104-106, each of which is linked
to SEQ ID NO. 99. III SEQ ID NO. 100 linked to any of SEQ ID NOS.
75-84 or 94-98; SEQ ID NO. 101 linked to either SEQ ID NO. 74 or
SEQ ID NO. 93; or SEQ ID NO. 102 linked to SEQ ID NO. 74 or SEQ ID
NO. 93; or any of SEQ ID NO. 72, 103, 73, or 63-71. IV Any of SEQ
ID NOS. 104-106. V SEQ ID NO.99.
[0304] Specific examples of such TBAs are SEQ ID NOS. 109-116,
assembled as follows:
5 Set SEQ ID NO. Link SEQ IDS A 109 85 + Met Ser + 104 + 99 + 100 +
94 A 110 85 + Met Ser + 104 + 99 + 72 A 111 86 + Met Ser + 105 + 99
+ 102 + 74 A 112 86 + Met Ser + 106 + 99 + 73 A 113 89 + Met Ser +
106 + 99 + 63 C 114 106 + 64 C 115 105 + 64 B 116 106 + 99 + 73
[0305] In this fashion, choosing between appropriate asymmetry
sequences, assembly sequences, and DNA recognition units, many
different TBAs may be formed. Furthermore, sets of these, such as
SEQ ID NOS. 114 and 115, will associate with each other but dimers
of SEQ ID NO. 114 or 115 will not form due to charge repulsion in
the mutated assembly sequences (SEQ ID NO. 104 is cro; SEQ ID NO.
105 is a novel mutated, negatively charged cro, and SEQ ID NO. 106
is a novel mutated, positively charged cro).
[0306] Naturally, given the amino acid sequence of these TBAs, one
of ordinary skill could produce recombinant nucleic acid clones
encoding these, and such recombinant clones naturally form an
integral part of this invention.
EXAMPLE 10
HIV Test Using "HIV-LOCK"
[0307] In much the same method as used in Example 6, the "HIV-LOCK"
produced according to Example 9 is used as the TBA, reagent 2, with
similar results.
EXAMPLE 11
HIV Test Using "HIV-LOCK" When Testing Blood for Donation
[0308] When the quantity of blood to be tested is not limiting, as
when samples of blood for donation are to be tested for HWV
contamination, tests similar to Example 6 are run, but for each of
tubes A-C, about 5 nL of blood is pelleted in a tabletop
centrifuge. Other reagents are scaled up as necessary to handle the
larger quantity of TNA present in the sample.
EXAMPLE 12
"HIV-LOCK" as an Anti-HIV Therapeutic Agent
[0309] "HIV-LOCK" produced according to Example 9 is formulated as
a 1 mg/mL solution in liposomes and injected intravenously into a
subject who has been tested and confirmed to be infected with HIV.
A dose of about 0.1 mg to 100 mg of "HIV-LOCK"/kilogram body mass
is infused over a twenty-four hour period and the concentration of
HIV p24 in the patient's serum monitored. The treatment is repeated
as often as necessary, such as when elevations in the serum p24
occur.
EXAMPLE 13
Use of an HIV-TBA Construct as a Therapeutic
[0310] A recombinant retroviral or like vector is used to deliver a
construct encoding an HIV-LTR binding TBA to an infected patient.
The vector encodes a chaperone, such as cro, and sequences DNA for
binding portions of p50. The same vector also encodes a chaperone
on which an SP1 TBA folds. Asymmetry sequences are provided such
that upon co-expression of the p50-TBA and the SP1-TBA in a single
HWV infected cell in vivo, an immediate association occurs between
these TBAs, while at the same time preventing any association
between the DNA binding portion of p50 and endogenous p50 or p65
monomers. NLS sequences are also provided in the TBAs so that, upon
dimer formation, the TBA immediately relocates to the nucleus of
the cell and binds specifically to integrated HIV sequences, thus
preventing any transcription from that locus.
[0311] For this purpose, it is desirable to select sequences
encoding DNA binding domains such that the expressed monomers are
assembled into a TBA which does not bind to natural human
sequences. Thus, it is only upon binding of the TBA components to
their target sequences that association between all components of
the TBA occurs to form a complex which tightly and specifically
binds the HIV LTR.
EXAMPLE 14
Diagnostic Test Kit for Human Papillomavirus
[0312] This diagnostic for human papillomavirus takes advantage of
the known differential between benign and carcinogenic HPV to
provide a test which indicates the susceptibility to malignancy in
a patient. The papillomaviruses are a group of small DNA viruses
associated with benign squamous epithelial cell tumors in higher
vertebrates. At least 27 distinct human types of papillomaviruses
(HPVs) have been found; many of these have been associated with
specific clinical lesions. Four of these, HPV-6, HPV-11, HPV-16,
HPV-18, and HPV-33 have been associated with human genital tract
lesions. In general, HPV-6 and HPV-11 DNAs have been found
associated with benign lesions of the genital tract. HPV-16,
HPV-18, and HPV-33 have also been found associated with
premalignant and malignant lesions and are transcribed in most cell
lines established from cervical carcinomas. HPV-16, HPV-18, and
HPV-33 are likely to be only two members of a large set of HPV DNAs
associated with malignant human cervical carcinomas.
[0313] Animal models have shown that benign papillomavirus lesions
can progress to malignant lesions in the presence of a
co-carcinogen. HPV DNA has been found in metastases of cervical
carcinomas. In malignant cervical lesions, HPV DNA is usually
integrated into the human genome, but there may also be
extrachromosomal HPV DNA present. Integration of HPV to form the
provirus usually results in the disruption of the viral E2 open
reading frame (ORF). Despite disruption of the E2 ORF, and
examination of cell lines from several cervical carcinomas has
shown transcriptionally active and integrated HPV-16 and HPV-18.
When HPV-16 genomes which are present in the human cervical
carcinoma cell lines SiHa and CaSki have been examined, there are
differences found in the integration of HPV-16. In the SiHa line,
the single HPV-16 genome integration occurred at bases 3132 and
3384, disrupting the E1 and E2 ORFs with a deletion of 0.3 kb. An
additional 50-basepair deletion of HPV-16 DNA resulted in the E2
and E4 OFRs being fused. The 5' portion of the HPV-16 DNA,
consisting of the disrupted E2 ORF, is ligated to continuous human
right flanking sequences. In addition, a single additional guanine
is detected at nucleotide 1138 in the middle of the E1 ORF. This
basepair addition results in the fusion of the E1a and E1b ORFs to
a single E1 ORF.
[0314] The complete genome of HPV-1 6 is available on GenBank as
accession number K02718; the complete genome of HPV-33 is available
on GenBank as accession number M12732; the complete genome of
HPV-18 is available on GenBank as accession number X05015.
[0315] As a preliminary screen, the fact of an HPV infection is
established for a given cervical biopsy sample by a simple "yes/no"
type of analysis using, for example, any or all of the PNAs SEQ ID
NOS. 46-53 and an E2 TBA as described above (i.e., fragment DNA,
binding the PNA, immobilize with the TBA, and detect signal with
BNAs and BBAs).
[0316] Once a biopsy sample is found to be positive for HPV,
additional information is obtained as to the malignancy potential
of the HPV by analyzing the integration status of the virus in the
human genome.
[0317] 1. Fragment the DNA in the cervical biopsy sample and
hybridize to a blocking probe having the sequence, SEQ ID NO. 60.
This probe will bind to all the fragments in the DNA which have not
spliced out the 0.3 kb fragment.
[0318] 2. Expose the DNA in the biopsy sample to a PNA having the
sequence, SEQ ID NO. 61. This probe will only bind to fragments
which have deleted the 0.3 kb fragment (the blocking probe will
prevent the looping out of the large deletion segments if
present).
[0319] 3. A PNA having SEQ ID NO. 62 is hybridized with SEQ ID NO.
41 to form a BBR which will bind to cro or .lambda. CI repressor as
a BBA, leaving a single-stranded portion capable of hybridizing
with the TATA site on SEQ ID NO. 61. This added to form a TBR on
the 5' end of the large deletion.
[0320] 4. The TBR is immobilized by a TBA having a TATA binding
protein DNA recognition unit.
[0321] 5. The bound fragments are detected by adding BNAs and BBAs
as described above.
[0322] Detection of signal in this assay indicates that the large
fragment is deleted in HPV present in the TNA. Since this deletion
is correlated with malignancy, this assay provides insight into the
malignancy potential of the HPV infection. This conclusion can be
confirmed by performing an analogous assay based on the deletion of
the 52-basepair fragment which is also correlated with HPV-induced
malignancy.
[0323] The TBP recognition unit used in the TBA for this assay may
be chosen, for example, from a sequence such as SEQ ID NO. 70 or
SEQ ID NO. 93.
EXAMPLE 15
Recombinant HIV-LOCK.TM. Production.
[0324] Phase One--Preparation of DNA to Produce the HIV-Lock.TM..
In vitro mutagenesis of the coding regions of the naturally
occurring, cloned components of the HV-Lock.TM. which need to be
modified is performed with a MutaGene Phagemid kit. The modified
protocol includes the use of a Blue-script plasmid containing each
of the binding components of HIV-Lock.TM.. These are transformed
into competent cells and uracil-containing phagemids are grown.
Single stranded DNA is extracted and used as a template for the
mutagenic strand. Oligonucleotides containing the desired
mutations, including the incorporation of a novel restriction site,
are synthesized and treated with polynucleotide kinase and ATP. The
kinase treated oligonucleotides are annealed to the single-stranded
template, and a mutagenic strand is synthesized and ligated
according to the MutaGene protocol, with the exception that
Sequenase 2.0 provides the polymerase. Libraries are screened using
both g-.sup.32P end-labeled nucleotides containing sequences
complementary to the introduced mutations and by isolating the
plasmid DNA and identifying the mutants by the presence of the
introduced restriction site. The mutations are also confirmed by
sequencing with a Sequenase kit. The HV-Lock.TM. DNA is cloned into
the baculovirus expression system with a polyhedron promotor.
[0325] Phase Two--Production of HIV-Lock.TM. Proteins Using
Baculovirus. Sf-9 cells are cultured to a pre-determined density
(about 1.times.10.sup.6 cells/ml, log phase), infected with the
baculovirus containing the HIV-Lock.TM. instructions and harvested
to recover the recombinant proteins comprising the HIV-Lock.TM.. In
the scale-up process, cultures are expanded from flasks to spinners
and subsequently to bioreactors. Following infection the cells are
harvested at 12, 24, 36 and 48 hours for the protein. Indices of
viability are monitored throughout the entire process.
[0326] Phase Three--Purification of the HIV-Lock.TM. Proteins The
harvested proteins are first separated from particulates by
flow-through ultracentrifugation to facilitate downstream
purification. The centrifuged product is then sterile filtered.
Extracts are then centrifuged at 40,000 rpm at 4.degree. C. for 30
minutes and aliquots are immunoprecipitated with polyclonal rabbit
antibody against one of the HIV-Lock.TM. components.
Immunoprecipitated proteins are run on an SDS-10% PAGE gel.
[0327] Phase Four--Test of HIV-Lock.TM.Proteins Against HIV DNA
Mobility shift assays are carried out using an oligonucleotide
probe comprising elements of the HIV long terminal repeat and
fragments containing NFKB binding DNA associated with kappa light
chain and microglobulin regulation. The oligonucleotide is annealed
to its complimentary strand and end-labeled with g-.sup.32P
ATP.
[0328] Footprinting is accomplished by combining small (10.sup.-15
M) of radiolabeled HIV LTR DNA with a slightly larger amount of
HIV-Lock.TM. in a buffer at room temperature for 10 minutes.
Dithiothreitol is added prior to the addition of protein. Iron
(II), EDTA, hydrogen peroxide and sodium ascorbate are added and
the reaction mixture is incubated. A quenching agent is added and
the products are analyzed suing denaturing gel electrophoresis.
This is done for different concentrations of protein. The resulting
gel is imaged using a phosphoimager scanner and the resulting high
resolution image file is analyzed to abstract the. binding affinity
of HIV-Lock.TM. for the HIV DNA relative to cellular DNA.
[0329] Multiple design and testing iterations may be used in order
to refind binding of HIV-Lock.TM. and other TBAs for HIV and other
organisms. This process makes it possible to design binding
assemblies such that the binding assembly is not competitive with
the wild type proteins for single binding sites in the genome
samples. The development of TBAs for other organisms and TNAs for
sequences within these organisms can be made using the
aforementioned method. This method is valid when producing binding
assemblies for all nucleic acid TBRs including DNA-DNA, DNA-RNA and
RNA-RNA hybrids and combinations of these hybrids.
EXAMPLE 16
Method for Identifying Nucleic Acid Binding Molecules for
Production of TBAs and BBAs of the Invention
[0330] In the method of this invention, target binding assemblies
and booster binding assemblies are assembled by identifying nucleic
acid binding molecules, and linking the nucleic acid binding
portions of the molecules in such a fashion as to achieve TBAs
which discriminate between particular target sequences and even
closely related sequences. One method for identifying the nucleic
acid binding molecules involves the following steps:
[0331] 1. Obtaining a biological sample containing the target
nucleic acid. This could be, for example, an organism or a tissue
extract infected with a pathogen.
[0332] 2. Fragmenting the sample so as to expose the nucleic acids
and to reduce the size complexity of the nucleic acids contained in
the sample.
[0333] 3. Contacting a first aliquot of the fragmented nucleic
acids with a control buffer medium and contacting a second aliquot
of the fragmented nucleic acids with the control buffer medium
containing a known profile of nucleic acid binding molecules.
[0334] 4. Analyzing the two aliquots to identify fragments which
have altered behavior in the aliquot contacted with the target
binding molecules as opposed to the control aliquot. This is
accomplished by single dimension gel electrophoresis, two dimension
gel electrophoresis, high performance liquid chromatography, paper
chromatography or any other means which reveals a different
behavior of the nucleic acid fragments when bound to a nucleic acid
binding molecule as opposed to when the nucleic acid fragment is
unbound.
[0335] 5. Identifying and isolating fragments which do exhibit
altered behavior when contacted with the nucleic acid binding
molecule and either sequencing the nucleic acid fragment to
determine whether known nucleic acid binding molecule motifs are
present, or directly identifying the nucleic acid binding molecule
bound to the nucleic acid. The latter can be achieved, for example,
by contacting a two dimensional grid of the electrophoresed nucleic
acids with differentially labeled antibodies which bind to the
various nucleic acid binding molecules.
[0336] In this method, preferably nucleic acid motifs are used for
either diagnostic or therapeutic purposes wherein the target
nucleic acid has more than a single utilizable nucleic acid binding
molecule target. In this way, a complex target binding assembly can
be generated which takes advantage of the proximity of different
nucleic acid binding molecular motifs to enhance the specificity of
the TBA assembled from the individual nucleic acid binding
components identified. The various nucleic acid binding portions of
the nucleic acid binding molecules are then assembled into the
complete TBAs as described above, for example, for
HIV-LOCK.TM..
EXAMPLE 17
Method of Identifying Specific RNA Sequences in a Sample
[0337] According to the methods and compositions taught in this
invention, any nucleic acid sequence can be specifically
identified. Identification of target HIV RNA in a sample is
achieved by obtaining a sample of a patient's blood or other
biological fluid or extract which may contain the HIV RNA, and
testing for the presence of TAR binding sites. Tat is a positive
regulator of HIV replication which binds to the TAR region of the
HIV RNA. The smallest naturally occurring, fully active form of
HIV-Tat is 72 amino acids in length, SEQ Id. 118 herein. Tat
contains at least two functional domains, and transactivates gene
expression from the HIV long terminal repeat (HIV LTR). Tat binds
to an RNA stem loop structure formed from the self-hybridization of
sequences in TAR, which is just 5' to the HIV LTR. HIV TAR RNA
forms a dinucleotide bulge and two stem-loop structures (Rhim et
al. 1994 Virology:202, 202-211). The Tat (SEQ. Id. 118) binds to
this structure with lower avidity than does Tat variants wherein
Ala58 is a threonine or where His65 is an Asp residue. (Derse et
al., 1993 Virology:194,530-536). Utilizing these facts in the
instant method is accomplished by:
[0338] 1. Fragmenting a biological sample to expose the nucleic
acids and reduce the size complexity of the nucleic acids.
[0339] 2 Contacting a TBA with the sample which identifies a hybrid
TAR binding protein sequence and a proximate flanking sequence in
the HIV genome. The TBA used for this purpose is assembled on cro
as the chaperone using Tat as the HIV RNA specific binding
molecule. To provide specificity such that cross-talk between the
HIV TAR site and closely related TAR sites which may be present due
to such other pathogens as cytomegalovirus, the TBA also has an
antibody component which recognizes the DNA-RNA hybrid target
binding region formed when a probe nucleic acid binds to the HIV
LTR RNA.
[0340] 3. Eliminating any "cross-talk" produced by binding of Tat
to the TAR region of the HIV RNA due to such contaminants (cousin
RNAs) as the CMV TAR sequence by contacting the reaction with
excess Tat variant (either the AlaS58 to Thr or the His65 to Asp
variants) which bind more avidly. In this way, single binding
events due to the TBA binding to a cousin RNAs are competed from
the nucleic acid sample by the Tat variant. On the other hand, by
appropriately selecting the affinity of the double binding achieved
as a result of the antibody and Tat, the TBA is not displaced from
true targets. This process is illustrated in FIG. 16. In another
aspect of this same method, the TBA could be one in which, rather
than using a variant of Tat, an antibody is used which recognizes
this nucleic acid segment, and the TBA used is a double antibody
TBA.
[0341] In an alternate version of this method, a probe nucleic acid
may be used which hybridizes with the HIV LTR RNA. Accordingly, a
duplex segment of the LTR sp1 sites can be created as part of the
target binding region. This region of the HIV RNA flanks the TAR
region which is 5' to the LTR but is in close proximity thereto. A
TBA containing Tat and two Sp1 binding units is chaperoned to
provide Tat binding to TAR and Sp1 binding to the Sp1 binding
sites. Amplification and detection is then carried out by adding
appropriate BNAs, BBAs and ITNAs. In yet another alternative, PNAs
having Seq. ID. 38 and Seq. ID. 39 (see FIG. 7) could be used. A
TBA is used which contains one or more Sp1 binding units and an
antibody unit which binds to the DNA-RNA hybrid produced from
sample RNA and the Seq. Id. 38 PNA. Appropriate BNAs, BBAs and
ITNAs are then added to amplify the signal.
[0342] Naturally, those skilled in the art will recognize that
other TBA and TNA combinations could be used to optimize the
methods exemplified herein.
[0343] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. It will be understood that sequences provided
herein are exemplary only and that other like sequences suggested
by these could be used in the methods of this invention. It will
also be understood that although any sequence provided herein might
be designated as linear, it could be used in a circularly or
otherwise permuted form and although designated as not being
anti-sense, it could be used in the coding or non-coding form or to
bind to coding or non-coding complementary sequences.
Sequence CWU 1
1
* * * * *