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.
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The instant invention concerns methods for detecting nucleic acids.
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.
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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
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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.
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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.
In some constructs, Groups I and II can be placed in a terminal position of the PNA chain. One of these groups can be placed on the PNA that is attached to a surface and the other group can be placed on the PNA that is free in solution. Typically, a reporter group is attached to the PNA that is free in solution.
As used herein, the term “reporter molecule” is to be understood to mean any group which is detectable by analytical means in vitro and/or in vivo and which confers this property to his property to the conjugate. Some reporter molecules are a fluorescent molecule having fluorescence properties which are a function of the concentration of the molecule. Other reporter molecules have a absorbance spectra that can be monitored for detection. Numerous reporter molecules are known to those skilled in the art and are suitable for use with the present invention. One preferred reporter molecule is biotin.
The term “cross reactive groups” refers to at least two groups that are capable of reacting to form a covalent bond linking the first and second PNAs.
Cross-linking reactive groups can be incorporated into PNAs by known techniques. In some embodiments, the cross-linking functional groups are attached in a terminal position in the PNA.
HRP Streptavidin consists of streptavidin protein that is covalently conjugated to horseradish peroxidase (HRP) enzyme. Streptavidin binds to biotin and the conjugated HRP provides a high level of activity for detection using an appropriate substrate system. In some preferred embodiments, the HRP can be a polymerized form of HRP. The commercially available complex is known to specifically react with biotin.
Some preferred PNAs contain trans-1,2-diaminocyclopentane which can potentially impact a broad range of scientific disciplines. Recent advances have improved the synthesis of trans-1,2-diaminocyclopentane. See, PCT Patent Application No. PCT/US2007/020466. These methods allow each nitrogen atom of cyclopentanediamine to be easily derivatized with identical or dissimilar groups.
Incorporation of trans-1,2-diaminocyclopentane into Peptide Nucleic Acids (PNAs) has a beneficial effect on the recognition of DNA and RNA sequences. As shown herein, this compound can be used in the development of nucleic acid detection kits for various pathogens.
Trans-1,2-diaminocyclopentanes can be prepared by ring-opening of an appropriate aziridine with an azide nucleophile in the presence of a promoter. In this way, two amine groups or its equivalents are installed in one step, thus circumventing the tedious functional group transformations. One such compound is trans-tert-butyl-2-aminocyclopentylcarbamate (1).
A scheme for producing rac-1 is illustrated by Scheme I.
In one embodiment, the synthesis begins with ring opening of tosyl-activated aziridine 2 (Scheme I, Table 1), which is readily accessible in one step from commercially available cyclopentene.
Without further purification, 3 can be reduced to the corresponding amine by Pd-catalyzed hydrogenation or Staudinger reduction (PPh3, THF/H2O). Those skilled in the art appreciate that other reactions may be used to convert 3 to the corresponding amine. Subsequent Boc protection of the resulting amine yielded 4 in 92% yield for two steps. Each of the aforementioned reactions are well known to those skilled in the art.
While this method is illustrated with the Boc protecting group, it is understood by those skilled in the art that other suitable protecting groups can be substituted. Additional protecting groups include any carbamate-based nitrogen protecting group. Examples of suitable protecting groups include fluorenyl-methoxy-carbonyl (Fmoc), carbobenzyloxy (Cbz), and allyloxycarbonyl (Alloc).
Generally, the major drawback of tosyl-activated aziridine chemistry is that harsh conditions are required for the cleavage of the sulfonamide bond at a later stage of the synthesis. Recently, milder conditions have been developed in this context, and magnesium in methanol under ultrasonic conditions has been successfully applied to a variety of substrates. Under these conditions, 4 underwent clean but very sluggish conversion. After considerable experimentation, the detosylation was achieved with lithium and naphthalene in dimethoxyethane (DME) or tetrahydrorfuran (THF). The reaction was temperature-dependent: at low (−78° C.) or room temperature, either very slow conversion (10%) was observed or low yield (40%) resulted. In one embodiment, the reaction was best performed at 0° C. for 5 h to afford 1 in 72% yield.
The resolution of primary amines with similar structures to 1 has been typically performed with tartaric acid or mandelic acid. Our initial attempts to resolve 1 with these two acids did not give precipitate under various conditions. Therefore, twenty other chiral resolving acids were screened. The resolution results were rapidly examined by 1H NMR method as follows (Scheme II): the precipitated salts were converted to amine 1 and subsequently treated with optically pure R-(+)-1-phenylethylisocyanate in CDCl3, to give corresponding urea disastereomers 5. The Boc groups of the two disastereomers 5 showed separated peaks at 1.30 and 1.44 ppm in the 1H NMR. Among the different chiral acids that were screened, di-p-toluoyl-tartaric acid, 2-phenylpropionic acid, and menthyloxyacetic acid showed partial resolution. Fortunately, optimal results were obtained when 10-camphorsulfonic acid (CSA) was used as a resolving agent. The precipitate from rac-1 and CSA (1:1 or 1:0.5) in acetone showed approximately 60% ee.
After crystallizations from acetonitrile, the optical purity of 1 was enhanced to over 99% enantiomeric excess (ee), as determined by HPLC analysis (on a chiral stationary phase) of the benzoylated derivative 6. The configuration of 1 obtained from the resolution was assigned based on the comparison of HPLC data of 6 (obtained on a chiral stationary phase) to material obtained from previous syntheses performed in our laboratory.
Among the end uses of compound 1 is incorporation into the backbones of PNAs. The PNAs of the instant invention can be used in methods for detecting a nucleic acid comprising contacting a sample suspected of containing the nucleic acid with a peptide nucleic acid prepared in accordance with the invention. These PNAs can be used in a kit for detecting a nucleic acid comprising at least one peptide nucleic acid prepared in accordance with the invention. The kits can be adapted for detecting an infectious agent, the infectious agent such as anthrax, avian flu, tuberculosis, severe acute respiratory syndrome (SARS), human papilloma virus (HPV), or human immunodeficiency virus (HIV). This list of agents is illustrative only and those skilled in the art are aware of other infectious agents that can be detected with the use of PNAs comprising 1,2-diaminopentane residues in the PNA backbone.
Chemical modification in the backbone of a peptide nucleic acid (PNA) lowers the detection limit of anthrax DNA by six orders of magnitude compared to the regular, unmodified PNA. Furthermore, the modified-PNA has improved sequence specificity compared to regular PNA, and is a key component of a colorimetric detection system for anthrax DNA. These findings make PNA a highly desirable probe for incorporation into DNA detection devices, and should stimulate the replacement of traditional DNA probes.
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. Direct detection methods that eliminate the requirement for a PCR step could afford faster and simpler devices that can be used outside of a laboratory. Devices based on nanotechnology have yielded impressive results, yet the use of PCR is still predominant in most applications. However, replacing DNA probes with a class of synthetic nucleic acids, such as peptide nucleic acids (PNAs), can significantly improve detection devices. There are numerous advantages to using PNA instead of DNA probes in hybridization assays, including: complete resistance to degradation by enzymes, increased sequence specificity to complementary DNA, and higher stability when bound with complementary DNA. Despite these advantages, the use of PNA in DNA detection systems has received sparse attention, and has not replaced the use of DNA probes. We believe that one reason PNA does not dominate in this area is due to the lack of backbone modifications that can be used to adjust the properties of a probe sequence. Without the ability to improve and fine-tune the basic properties of PNA, it is likely not worth the effort and/or funds for researchers to switch from DNA to PNA probes. We have developed a system of chemical modifications for PNAs, using cyclopentane groups, to predictably improve the melting temperature and sequence specificity of PNA-DNA duplexes. To demonstrate the utility of our chemical strategy and to highlight the importance of PNA in detection, we developed a simple, colorimetric sandwich-hybridization assay to detect anthrax lethal factor DNA using PNA as capture and detection probes (FIGS. 1 and 2). Our system has been developed into a convenient 96-well plate format in which the capture probe (PNAα) is covalently attached to a DNA-Bind® plate. A biotin-labeled PNA detection probe (PNAβ), in combination with commercially available avidin-horseradish peroxidase conjugate (avidin-HRP) and tetramethylbenzidine (TMB), is used to generate a signal if the target DNA is present. If a sandwich complex forms on the plate, the strong interaction between biotin and avidin will retain avidin-HRP on the plate. The HRP will then catalyze oxidation of TMB, and after stopping the reaction with sulfuric acid a colored product that absorbs at 450 nm is generated. These items are accessible to most biomedical research facilities. Incorporation of a cyclopentane-modified PNA residue (tcyp) into the capture probe (PNAα(1)) affords a system with a detection limit of 50 zeptomoles for lethal factor DNA, which is 6 orders of magnitude lower compared to the same system that uses regular, unmodified PNA (PNAα(2)) (FIG. 3 and Table 5). Furthermore, the sequence specificity is improved in the tcyp-modified PNA compared to the regular PNA.
Finally, we have demonstrated that our detection system effectively identities lethal factor DNA from a whole cell extract of B. anthracis DNA, giving a colored signal visible to the naked eye (FIG. 2). The stability of PNAs, and the ability to synthesize PNAs of different sequences should allow the development of effective detection systems for numerous bacterial and viral pathogens (such as HIV and Avian flu), as well as single nucleotide polymorphisms associated with several diseases (such as cancer and Alzheimer\'s disease). Furthermore, the ability to introduce chemical modifications with predictable effects into the PNA should allow researchers to fine-tune the properties of PNA for a specific application, and this flexibility could provide robust genomic detection devices available to healthcare workers in the field and to the general public.
2-Azido-N-tosyleyclopentanamine(3). To a mixture of 6-tosyl-6-aza bicyclo[3,1,0]hexane 2 (20.0 g, 84.4 mmol) and NaN3 (5.5 g, 84.4 mmol) in dry THF (300 mL) was added TMSN, (2.9 g, 3.0 mL, 25.3 mmol), followed by the addition of TBAF (25.3 mL, 1M in THF, 25.3 mmol). The solution was stirred at 40° C. for 20 h. The reaction solution was cooled to room temperature, saturated NaHCO3 aqueous solution (200 mL) was added. The aqueous layer was extracted with diethyl ether (100 mL×3). Combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under vacuum. The oil residue was filtered through a pad of silica gel and washed with a mixture of ethyl acetate/hexanes (1:2, 2000 mL). Solvents were removed under vacuum to afford 3 (22.4 g, 95%) as a colorless oil. Spectroscopic data of 3 were consistent with the literature data for this compound.
The reaction to produce 3 was repeated with a variety of conditions. These are summarized in the table below.