METHOD OF DETECTION OF NUCLEIC ACIDS WITH A SPECIFIC SEQUENCE COMPOSITION
First Claim
1. A probe nucleic acid (PNA) comprising:
- (a) a single-stranded sequence, ½
TBR, which is capable of forming under hybridizing conditions, a hybrid, TBR, with a ½
TBR present in a target nucleic acid (TNA);
(b) a single stranded sequence, ½
BBR, which is capable of forming, under hybridizing conditions, a hybrid BBR, with about 0-10 ½
BBR present in a booster nucleic acid (BNA); and
(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;
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.
1 Assignment
0 Petitions
Accused Products
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.
47 Citations
51 Claims
-
1. A probe nucleic acid (PNA) comprising:
-
(a) a single-stranded sequence, ½
TBR, which is capable of forming under hybridizing conditions, a hybrid, TBR, with a ½
TBR present in a target nucleic acid (TNA);
(b) a single stranded sequence, ½
BBR, which is capable of forming, under hybridizing conditions, a hybrid BBR, with about 0-10 ½
BBR present in a booster nucleic acid (BNA); and
(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;
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. - View Dependent Claims (4, 5, 6, 7, 8, 42)
-
-
2. A booster nucleic acid (DNA) comprising:
-
(a) a ½
BBR which has a sequence which is complementary to a ½
BBR sequence in a PNA or another BNA and which is capable of forming, under hybridizing conditions a hybrid, BBR, with the PNA;
(b) an OSA, which is no attached support 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; and
(c) additional hybridization sites, ½
BBRs, for hybridization with additional BNAs;
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.
-
-
3. A Hairpin Nucleic Acid (HNA) comprising a single-stranded sequence, ½
- BBR, which under hybridizing condition 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 being a substance capable of discriminating between a perfect BBR and a BBR having impaired nucleotides.
-
9. A method of detecting the presence in a sample of a specific Target Nucleic Acid, TNA, which comprises:
-
(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; and
(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. - View Dependent Claims (39, 40, 41)
-
-
10. A method for detecting or localizing specific nucleic acid sequences with a high degree of sensitivity and specificity which comprises:
-
(a) adding PNAs containing a ½
BBR and a ½
TBR to a sample containing or suspected of containing TNAs containing ½
TBR sequences, to form a complex having target binding regions, TBRs, formed by the hybridization of complementary ½
TBRs present in the PNAs and TNAs respectively;
(b) binding the TBRs formed in step (a) to an immobilized TBA to form a TBA-TNA-PNA complex;
(c) adding Booster Nucleic Acids, BNAs, containing booster binding regions, ½
BBRs, to the complex formed in step (b) such that the ½
BBRs in the BNAs hybridize with the ½
BBR present in the PNAs or to ½
BBRs present in BNAs already bound to the PNA, to form BBRs, such that TBA-TNA-PNA-(BNA)n complexes are formed;
(d) adding Hairpin Nucleic Acids, HNAs, containing ½
BBR sequences, to the complex formed in step (c) such that the ½
BBRs in the HNAs hybridize with any available ½
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)n complexes of step (c) to form TBA-TNA-PNA-(BNA)n-HNA complexes;
(e) adding Booster Binding Assemblies, BBAs, linked to indicator moieties, to the TBA-TNA-PNA-(BNA)n-HNA complexes formed in step (d) to form TBA-TNA-PNA-(BNA-BBA)n-HNA complexes; and
(f) detecting the signals produced by the indicator moieties linked to the TBAs, PNAs, BNAs, BBAs or HNAs in the TBA-TNA-PNA-(BNA-BBA)n-HNA complexes of step (e);
wherein the TNA comprises;
(i) one or more specific ½
TBR nucleic acid sequences, the presence or absence of which in a particular sample is to be confirmed;
the PNA comprises;
(i) a single/stranded sequence, ½
TBR, which is capable of forming, under hybridizing conditions, a hybrid, TBR, with a ½
TBR present in a target nucleic acid (TNA);
(ii) a single stranded sequence, ½
BBR, which is capable of forming, under hybridizing conditions, a hybrid BBR with a ½
BBR present in a booster nucleic acid (BNA); and
(iii) an OSA, which is no attached support and/or indicator, or an attached support or other means of localization, including, but not limit to, attachment to beads, polymers, and surfaces, and/or indicators;
the BNA comprises;
(i) a ½
BBR, as shown in FIG. 1(IIb), which has a sequence which is complementary to a ½
BBR sequence in a PNA and which is capable of forming, under hybridizing conditions, a hybrid, BBR, with the PNA;
(ii) an OSA, which is no attached support 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;
(iii) additional hybridization sites, ½
BBRs, for other BNAs; and
(iv) sequences, ½
BBRs, which can hybridize to BNAs already hybridized to the PNA;
the BBA comprises;
(i) a molecule or a portion of a molecule which is capable of selectively binding to a BBR; and
(ii) 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;
and the TBA comprises;
(i) a molecule or a portion of a molecule which is capable of selectively binding to a TBR; and
(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.
-
-
11. In 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;
(a) using a Target Binding Assembly, TBA, as the means for achieving immobilization of said target polynucleotide, wherein said TBA binds only to a unique 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
(b) including in the PNA a single stranded sequence, ½
BBR, capable of binding a Booster Nucleic Acid, BNA, containing a single stranded complementary ½
BBR which, upon hybridization with the ½
BBR in the PNA, forms a BBR capable of binding labeled Booster Binding Assemblies, BBAs.
- 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;
- 12. A target binding assembly, TBA, or a booster binding assembly, BBA, comprising at least one nucleic acid recognition unit, and optional one or all of the sequences selected from the group consisting of a linker sequence, an assembly sequence, an asymmetry sequence, a nuclear localization signal sequence (NLS) and an OSA.
-
31. A diagnostic or forensic test kit for the detection in a sample of nucleic acid having a specific sequence composition, which comprises:
-
(a) a first nucleic acid probe complementary to nucleic acid with specific sequence composition, the presence of which is to be ascertained in a test sample, wherein said first nucleic acid probe and said nucleic acid with specific sequence composition forming, upon hybridization, a binding site for a first nucleic acid binding protein, and wherein said first nucleic acid probe further comprises additional sequence complementary to a second nucleic acid probe;
(b) a first nucleic acid binding protein specific for the duplex formed by hybridization of said first nucleic acid probe and said nucleic acid with specific sequence composition;
(c) a second nucleic acid probe complementary to said additional sequence in said first nucleic acid probe, wherein, upon hybridization of said first and second nucleic acid probes, a binding site for a second nucleic acid binding protein is formed;
(d) a second nucleic acid binding protein which binds specifically to the duplex formed upon hybridization of said first nucleic acid probe and said second nucleic acid probe, wherein said second nucleic acid binding protein is labeled with a detetable label. - View Dependent Claims (32, 33, 34, 35)
-
-
36. A composition comprising HIV-Lock or a recombinant vector encoding HIV-Lock and a pharmaceutically acceptable carrier.
-
37. A method of differentially binding a nucleic acid binding protein to a nucleic acid sequence correlated with a pathogenic condition which comprises:
-
(a) selecting a particular configuration of nucleic acid binding protein sequences present in the nucleic acid sequence correlated with a pathogenic condition as a target sequence for designing a probe nucleic acid which will hybridize to that particular configuration of nucleic acid sequences if present in a test sample, and further, ensuring that a binding site for an available nucleic acid binding protein is formed upon hybridization of said probe nucleic acid and said particular configuration of nucleic acid sequences chosen as a target;
(b) selecting a nucleic acid binding protein which specifically binds to the selected particular configuration of nucleic acid binding protein sequences correlated with a pathogenic condition, but which does not bind to sequences not correlated with said pathogenic condition;
(c) hybridizing said probe nucleic acid with a test sample suspected of containing said particular configuration of nucleic acid binding protein sequences present in nucleic acid sequences correlated with a pathogenic condition;
(d) contacting said nucleic acid binding protein with any hybrids formed in step (b); and
(e) detecting any binding of said nucleic acid binding protein with said hybrids. - View Dependent Claims (38)
-
-
43. A method of assembling a nucleic acid binding complex which comprises using asymmetry sequences to direct the association or non-association of components of the nucleic acid binding complex.
-
44. A method of assembling a nucleic acid binding complex which comprises using assembly sequences derived from bacteriophage lambda cro or CI to assemble associated components of the nucleic acid binding complex.
-
45. A method of using assembly, asymmetry, or piloting sequences to assemble a multimeric protein complex which comprises linking subunits to be incorporated into the multimeric protein complex to said assembly, asymmetry, or piloting sequence, and recovering said multimeric complex.
-
46. A composition comprising SEQ ID NO. 105, SEQ ID NO. 106, or SEQ ID NO. 108.
-
49. A method of assembling multimeric TBAs in vivo or in situ which comprises introducing component TBAs into a cell utilizing a covalently or non-covalently attached protein or bi-layer vesicle or by introducing nucleic acids encoding component TBAs into a cell, said component TBAs each comprising a DNA recognition unit, assembly sequences, asymmetry sequences, nuclear localization signal sequences, and optional linker sequences, such that upon proximal binding via the DNA recognition unit of each component TBA to nucleic acid sequences encountered in the nucleus or elsewhere in the cell, component expressed TBAs assemble via said assembly and asymmetry sequences into multimeric TBAs.
-
50. A method for identifying nucleic acid binding molecules for preparation of a target binding assembly or a booster binding assembly comprising:
-
a. Obtaining a sample containing the target nucleic acid;
b. Fragmenting the sample so as to expose the nucleic acids and to reduce the size complexity of the nucleic acids contained in the sample;
c. 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;
d. Analyzing the two aliquots to identify fragments which have altered behavior in the aliquot contacted with the target binding molecules as opposed to the aliquot contacted with the control buffer medium;
e. 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; and
f. synthesizing TBAs comprising the nucleic acid binding molecules which produced the altered behavior using assembly, asymmetry nuclear localization and, optionally, linker sequences.
-
-
51. A method for identifying specific nucleic acid sequences in a sample comprising:
-
a. Fragmenting the nucleic acids in said sample to expose the nucleic acids and reduce the size complexity of the nucleic acids;
b. Contacting a TBA with the sample, said TBA comprising two or more nucleic acid binding components each of which has a relatively weak binding for its nucleic acid recognition unit within the TBR but which in combination provides strong binding for the complete TBR; and
c. Eliminating any “
cross-talk”
produced by binding of the TBA to cousin nucleic acids that contain individual recognition units, which comprises contacting the sample with excess nucleic acid binding components with relatively strong binding affinity for cousin nucleic acids that contain the individual recognition units but relatively weak binding relative to the TBA'"'"'s affinity for binding to the complete TBR having said two or more nucleic acid binding components.
-
Specification