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Cross-coupled peptide nucleic acids for detection of nucleic acids of pathogens

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Title: Cross-coupled peptide nucleic acids for detection of nucleic acids of pathogens.
Abstract: The present invention concerns methods for detecting a nucleic acid comprising (i) contacting a solution comprising a first PNA having a first cross-reactive functional group with a substrate having a second PNA affixed thereto, the second PNA having a second first cross-reactive functional group, wherein the first PNA has a reporter molecule attached thereto and the first and second PNAs being complementary to different portions of a target DNA; (ii) contacting a sample suspected of containing the nucleic acid with the first and second PNAs; and (iii) determining the presence of the reporter molecule on the substrate. ...


Inventors: Daniel H. Appella, Christopher Micklitsch
USPTO Applicaton #: #20120107794 - Class: 435 5 (USPTO) - 05/03/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Virus Or Bacteriophage

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The Patent Description & Claims data below is from USPTO Patent Application 20120107794, Cross-coupled peptide nucleic acids for detection of nucleic acids of pathogens.

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CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation in part of U.S. patent application Ser. No. 12/441,925 filed Mar. 19, 2009, which claims benefit to International Patent Application No. PCT/US2007/020466, filed Sep. 21, 2007, which claims benefit of U.S. Provisional Application Nos. 60/846,354, filed Sep. 22, 2006 and 60/896,667, filed Mar. 23, 2007, each of which is incorporated herein by reference in their entirety; and the disclosures of which are incorporated herein in their entirety.

TECHNICAL FIELD

The instant invention concerns methods for detecting nucleic acids.

BACKGROUND

Polymerase chain reaction (PCR) is a widely used technique for the detection of pathogens. The technique uses a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. The PCR process generates DNA that is used as a template for replication. This results in a chain reaction that exponentially amplifies the DNA template.

Technologies for genomic detection most commonly use DNA probes to hybridize to target sequences. To achieve required sensitivity, the use of PCR to amplify target sequences has remained standard practice in many labs. While PCR has been the principle method to identify genes associated with disease states, the method has remained confined to use within a laboratory environment. Most current diagnostic applications that can be used outside of the laboratory are based on antibody recognition of protein targets and use ELISA-based technologies to signal the presence of a disease. These methods are fast and fairly robust, but they can lack the specificity associated with nucleic acid detection

Recently, it was reported that incorporating trans-1,2-diaminocyclopentane into aminoethylglycine peptide nucleic acids (aegPNAs) significantly increases binding affinity and sequence specificity to complementary DNA. See, Pokorski, et al, J. Am. Chem. Soc. 2004, 126, 15067-15073 and Myers, et al, Org. Lett. 2003, 5, 2695-2698. Despite the promise of PNAs with 1,2-diaminocyclopentane residues in the backbone, commercially viable uses of such PNAs have not been realized.

There is a need for pathogen detection methods that are highly specific and robust for use outside of a laboratory environment.

SUMMARY

In some aspects, the invention concerns methods of detecting a nucleic acid comprising:

contacting a solution comprising a first PNA having a first cross-reactive functional group with a substrate having a second PNA affixed thereto, said second PNA having a second first cross-reactive functional group, wherein the first PNA has a reporter molecule attached thereto and the first and second PNAs being complementary to different portions of a target DNA;

contacting a sample suspected of containing the nucleic acid with said first and second PNAs;

determining the presence of said reporter molecule on said substrate. The substrate can be washed prior to determining the presence of said reporter molecule.

In some aspects, the substrate is visually observed to detect the appearance of color from the reporter molecule. The detecting can be performed visually by an observer.

Preferred PNAs for the first and second PNAs include trans-cyclopentane-containing PNAs.

Any suitable cross-reactive functional groups may be used. For example, pyrrole-2,5-dione and a thiol functionality can be used as the functional groups. In some embodiments, the first cross-reactive group comprises a pyrrole-2,5-dione functionality. In certain embodiments, the second cross-reactive group comprises a thiol functionality. In addition, in some preferred embodiments, the reporter molecule is biotin.

While the instant methods can be used to detect a wide variety of samples, particularly useful samples include anthrax, avian flu, severe acute respiratory syndrome (SARS), tuberculosis (TB), human papilloma virus (HPV), or human immunodeficiency virus (HIV).

In one aspect, the invention concerns methods where the detection is performed by a method comprising:

contacting a solution comprising a first PNA with a substrate having a second PNA affixed thereto, wherein the PNA has a reporter molecule attached thereto and the first and second trans-cyclopentane PNAs being complementary to different portions of a target DNA;

contacting DNA with the first and second cyclopentane-containing PNAs;

visually observing the substrate to detect the appearance of color from the reporter molecule.

The invention also concerns kits for detecting a nucleic acid comprising:

solution comprising a first PNA having a first cross-reactive functional group; and

a substrate having a second PNA affixed thereto, said second PNA having a second first cross-reactive functional group,

wherein the first PNA has a reporter molecule attached thereto and the first and second PNAs being complementary to different portions of a target DNA;

Some kits may be adapted for detecting an infectious agent such as anthrax, avian flu, severe acute respiratory syndrome (SARS), tuberculosis (TB), human papilloma virus (HPV), or human immunodeficiency virus (HIV).

In yet other embodiments, the kit further comprises biotin-labeled PNA detection probe, such as an avidin-horseradish peroxidase conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a PNA-based sandwich-hybridization assay. PNAα is the capture probe, and PNAβ is the detection probe.

FIG. 2 illustrates signal amplification from PNA-based sandwich-hybridization using PNAα(2) and PNAβ(2) with 103 fmol DNA and HRP-avidin. Four wells of a 96-well plate are shown, and each column represents identical conditions. Blue color results from initial oxidation of 1-Step TurboTMB, and yellow color is produced once the enzymatic reaction is stopped.

FIG. 3 shows colorimetric detection of protective antigen DNA (PA) from Bacillus anthracis Ames 35 strain (+PA) and Ames 33 strain (−PA). The signal is obtained from PNA-based sandwich-hybridization using PNAα(2) and (2) with poly-HRP-avidin.

FIG. 4 illustrates the covalent cross-linking approach. A solution comprising a first PNA containing a first cross-reactive functional group and a reporter molecule and a substrate to which a second PNA containing a second cross-reactive functional group is attached, is contacted with a sample suspected of containing a nucleic acid. Once the two PNAs are present next to one another, cross-reactive functional groups form a covalent bond so that the both PNAs are now attached to the surface.

FIG. 5 illustrates use of horseradish peroxidase (HRP)-streptavidin to increase detection limits. Detection Limits were determined to be 10 fmol (10·10−15 mol) DNA with regular aegPNA and 10 zmol (10·10−21 mol) DNA with tcypPNA.

FIG. 6 presents results from an isothermal temperature control experiment. Synthetic Target DNA: 5′-GGA-TTA-TTG-TTA-TAG-GAA-TAG-TTA-AAT-3′; (SEQ ID NO:9); Surface Probe (SP1) H2NCO-Lys-(mPEG-Cys-Ac)-TTA-TAA-CTA-TTC-CTA-mPEG2-Ac (SEQ ID NO:10); Reporter Probe (RP1): H2NCO-Lys-(mPEG-Ac)-CCT-AAT-AAC-AAT-mPEG5-Mal (SEQ ID NO:11).

FIG. 7 presents results from immobilized PNA/DNA sandwich hybridization and capture experiments.

FIG. 8 presents results from experiments using different DNA concentrations.

DETAILED DESCRIPTION

OF ILLUSTRATIVE EMBODIMENTS

The present invention concerns diagnostic methods for detection of nucleic acids, without using PCR, that also is very stable. Using peptide nucleic acids (PNAs) we have engineered a system in which two PNAs with cross-reactive functional groups target adjacent sites of an oligonucleotide sequence associated with infection (anthrax, for example). One of the PNAs is covalently attached to a surface of a substrate while the other is free in solution and also bears a reporter molecule (biotin, for example). A sandwich-complex forms on the substrate surface only if the complementary DNA is present. Once the two PNAs are present next to one another, cross-reactive functional groups form a covalent bond so that the both PNAs are now attached to the surface. Once the both PNAs are attached to the surface, the surface may be washed extensively to remove impurities before a signal from the reporter molecule is developed.

Use of a DNA-promoted cross link to the surface is an advantageous aspect of this technology. In the non-covalent complexes (where the complex is held together only by hydrogen bonds), washing the surface is inherently tricky: too little washing and impurities interfere with the detection signal, too much washing and the complex is removed from the surface. In our cross-linked technology, excess washes will not remove the second PNA from the surface and this helps to improve the quality of signal over the non-covalent versions of nucleic acid detection.

The benefits of using Peptide Nucleic Acids (PNAs) include: (i) neutral backbone leads to increased duplex stability over DNA, (ii) enzyme degradation minimized by nonnatural backbone, (iii) synthesis from well established and efficient SPPS procedures (Boc- or Fmoc-) is available, and (iv) greater sequence selectivity over DNA. Numerous PNA variations are know in the art. These include compositions represented by the following structures.

Natural and non-natural bases can be used in these structures and are well known by those skilled in the art.

Many cross-reactive functional groups are known in the art and can be used with the present invention. In some embodiments, the cross-reactive functional groups can be of the formulas I and II. Reaction of a molecule of Group I with Group II produces a linkage shown by formula III.



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stats Patent Info
Application #
US 20120107794 A1
Publish Date
05/03/2012
Document #
12409159
File Date
03/23/2009
USPTO Class
435/5
Other USPTO Classes
435/611
International Class
/
Drawings
7



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