EP1639093A2 - Modified molecular beacons - Google Patents

Abstract

Nucleic acid reporters and methods of their use are provided. The nucleic acid reporters include molecular beacons modified with protein transduction domains to facilitate translocation of the nucleic acid reporter across cellular membranes. The nucleic acid reporters are also optionally modified with a targeting signal to direct the nucleic acid reporter to a specific cell, tissue, organ, intracellular region, organelle or vesicle.

Description

TKHR DOCKET NO. 820701.1135

MODIFIED MOLECULAR BEACONS

CROSS REFERENCE TO RELATED APPLICATION This application claims benefit of and priority to U.S. Provisional Patent

Application No. 60/482,648 filed on June 25, 2003. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Aspects of the work disclosed in this application were funded, in part, under Grant No. BSE-0222211 awarded by the National Science Foundation,

Grant No. F49620-03-0320 awarded by the Defense Advanced Research Projects Agency, and Grant No. R01 GM060562 awarded by the National

Institutes of Health. Therefore, the U.S. government may have certain rights in the claimed subject matter. BACKGROUND

1. Technical Field The disclosure is generally related to molecular beacons, for example, dual-labeled oligonucleotide probes with a fluorophore at one end and a quencher at the other end, more particularly to molecular beacons modified to translocate across membranes.

2. Related Art Quantitative methods to measure gene expression in vitro, such as real-time PCR and microarray analysis, have revolutionized molecular biology and drug development. These approaches, however, are generally used with purified DNA or RNA from cell lysates. The ability to detect and quantify the expression of specific endogenous mRNA in living cells and tissues in real time will offer tremendous opportunities for biological and disease studies, and will significantly impact drug discovery and medical diagnostics. However, currently available technologies for gene detection in intact cells such as in-situ hybridization (ISH) cannot be used in vivo or in living cells, since ISH studies rely on the use of fixed cells and washing away unbound probes to reduce background signal (Femino, A.M. et al. (1998) Science 280, 585-590; Levsky, J.M. et al. (2002) Science 297, 836-840). To perform gene TKHR DOCKET NO. 820701.1135

detection in living cells, the probes must be able to recognize the target with high specificity, convert target recognition directly into a measurable signal with high signal-to-background ratio, and allow for differentiation between true and false-positive events (Molenaar, C. et al. (2001 j Nucleic Acids Res. 29, E89-9). Probes must also be delivered into living cells with high efficiencies. Among the technologies currently under development for living cell gene detection and quantification, the most promising one is perhaps molecular beacons. Molecular beacons are dual-labeled antisense oligonucleotide (ODN) probes with a fluorophore at one end and a quencher at the other end (Tyagi, S., Kramer, F.R. (1996) Nat. Biotechnol. 14, 303-308; Tyagi, S. et al. (1998) Nat. Biotechnol. 16, 49-53). In contrast to fluorescently labeled linear ODN probes, molecular beacons are designed to form a stem- loop (hairpin) structure in the absence of complementary target so that fluorescence of the fluorophore is quenched. Hybridization with target mRNA opens the hairpin and physically separates the reporter form the quencher, allowing a fluorescence signal to be emitted upon excitation. Thus, molecular beacons enable a homogenous assay format where background is low without the need to wash away free probes. However, to detect mRNA in vivo, one needs to deliver molecular beacons into living cells with high efficiencies and fast kinetics. Conventional delivery approaches are based on DNA transfection techniques, such as those employing liposomes or dendrimers. These approaches are inefficient (<80%) (Barton, G.M., Medzhitov, R. (2002) Retroviral delivery of small interfering RNA into primary cells. Proc. Natl. Acad. Sci. U.S.A. 99, 14943-14945) slow (delivery times ~4 h), and can potentially trap molecular beacons in the endosomes and degrade them in the lysosomes, significantly increasing the background signal (Cheung, C. et al. (2001) Bioconjug. Chem. 12, 906-910). The long delivery time also gives rise to significant degradation of cytoplasmic molecular beacons by nuclease, further increasing false positive detection (Mitchell, P. (2001) Nat. Biotechnol. 19, 1013-1017). Although the reversible membrane permeabilization method using streptolysin O (SLO) is faster (~ 2 h) and not based on endocytosis (Faria, M. (2001) Nat. Biotechnol. 19, 40-44) it can only TKHR DOCKET NO. 820701.1135

be used in ex-vivo cellular assays. Further, this reversible permeablization method typically requires removal of serum/other growth factors in the media for 10-15 min, which may trigger a signaling cascade in cells, resulting in altered gene expression. Other delivery methods, such as microinjection (Sokol, D.L. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11538-11543) and electroporation (Yin, D., Tang, J.G. (2001) FEBS Lett. 495, 16-20) are invasive and may cause severe damage to cells. Further, microinjection is labor-intensive and only practical for studying a small number of cells. SUMMARY Aspects of the present disclosure provide compositions and methods for detecting, real time imaging and/or quantifying a target nucleic acid with high signal to background ratio. One aspect provides compositions that can non-invasively detect or quantify a target nucleic acid in a living cell. It has been discovered that nucleic acid reporters can be modified to translocate across membranes and thereby avoid invasive delivery techniques such as microinjection or poration of membranes. For example, the present disclosure provides modified nucleic acid reporters, including but not limited to, molecular beacons operably linked to a protein transduction domain. In other aspects, the nucleic acid reporters linked to a protein transduction domain can be further modified to be operably linked to a targeting signal, including for example, a nuclear localization signal. Some embodiments of the disclosed compositions can quickly and efficiently enter living cells without the need of any other delivery reagent, and enter specific membrane bound organelles or localize to specific intracellular regions. A particular aspect provides molecular beacons conjugated to a protein transduction domain (PTD) peptide TAT-1 providing a multifunctional probe that can enter into living cells with nearly 100% efficiencies, fast (-30 min) delivery kinetics, and the ability to localize in cell cytoplasm. Further, the disclosed nucleic acid reporters function well (with much better signal to background ratio) for mRNA detection in living cells as compared with the results of in situ hybridization. This nucleic acid reporter design provides a TKHR DOCKET NO. 820701.1135

powerful means for rapid detection of gene expression in living cells and tissues in real time, with high specificity and sensitivity. Another aspect provides a nucleic acid reporter having a streptavidin- biotin linkage. The nucleic acid reporter includes a modified nucleotide, for example biotin-dT, in the quencher arm of the stem. The biotin moiety can be linked to the modified nucleotide via a linker, for example an alkyl linker. A biotin-modified PTD is linked to the biotin modified nucleic acid reporter through a streptavidin molecule, which has four biotin-binding sites. In another aspect, the disclosed nucleic acid reporter includes a thiol- maleimide linkage in which the nucleic acid reporter is modified by adding a thiol group which can react with a maleimide group placed to the C terminus of the PTD to form a direct, stable linkage. Yet another aspect provides a nucleic acid reporter linked to a PTD or targeting signal with a cleavable disulfide bridge. The PTD can be modified by adding a cysteine residue at the C terminus which forms a disulfide bridge with the thiol-modified nucleic acid reporter. This disulfide bridge design allows the PTD to be cleaved from the nucleic acid reporter by the reducing environment of the cytoplasm. In another aspect, the delivery peptide sequence may be synthesized along with the nucleic acid reporter, for example, in the case of a PNA probe, where both the delivery peptide and the nucleic acid probe sequence can be generated using a single peptide. Further, the linkage between the delivery peptide and the nucleic acid probe can be tailored to allow for specific cleavage, for example by using a reducing disulfide bridge or using enzymatic cleavage sites in the linkage. Another aspect of the design allows for indirect linkage of the nucleic acid reporter with a delivery peptide, e.g., the delivery vehicle itself may be dendrimer-based or lipid-based such as liposomes/polymeric or any combination of the above, in which nucleic acid probes are packaged inside the delivery vehicle, with delivery peptides attached to the surface of such construct. The attached delivery peptides (single or multiple types) may allow TKHR DOCKET NO. 820701.1135

efficient delivery of the probes to a specific organ, tissue, cell type or subcellular compartment. Another aspect provides compositions and methods to detect a target nucleic acid, for example nuclear RNA, in living cells with high specificity and signal-to-background ratio than existing methods. This approach combines the ability of site directed delivery, for example, nucleus-specific delivery of probes using a toxin-based reversible permeablization of cells and a targeting signal, for example NLS peptide, and the sensitive detection of target nuclear RNA using molecular beacons. Other aspects include nucleic acid reporters having both a targeting signal and cell permeating peptides (e.g., Tat, poly- Arginine) for faster and more efficient delivery of probes into specific regions of a cell, for example the cell nucleus. Still another aspect provides a method for determining the effects of an agent on gene expression in a host by contacting one or more cells of the host with an agent, contacting the cells with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon is specific for a target nucleic acid, irradiating the cells with an exciting amount of radiation, detecting the electromagnetic emissions in response to the exciting amount of radiation, and comparing the emissions in cells treated with the agent to emissions from a control sample. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-D are schematic illustrations of three exemplary embodiments for linking the delivery peptide to molecular beacons. Figure 2A is a line graph of normalized fluorescence intensity as a function of time for unmodified molecular beacons, and for the three types of peptide-Iinked molecular beacons. Figure 2B is a bar graph of signal-to-background ratios of probe-target hybridization for exemplary peptide-Iinked molecular beacons linked by different conjugation methods. Figures 3A-G are fluorescence micrographs of HDF cells incubated with exemplary molecular beacons. TKHR DOCKET NO. 820701.1135

Figures 4A and B are fluorescence in situ hybridization micrographs of HDF cells showing GAPDH mRNA. Figures 5A and B are fluorescence micrographs showing the detection of survivin mRNA in live HDF and MiaPaca-2 cells. Figures 6A-F are fluorescence micrographs of HDF cells comparing cellular delivery of molecular beacons using conventional techniques. Figures 7A-D are fluorescence micrographs of HDF cells showing nuclear delivery of an exemplary embodiment of the disclosed nucleic acid reporters. Figure 8 is a panel of fluorescence micrographs of HDF cells showing nuclear and cytoplasmic localization of U1 snRNA using a U1 snRNA targeted molecular beacon linked with Tat-1 peptide. Shown in the figure are the Z- stacks (confocal slices at respective z positions). U1 snRNA is detected in the peri-nuclear region of cytoplasm and has a distinct spot like distribution in nucleus. DETAILED DESCRIPTION 1. Definitions In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below. The term "polypeptides" includes proteins and fragments thereof.

Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V). "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A TKHR DOCKET NO. 820701.1135 typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties. In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine ^• (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn TKHR DOCKET NO. 820701.1135

defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within + 0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set TKHR DOCKET NO. 820701.1135

forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest. "Identity," as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present invention. By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said TKHR DOCKET NO. 820701.1135

alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide. As used herein, the term "purified" and like terms relate to the isolation of a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components normally associated with the molecule or compound in a native environment. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. As used herein, the term "treating" includes alleviating the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. "Operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence, and an organelle localization sequence operably linked to protein will direct the linked protein to be localized at the specific organelle. "Localization Signal or Sequence or Domain" or "Targeting Signal or Sequence or Domain" are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, or intracellular region. The signal can be polynucleotide, polypeptide, or carbohydrate moiety or can be an organic or inorganic compound sufficient to direct an attached molecule TKHR DOCKET NO. 820701.1135

to a desired location. Exemplary organelle localization signals include nuclear localization signals known in the art and other organelle localization signals known in the art such as those provided in Tables 1 and 2 and described in Emanuelson et al., Predicting Subcellular Localization of Proteins Based on Their N-terminal Amino Acid Sequence. Journal of Molecular Biology.

300(4): 1005-16, 2000 Jul 21 , and in Cline and Henry, Import and Routing of Nucleus-encoded Chloroplast Proteins. Annual Review of Cell & Developmental Biology. 12:1-26, 1996, the disclosures of which are incorporated herein by reference in their entirety. It will be appreciated that the entire sequence listed in Tables 1 and 2 need not be included, and modifications including truncations of these sequences are within the scope of the invention provided the sequences operated to direct a linked molecule to a specific organelle. Organelle localization signals of the present invention can have 80 to 100% homology to the sequences in Tables 1 and 2. In some embodiments the organelle localization signals include signals having or conferring a net charge, for example a positive charge. Positively charged signals can be used to target negatively charged organelles such as the mitochondria. Negatively charged signals can be used to target positively charged organelles or regions. "Protein Transduction Domain" or PTD refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compounds that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing membranes, for example going from extracellular space to intracellular space, or cytosol to within an organelle. Exemplary PTDs include but are not limited to HIV TAT YGRKKRRQRRR (SEQ ID NO. 1) or RKKRRQRRR (SEQ ID NO. 2); 11 Arginine residues, or positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues. As used herein, the term "exogenous DNA" or "exogenous nucleic acid sequence" or "exogenous polynucleotide" refers to a nucleic acid sequence TKHR DOCKET NO. 820701.1135

that was introduced into a cell or organelle from an external source. Typically the introduced exogenous sequence is a recombinant sequence. As used herein, the term "transfection" refers to the introduction of a nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus or chloroplast. The nucleic acid may be in the form of naked DNA or RNA, associated with various proteins or the nucleic acid may be incorporated into a vector. As used herein, the term "vector" is used in reference to a vehicle used to introduce a nucleic acid sequence into a cell. A viral vector is virus that has been modified to allow recombinant DNA sequences to be introduced into host cells or cell organelles. As used herein, the term "organelle" refers to cellular membrane bound structures such as the chloroplast, mitochondrion, and nucleus. The term "organelle" includes natural and synthetic organelles. As used herein, the term "non-nuclear organelle" refers to any cellular membrane bound structure present in a cell, except the nucleus. As used herein, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The terms "nucleic acid," "nucleic acid sequence," or "oligonucleotide" also encompasses a polynucleotide as defined above. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically TKHR DOCKET NO. 820701.1135

involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are

"polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. "Oligonucleotide(s)" refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and double- stranded DNAs, among others. Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1 ,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofIuorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5- FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5- Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7- Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4- methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); TKHR DOCKET NO. 820701.1135

AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO™ -1 ; BOBO™ -3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FI; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1 ; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1 Ca²⁺ Dye ; Calcium Green-2 Ca²⁺ ; Calcium Green-5N Ca²⁺ ; Calcium Green-C18 Ca²⁺ ; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP - Cyan Fluorescent Protein; CFP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl TKHR DOCKET NO. 820701.1135

Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3' DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di- 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD - Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer -1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; lndo-1 , high calcium; lndo-1, low calcium;

Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1 ; JO- JO-1 ; JO-PRO-1 ; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1 ; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); TKHR DOCKET NO. 820701.1135

Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD

Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red;

Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green

488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7;

PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red);

Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;

PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma);

PKH67; PMIA; Pontochrome Blue Black; POPO-1 ; POPO-3; PO-PRO-1 ; PO- PRO-3; Primuline; Procion Yellow; Propidium lodid (PI); PyMPO; Pyrene;

Pyronine ; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine

Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine;

Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G;

Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine

Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine;

R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP;

SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron

Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS (Primuline);

SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1 ; SNAFL-2;

SNARF calcein; SNARF1 ; Sodium Green; SpectrumAqua; SpectrumGreen;

SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3- sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 11 ; SYTO 12; SYTO 13; SYTO 14; SYTO 15;

SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23;

SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44;

SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64;

SYTO 80; SYTO 81 ; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine

(TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine

(DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; TKHR DOCKET NO. 820701.1135

Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1 ; TO-PRO-3; TO-PRO-5; TOTO-1 ; TOTO-3; Tricolor (PE- Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781 ; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1 ; YO-PRO- 3; YOYO-1 ;YOYO-3 ,Sybr Green, Thiazole orange (interchelating dyes), semiconductor nanoparticles such as quantum dots, or caged fluorophore (which can be activated with light or other electromagnetic energy source) or a combination thereof. The term "nucleic acid reporter" means a compound, molecule, or polymer that specifically detects or can be used to detect a specific nucleic acid sequence. Exemplary nucleic acid reporters include, but are not limited to, labeled polynucleotides, for example labeled antisense polynucleotides. The nucleic acid reporters can be single or multistranded. Exemplary nucleic acid reporters also include molecular beacons. The term "molecular beacon" generally means dual-labeled antisense oligonucleotide (ODN) probes. Exemplary backbones include, but are not limited to, unmodified oligodeoxyribonucleotides, 2'-O-methyl oligoribonucleotides, phosphorothioate oligodeoxynucleotides, and oligodeoxynucleotides containing methylphosphonate, phosphoramidate, methythiophosphonate, or methythiophosphotriester. Exemplary labels include, but are not limited to, a fluorophore at one end and a quencher at the other end. A quencher can be any molecule or substance that reduces or eliminates the detectable single from the other label. Exemplary quenchers include but are not limited to, metal particles less than about 100 nm in diameter, typically less than about 10 nm in diameter, such as gold or silver or other dyes including but not limited to DABCYL (4-{[4- (dimethylamino)phenyl]diazenyl}benzoyl, BHQ-1, and BHQ-2. The quenchers can have no fluorescence themselves. Such quenchers are typically referred to as dark quenchers. Alternatively, the quenchers can be fluorescent themselves. TKHR DOCKET NO. 820701.1135

2. Modified Molecular Beacons One of several embodiments of the present disclosure provides nucleic acid reporter constructs that can non-invasively report the presence of a target nucleic acid either in vivo as well as in vitro. Non-invasive delivery refers to delivery without significant physical damage to a cell or tissue using for example, a mechanical device such as needle or other mechanical or physical means such as poration that may cause significant cellular or tissue damage. Embodiments of the disclosure provide nucleic acid reporters modified with a protein transduction domain to facilitate translocation of the nucleic acid reporter from the extracellular space to intracellular space. The nucleic acid reporter can translocate to any region of the interior of a cell including the interior of membrane bound organelles such as the nucleus, mitochondrion, or chloroplast. It will be appreciated by those of skill in the art that any membrane organelle is included within the scope of the disclosure. It will be further appreciated that any cell having a membrane is within the scope of this disclosure including, but not limited to animal cells such as human cells, or plant cells. Other embodiments provide nucleic acid reporters that are further modified to include targeting signals such as intracellular targeting signals, organelle targeting signals, cellular targeting signals, tissue targeting signals, or organ targeting signals. Generally, such targeting signals are known in the art. Targeting signals include, but are not limited to, amino acid or nucleic acid sequences, as will as lipids or carbohydrates that target the nucleic acid reporter to a specific cell, tissue, organ or intracellular region of a cell. Such targeting can be accomplished through receptor: ligand interactions or by using a targeting signal that modifies the polarity, hydrophobicity, hydrophilicity, or any combination thereof, of the nucleic acid reporter. Thus, the targeting signal can confer a positive or negative charge to the nucleic acid reporter as needed. Exemplary targeting signals include, but are not limited to, growth factors, growth factor receptors, antibodies or fragments thereof specific for extracellular eptiopes, carbohydrates, lipids, peptides, TKHR DOCKET NO. 820701.1135

nucleic acids, nuclear localization signals, mitochondria localization signals, polar or non-polar small molecules, co-factors and vitamins. It will be appreciated by those of skill in the art that the disclosed nucleic acid reporters can include a PTD, a targeting signal, or a combination thereof. The PTD, the targeting signal, or both can be releaseably linked to the nucleic acid reporter, for example through cleavable bonds, so that the nucleic acid reporter is released from the PTD or targeting signal when the nucleic acid reporter arrives at a desired location. In one aspect, the PTD is cleaved when the nucleic acid reporter enters the cytosol and the targeting signal remains linked to the nucleic acid reporter. In another aspect, the targeting signal is removed from the nucleic acid reporter when the nucleic acid reporter reaches the desired location. 2.1 Molecular Beacons The disclosed nucleic acid reporters include, but are not limited to, labeled oligonucleotides such as molecular beacons. Molecular beacons are polynucleotides generally having a pair of labels, for example a label at each end of the molecule. The polynucleotides include a sequence that is complementary to a target nucleic acid (target recognition sequence). The degree of complementarity is sufficient to enable sequence specific interactions between the nucleic acid reporter and the target nucleic acid. Some embodiments can detect single base differences or single nucleotide polymorphisms in a target nucleic acid. The disclosed nucleic acid reporters may have target recognition sequences 7-140 nucleotides, but it will be appreciated that the target recognition sequence can be of any length that permits sequence specific association with the target nucleic acid. In some embodiments, the sequences flanking the target recognition sequences form a stem hybrid, or "stem duplex" 3-25 nucleotides in length. Modified nucleotides and modified nucleotide linkages may be used to produce the disclosed nucleic acid reporters and are described more fully below. Such modifications are known in the art and include modifications to increase resistance to the enzymatic degradation. In non-limiting embodiments, labile phosphodiester or phosphoester linkages may be replaced with more stable TKHR DOCKET NO. 820701.1135

linkages, such as phosphorothioate or thioester linkages. For example the disclosed nucleic acid reporters may include, for example, peptide nucleic acid ("PNA") linkages. The disclosed compositions and target nucleic acids can be DNA, RNA including rRNA, nuclear RNA, mRNA, cDNA, genomic DNA, or combinations thereof. 2.1.1 Modified Nucleotide Linkages Some embodiments provide nucleic acid reporters including a plurality of nucleic acids or oligonucleotides containing modified backbones or non- natural intemucleoside linkages. Exemplary modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Representative modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonat.es, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Some oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; TKHR DOCKET NO. 820701.1135

5,536,821 ; 5,541 ,306; 5,550,111 ; 5,563,253; 5,571 ,799; 5,587,361 ; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050. Some oligonucleotide backbones do not include a phosphorus atom therein and have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methytenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541 ,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439. In still other embodiments provide nucleic acid reporters containing oligonucleotide mimetics in which both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States TKHR DOCKET NO. 820701.1135

patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al. (1991) Science 254:1497-1500. Some embodiments provide nucleic acid reporters having oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH₂-NH-O-CH₂-, -CH₂-N(CH₃)- O-CH₂- [known as a methylene (methylimino) or MMI backbone], -CH₂-O- N(CH₃)-CH₂- -CH₂-N(CH₃)-N(CH₃)-CH2- and -O-N(CH₃)-CH₂-CH₂- [wherein the native phosphodiester backbone is represented as -O-P-O- CH₂-] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. In other embodiments, the nucleic acid reporters may comprise modified oligonucleotides containing one or more substituted sugar moieties. Other modified oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C₁₀ alkyl or C₂ to C-io alkenyl and alkynyl. Particularly preferred are O[(CH₂)_nO]_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)nON[(CH₂)_nCH₃]₂, where n and m are from 1 to about 10. Other oligonucleotides comprise one of the following at the 2' position: Ci to C-io lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of the nucleic acid reporter and other substituents having similar properties. Another modification includes 2'-methoxyethoxy (2'-O- CH₂CH₂OCH₃, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al. (1995) Helv. Chim. Acta, , 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2'-DMAOE, and I'll TKHR DOCKET NO. 820701.1135

dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino- ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH₂-O-CH₂-N(CH₃)₂. Other modifications include 2'-methoxy (2'-O-CH₃), 2'-aminopropoxy (2'-OCH₂CH₂CH₂NH₂), 2'-allyl (2'-CH₂-CH=CH₂), 2'-O-allyl (2'-O-CH₂- CH=CH₂) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. An exemplary 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981 ,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811 ; 5,576,427; 5,591 ,722; 5,597,909; 5,610,300;

5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. A further modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (-CH₂-)_n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in U.S. Patent No. 6,268,490 and WO 99/14226. Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and TKHR DOCKET NO. 820701.1135

guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-haIo, 8-amino, 8-thiol, 8-thioalkyI, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8- azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1 H-pyrimido[5,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1 H-pyrimido[5,4-b][1 ,4]benzothiazin-2(3H)-one), G- clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)- H-pyrimido[5,4-b][1 ,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pa No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases may be particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, TKHR DOCKET NO. 820701.1135

pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No.

3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;

5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;

5,502,177; 5,525,711 ; 5,552,540; 5,587,469; 5,594,121 , 5,596,091 ;

5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681 ,941 , certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692. 2.1.1 Fluorescence Resonance Energy Transfer Some embodiments provide compositions that contain a reporter moiety whose reporting ability changes depending on whether the nucleic acid reporter is bound to its complement or target nucleic acid. For example, some nucleic acid reporters are designed to take advantage of quenching by fluorescence resonance energy transfer (FRET) or LRET (luminescence resonance energy transfer) to detect and report binding to target molecules.

FRET is a highly distance-dependent interaction between a fluorescent reporter dye in an excited state and a quencher in its ground state. Energy is transferred from one molecule (the fluorophore) to the other (the quencher) without the emission of a photon. Additional examples of reporting abilities may also include the use of interchelating dyes conjugated to nucleic acid probes, such that there is significant increase/decrease in signal upon binding to target nucleic acid. Further examples may include the use of reporters which change the polarization state of emission energy upon binding to target nucleic acid. As noted above, some embodiments of the disclosed nucleic acid reporters include a pair of labels. Representative pairs of labels include, but are not limited to, at least one donor/quencher pair, for example a dye pair, or a dye and a non-dye quencher. The pair of labels typically includes a fluorescent donor dye and a quencher for the donor fluorophore. In one TKHR DOCKET NO. 820701.1135

embodiment, the labels are linked to a sequence or structure in the nucleic acid reporter which does not hybridize directly to the target sequence. The disclosed nucleic acid reporters include any nucleic acid sequence or structure which can be labeled such that the presence of its complement or target nucleic acid indicates the presence of the target sequence. In one embodiment, the nucleic acid reporter moiety is labeled with a donor/quencher dye pair such that donor fluorescence is quenched prior to the sequence specific binding of the nucleic acid reporter to the target nucleic acid, and such that quenching of donor fluorescence is reduced as an indication of the presence of the target. The nucleic acid reporter may have a secondary structure such as a stem-loop (or hairpin) as described in U.S. Pat. No. 5,925,517 or a G-quartet as described in U.S. Pat. No. 5,691 ,145. The secondary structure is labeled such that the donor and quencher are in close proximity when the secondary structure is folded, resulting in quenching of donor fluorescence. In the presence of a target, the secondary structure is unfolded in a target-dependent reaction so that the distance between the donor and quencher is increased. This decreases quenching and produces an increase in donor fluorescence which can be detected as an indication of the presence of the target sequence. For efficient FRET quenching to take place the fluorophore and quencher molecules are typically less than about 100 A apart. In some embodiments, the absorption spectrum of the quencher overlaps with the emission spectrum of the fluorophore. Many donor/quencher dye pairs known in the art are useful in some embodiments of the present invention. These include, for example, fluorescein isothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TRITC), FITC/Texas Red™ (Molecular Probes), FITC/N- hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS)/FITC, FITC/Rhodamine X, FITC/tetramethylrhodamine (TAMRA), and others. For energy transfer quenching mechanisms donor/quencher pairs can be selected so that the emission wavelengths of the donor fluorophore overlap the excitation wavelengths of the quencher, i.e., there must be TKHR DOCKET NO. 820701.1135

sufficient spectral overlap between the two dyes to allow efficient energy transfer, charge transfer or fluorescence quenching. P-(dimethyl aminophenylazo) benzoic acid (DABCYL) is a non-fluorescent quencher dye which effectively quenches fluorescence from an adjacent fluorophore, e.g., fluorescein or 5-(2'-aminoethyl) aminonaphthalene (EDANS). Any dye pair which produces fluorescence quenching in the disclosed nucleic acid reporters are suitable for use in the disclosed methods, regardless of the mechanism by which quenching occurs. Terminal and internal labeling methods are also known in the art and may be routinely used to link the donor and quencher dyes at their respective sites in the nucleic acid reporter. 2.2 Protein Transduction Domains Embodiments of the present disclosure also include nucleic acid reporters operably linked to a protein transduction domain. Several small regions (9-16 amino acids) of proteins called protein transduction domains (PTDs) or cell penetrating peptides (CPPs) that confer the ability to traverse biological membranes efficiently. Without wishing to be bound by any one theory, it is believed that some PTDs are able to cross a cell membrane in a receptor-independent mechanism (Kabouridis, P. (2003) Trends in Biotechnology 21 (11 ):498-503). Although several of PTDs have been documented, the two most commonly employed PTDs are derived from TAT protein of HIV (Frankel and Pabo (1988) Cell 55(6): 1189-93) and Antennapedia transcription factor from Drosophila, whose PTD is known as Penetratin (Derossi et al.(1994) J Biol Chem. 271 (30):18188-93). The Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices. Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia.(Fenton et al. (1998) J Immunol Methods 212(1):41-8). TAT protein consists of 86 amino acids and is involved in the replication of HIV-1. The TAT PTD consists of an 11 amino acid sequence domain (residues 47 to 57; YGRKKRRQRRR (SEQ ID NO. 1) of the parent protein that appears to be critical for uptake (Vives et al. (1997) J Biol Chem. 272(25): 16010-7). TKHR DOCKET NO. 820701.1135

Additionally, the basic domain Tat(49-57) or RKKRRQRRR (SEQ ID NO. 2) (Wender et al. (2000) Proc Natl Acad Sci U S A. 97(24): 13003-8) has been shown to be a PTD. In the current literature TAT has been favored for fusion to proteins of interest for cellular import. Several modifications to TAT, including substitutions of Glutatmine to Alanine, i.e., Q to A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al. (2000) Proc Natl Acad Sci U S A. 97(24): 13003-8) to up to 33 fold in mammalian cells. (Ho et al. (2001) Cancer Res. 61(2):474-7) The most efficient uptake of modified proteins was revealed by mutagenesis experiments of TAT-PTD, showing that an 11 arginine stretch was several orders of magnitude more efficient as an intracellular delivery vehicle. Thus, some embodiments include PTDs that are cationic or amphipathic. Additionally exemplary PTDs include but are not limited to poly-Arg - RRRRRRR (SEQ ID NO. 3); PTD-5 - RRQRRTSKLMKR (SEQ ID NO. 4); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO. 5); KALA - WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO. 6); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO. 7), and RQIKIWFQNRRMKWKK (SEQ ID NO. 8). The disclosed nucleic acid reporters can have the protein transduction domain linked directly or indirectly to the reporter composition. One embodiment provides a nucleic acid reporter having a modified monomer, such as a nucleotide, to facilitate linking chemistries. The monomer can be modified with a reactive group such as an amine, carbonyl, carboxyl, and thiol or have a biotin group attached to the nucleotide. Such modified monomers are known in the art, and are commercially available and include modified nucleotides such as dT (Link Technologies, Scotland, UK). The protein transduction domain can be directly linked using conventional linking chemistry to the modified nucleotide or can be indirectly linked to the amine modified nucleotide using a linker or spacer group. The linker group can be linked to the modified nucleotide at one end, and linked to the PTD on the other end. TKHR DOCKET NO. 820701.1135

In another embodiment, the protein transduction domain is operably linked to the nucleic acid reporter using a streptavidin-biotin linkage. The nucleic acid reporter includes a modified monomer, for example biotin-dT, in the region of the nucleic acid reporter that is not complementary to the target nucleic acid, for example to the quencher arm of the stem. The linkage can be through a carbon linker. Protein transduction domains can be modified to include a biotin moiety. The modified PTD and the modified nucleic acid reporter can then be linked through a streptavidin molecule. In another embodiment, a thiol-maleimide linkage is used to link the PTD to the nucleic acid reporter. In one aspect, the non-complementary region of the nucleic acid reporter, for example, the stem region, in particular the stem region linked to a quencher, is modified by adding a thiol group to a monomer of the nucleic acid reporter. The thiol group can react with a maleimide group placed at the C terminus of the PTD or targeting signal to form a linkage, in particular a direct and stable linkage. Another embodiment incorporates a cleavable disulfide bridge in which the PTD or targeting signal is modified by adding a cysteine residue at the C terminus which forms a disulfide bridge with the thiol-modified nucleic acid reporter. This disulfide bridge design allows the PTD or targeting signal to be cleaved from the nucleic acid reporter by the reducing environment of the cytoplasm. Another embodiment allows for combined synthesis of the delivery peptide sequence along with the nucleic acid reporter, for example, in the case of a PNA probe, where both the delivery peptide and the nucleic acid probe sequence can be generated using a single peptide. Further the linkage between the delivery peptide and the nucleic acid probe can be tailored to allow for specific cleavage using reducing disulfide bridge or using enzymatic cleavage sites in the linkage. Another embodiment allows for indirect linkage of the nucleic acid reporter with delivery peptide, e.g., the delivery vehicle itself may be dendrimer-based or lipid-based such as liposomes/polymeric or any combination of the above, in which nucleic acid probes are packaged inside TKHR DOCKET NO. 820701.1135

the delivery vehicle, with delivery peptides attached to the surface of such construct. The attached delivery peptides (single or multiple types) may allow efficient delivery of the probes to a specific organ, tissue, cell type or subcellular compartment. 2.3 Linkers The disclosed nucleic acid reporters can be linked to a protein transduction domain or targeting signal via a linker or spacer. The linker can be one or more monomer units including atoms, amino acids, nucleic acids, sugars, or natural or synthetic polymer monomers. Generally, the linker is composed of monomers that are substantially inert or do not otherwise chemically react once coupled to the nucleic acid reporter. Representative linkers include alkyl linkers of 1 to 12 carbons, typically about 6 carbons. The alkyl groups of the linker can be substituted, for example with alkyl or aryl groups, heterocycles, halogens, and the like. The number of monomers of the linker can vary, however, the linker should have enough monomers to prevent the protein transduction domain or targeting signal from sterically interfering with the binding of the nucleic acid reporter to its target nucleic acid. Typically, the linker is modified to link the protein transduction domain or target signal via conventional linking chemistry. The linkers can be linked with or contain cleavable bonds, for example photo cleavable, thermally cleavable, or enzymatically cleavable bonds. Such bonds are known in the art. Upon entry into the cell, the cleavable bond can be cleaved. The cleavage of the bond can result in the separation of the PTD or targeting signal or both from the nucleic acid reporter. 2.4 Linking Chemistry Of the various linking chemistries that can be used to link molecules with other molecules or reagents, the most common are amine, carbonyl, carboxyl, and thiol. It will be appreciated by those of skill in the art, that any linking chemistry may be utilized. Indirect crosslinking of the amines in one molecule to the thiols in a second molecule is the predominant method for forming a heteroconjugate. If the nucleic acid reporter, the linker, or the TKHR DOCKET NO. 820701.1135

protein transduction domain does not already contain one or more thiol groups, the thiol groups can be introduce using a thiolation procedure. Thiol groups (also called mercaptans or sulfhydryls) are present in cysteine residues of proteins. Thiols can also be generated by selectively reducing cystine disulfides with reagents such as dithiothreitol (DTT) or - mercaptoethanol. Removal of DTT or -mercaptoethanol is sometimes accompanied by air oxidation of the thiols back to the disulfides. Reformation of the disulfide bond can be avoided by using the reducing agent tris-(2- carboxyethyl)phosphine (TCEP), which does not contain thiols. TCEP is generally impermeable to cell membranes and to the hydrophobic protein core, permitting its use for the selective reduction of disulfides that have aqueous exposure. The pH-insensitive and less polar phosphine derivative tris-(2-cyanoethyl)phosphine may yield greater reactivity with buried disulfides. Several methods are available for introducing thiols into molecules, including the reduction of intrinsic disulfides, as well as the conversion of amine, aldehyde or carboxylic acid groups to thiol groups. Disulfide crosslinks, for example of cystines in proteins, can be reduced to cysteine residues by dithiothreitol, tris-(2-carboxyethyl)phosphine or tris-(2- cyanoethyl)phosphine. Amines can be indirectly thiolated by reaction with succinimidyl 3-(2- pyridyldithio)propionate, followed by reduction of the 3-(2- pyridyldithio)propionyl conjugate with DTT or TCEP. Alternatively, amines can be indirectly thiolated by reaction with succinimidyl acetylthioacetate, followed by removal of the acetyl group with 50 mM hydroxylamine or hydrazine at near-neutral pH. Thiols can also be incorporated at carboxylic acid groups by an EDAC- mediated reaction with cystamine, followed by reduction of the disulfide with DTT or TCEP. Tryptophan residues in thiol-free proteins can be oxidized to mercaptotryptophan residues, which can then be modified by iodoacetamides or maleimides. TKHR DOCKET NO. 820701.1135

Thiol-reactive functional groups are primarily alkylating reagents, including iodoacetamides, maleimides, benzylic halides and bromomethylketones. Arylating reagents such as NBD halides react with thiols or amines by a similar substitution of the aromatic halide. Reaction of any of these functional groups with thiols usually proceeds rapidly at or below room temperature in the physiological pH range (pH 6.5-8.0) to yield chemically stable thioethers. Thiols also react with many of the amine-reactive reagents described in including isothiocyanates and succinimidyl esters. Although the thiol— isothiocyanate product (a dithiocarbamate) can react with an adjacent amine to yield a thiourea, the dithiocarbamate is more likely to react with water, consuming the reactive reagent without forming a covalent adduct. Iodoacetamides readily react with all thiols, including those found in peptides, proteins and thiolated polynucleotides, to form thioethers. Iodoacetamides can sometimes react with methionine residues. They may also react with histidine or tyrosine, but generally only if free thiols are absent. Although iodoacetamides can react with the free base form of amines, most aliphatic amines, except the -amino group at a protein's N-terminus, are protonated and thus relatively unreactive below pH 8. In addition, iodoacetamides react with thiolated oligonucleotide primers, as well as with thiophosphates and thiouridine residues present in certain nucleic acids, but usually not with the common nucleotides. Iodoacetamides are intrinsically unstable in light, especially in solution; reactions should therefore be carried out under subdued light. Adding cysteine, glutathione or mercaptosuccinic acid to the reaction mixture will quench the reaction of thiol-reactive probes, forming highly water-soluble adducts that are easily removed by dialysis or gel filtration. Although the thioether bond formed when an iodoacetamide reacts with a protein thiol is very stable, during amino acid hydrolysis the bioconjugate loses its fluorophore to yield S-carboxymethylcysteine. Maleimides are excellent reagents for thiol-selective modification, quantitation and analysis. The reaction involves addition of the thiol across the TKHR DOCKET NO. 820701.1135

double bond of the maleimide to yield a thioether. Maleimides apparently do not react with methionine, histidine or tyrosine. Reaction of maleimides with amines usually requires a higher pH than reaction of maleimides with thiols. Hydrolysis of maleimides to a mixture of isomeric nonreactive maleamic acids can compete significantly with thiol modification, particularly above pH 8. Furthermore, maleimide adducts can hydrolyze or they can ring-open by nucleophilic reaction with an adjacent amine to yield crosslinked products. This latter reaction can potentially be enhanced by raising the pH above 9 after conjugation. For example, a disulfide-containing linker or spacer, including but not limited to an alkyl linker or spacer of about 1 to about 12 carbon atoms, is photo- or thermally coupled to the target nucleobase or polynucleotide using conventional chemistry, for example azide chemistry. The disulfide bond is reduced, yielding a free thiol. A covalent bond is formed between the reagent thiol and a thiol-reactive linker, hapten, fluorochrome, sugar, affinity ligand, or other molecule. The linking of two molecules can be achieved using heterobifunctional crosslinkers. Representative heterobifunctional crosslinkers include, but are not limited to, p-maleimidophenyl isocyanate; succinimidyl acetylthioacetate; succinimidyI-trans-4(maleimidylmeyt yl)-cyclohexane-1 carboxylate (SMCC); succinimidyl acetylthioacetate (SATA); succinimidyl 3-(2- pyridyldithio)propionate (SPDP); Λ/-((2-pyridyldithio)ethyl)-4-azidosaIicylamide (PEAS; AET); 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (ATFB, SE); 4-azido-2,3,5,6-tetrafluorobenzoic acid, STP ester, sodium salt (ATFB, STP ester); 4-azido-2,3,5,6-tetrafluorobenzyl amine, hydrochloride; benzophenone-4-isothiocyanate; benzophenone-4-maleimide; 4- benzoylbenzoic acid, succinimidyl ester. The heterobifunctional crosslinkers can be photoreactive, amine and/or thiol reactive, or aldehyde/ketone reactive, or a combination thereof. 3. Targeting Signals The disclosed nucleic acid reporters can also include targeting signals or domains that target the nucleic acid reporter to a specific cell, tissue or TKHR DOCKET NO. 820701.1135

organ as well as specific intracellular locations or organelles. Such target signals are known in the art. For example, nuclear targeting signals can be found in the Nuclear Localization Signal Database at http://cubic.bioc.columbia.edu/db/NLSdb/ which is incorporated by reference herein in its entirety. Representative nuclear localization signals include, but are not limited to, SV 40 T antigen or a fragment thereof, such as PKKKRKV (SEQ ID NO. 9). The NLS can be simple cationic sequences of about 4 to about 8 amino acids, or can be bipartite having two interdependent positively charged clusters separated by a mutation resistant linker region of about 10-12 amino acids. Additional representative NLS include but are not limited to GKKRSKV (SEQ ID NO. 10); KSRKRKL (SEQ ID NO. 11); KRPAATKKAGQAKKKKLDK (SEQ ID NO. 12); RKKRKTEEESPLKDKAKKSK (SEQ ID NO. 13); KDCVMNKHHRNRCQYCRLQR (SEQ ID NO. 14); PAAKRVKLD (SEQ ID NO. 15); and KKYENWIKRSPRKRGRPRK (SEQ ID NO. 16). The targeting signal can also target the nucleic acid reporter to the mitochondria which are also known in the art. Representative mitochondrial targeting signals include, but are not limited to include the mitochondrial localization signal of subunit VIII of human cytochrome oxidase, the yeast cytochrome c oxidase subunit IV presequence and the amino-terminal leader peptide of the rat ornithine-transcarbamylase. The identification of the specific sequences necessary for translocation of a linked nucleic acid reporter into a chloroplast or mitochondria can be determined using predictive software known to those skilled in the art, including the tools located at http://www.mips.biochem.mpg.de/cqi- bin/proj/medgen/mitofilter. Targeting signals can also include vitamins such as folate to target the nucleic acid reporters to cells having a high number of folate receptors, including but not limited to, cancer cells. Asialoglycoprotein receptor ligands can also be linked to the disclosed nucleic acid reporters to target them to the liver. For example, N-acetylgalactosamine containing peptides, asialoorosomucoid, and galactoside-containing cluster ligands can be used. TKHR DOCKET NO. 820701.1135

Targeting sequences can also be synthetic small molecules/ligands with high affinity for specific compartments in addition to specific localizing sequences, e.g. mitochondria can be targeted using amphiphilic synthetic molecules. 4. Dual FRET/LRET Nucleic Acid Reporters Another embodiment of the present disclosure provides dual FRET nucleic acid reporters or luminesence resonance energy transfer (LRET) operably linked to a protein transduction domain, and optionally to a targeting signal. Dual FRET nucleic acid reporters include molecular beacons that operate in pairs. A first molecular beacon is labeled with a donor dye and a quencher and a second molecular beacon is labeled with an acceptor dye and a quencher. In the absence of their complements, the dyes are quenched by their respective quenchers. Each molecular beacon in the dual FRET pair includes sequences complementary to adjacent regions of a target nucleic acid such that FRET between the donor dye of the first molecular beacon and the acceptor dye of the second molecular beacon only occurs when both beacons are hybridized to the target nucleic acid. Generally, the beacons must hybridize to the target nucleic acid so that the donor dye and the acceptor dye are apart by about 6 nm or less. See Tsourkas et al (2003) Anal Chem 75 (3697-3703) and Santagelo, P. et al. (2004) Nucleic Acid Res. 32(6) e57 which are incorporated by reference herein in their entirety. 5. Methods of Use Embodiments of the present disclosure can be used to detect, localize, or quantify a target nucleic acid in a cell, in particular a living cell, tissue, or organ. Representative cells include plant and animal cells. Target nucleic acids can be any polymer of nucleotides, DNA, RNA or combinations thereof, genomic, mRNA, nuclear RNA, enzymatic RNA, enzymatic DNA, or ribosomal RNA. Embodiments of the disclosed methods provide real-time visualization of specific endogenous mRNA expression in vivo. One embodiment provides a method for detecting cells expressing a target nucleic acid including contacting a cell suspected of expressing the target nucleic acid with a molecular beacon operably linked to a protein TKHR DOCKET NO. 820701.1135

transduction domain. The molecular beacon includes a region complementary to the target nucleic acid. The molecular beacons can be added to a cell culture of immortalized cells, primary culture cells, transfected cells, or administered to a organism or tissue. Preferred tissues are those tissues with optical characteristics that enable the detection of the molecular beacons or it may involve the use of endoscopic instruments in combination with appropriate optical instrumentation for excitation and detection of electromagnetic emission in vivo. Preferred organisms include humans and zebra fish. When used with tissues, near-infrared labels are preferred. Once the cell is contacted with the nucleic acid reporter, the PTD on the nucleic acid reporter facilitates translocation of cell membranes including, but not limited to, lipid bilayers, micelles, vesicles, and organelles such as the nucleus, mitochondria or chloroplast. The PTD enables the nucleic acid reporter to travel from extracellular space to intracellular space including the interior of organelles. The nucleic acid reporters can be irradiated with an exciting amount of electromagnetic radiation. An exciting amount of radiation is that amount of radiation sufficient to enable the nucleic acid reporter to emit electromagnet radiation. The electromagnetic energy emitted by the nucleic acid reporter indicates that nucleic acid reporter has bound its complement and that the cell expresses the target nucleic acid. Another embodiment provides a method for sorting cells expressing a target nucleic acid. A plurality of cells suspected of expressing the target nucleic acid is contacted with at least one nucleic acid reporter, for example a molecular beacon, operably linked to a protein transduction domain, wherein the protein transduction domain facilitates translocation of the at least one molecular beacon to at least one of the plurality of cells' interior. The molecular beacon includes a region complementary to the target nucleic acid. The plurality of cells are irradiated with an exciting amount of electromagnetic energy and electromagnetic energy emitted in response to the exciting amount of electromagnetic radiation by the molecular beacon in the interior of at least of the plurality of cells can be detected. Cells emitting a detectable amount of electromagnetic radiation can then be separated from cells, which TKHR DOCKET NO. 820701.1135

are not emitting a detectable amount of electromagnetic radiation. Methods for separating cells are known in the art, for example, by using a fluorescence activated cell sorter, which are commercially available. Another embodiment provides a method for detecting a target nucleic acid in a host. This embodiment includes administering to the host a molecular beacon operably linked to a protein transduction domain, and optionally linked to a targeting signal. Generally, the molecular beacon is in the form of a pharmaceutical composition. The molecular beacons are irradiated with an exciting amount of electromagnetic radiation, and the electromagnetic radiation emitted by the molecular beacons in response to the exciting amount of electromagnetic radiation is detected. The detected electromagnetic radiation can be correlated with the presence, location, and quantity ofthe target nucleic acid in the host, cell, or tissue. Still another embodiment provides a method for detecting expression of a target nucleic acid in a living cell by contacting the cell with a molecular beacon operably linked to a protein transduction domain optionally linked to a targeting signal, wherein the molecular beacon is specific for the target nucleic acid; irradiating the molecular beacon with an exciting amount of electromagnetic radiation, and detecting the emission of electromagnetic radiation from the molecular beacon, wherein detectable emission from the molecular beacon is indicative of expression or location of the target nucleic acid in the cell. Still another embodiment provides an approach to study the transport of RNA in living cells and model organisms such as oocytes using molecular beacon operably linked to a protein transduction domain as well as a targeting signal such as a nuclear localization signal (NLS) whenever necessary. For these studies, molecular beacons may be delivered specifically to the nuclear compartment, where they hybridize with the target RNA molecules which may be subsequently transported to the cytoplasm. This real time imaging of RNA transport may be used to gain a better understanding of RNA biology and developmental biology. Further molecular beacon may be linked to a targeting sequence such as NLS and modified using caged fluorophore. In TKHR DOCKET NO. 820701.1135

the native state the modified molecular beacon does not emit much signal. Using controlled excitation in the nuclear compartment, the caged molecule may be released, emitting a signal that can be imaged for studying transport of RNA molecules from nucleus to cytoplasm. Another embodiment provides an approach in which co-localization of

RNA with subcellular organelles and the dynamics of RNA or DNA molecules interacting with proteins and can be studied. For this approach, a molecular beacon operably linked to a protein transduction domain optionally linked to a targeting signal is used in conjugation with fusion protein of interest (GFP- protein) or any flourescently labeled protein (fluorescent labeled antibody targeted protein) or subcellular organelle. The molecular beacons target the nucleic acid molecules of interest (RNA or DNA) and, upon hybridization with the target, provide electromagnetic emission in particular wavelength range, while the fusion protein or any flourescently labeled protein or organelle provides the non-overlapping wavelength upon excitation. This allows one to study the RNA-protein interactions and its dynamics in living cells. Yet another embodiment provides a method for identifying compounds that interfere with the expression of a target nucleic acid, for example a nucleic acid suspected to be involved with a pathological condition, for example cancer or other disease states. The method includes contacting a living cell or a plurality of living cells with compound suspected of modulating the expression a gene, for example a small organic molecule or antisense drug, contacting the cell with a nucleic acid reporter having a region complementary to a transcript of the gene, irradiating the nucleic acid reporter or the cell with an exciting amount of electromagnetic radiation, and detecting the emission of electromagnetic radiation from the nucleic acid reporter, wherein detectable emission from the nucleic acid reporter is indicative of expression or the location of the gene. The cell can be a primary culture, immortalized cell, or transfected cell. Typically, a cell is transfected with a gene of interest and the nucleic acid reporter is specific for transcripts of the gene of interest. A test compound can be selected based on its ability to decrease the expression of a gene or increase the expression of a gene when TKHR DOCKET NO. 820701.1135

compared to a control sample. A control sample includes a cell contacted with the nucleic acid reporter in the absence of the test compound. Decreased expression of a gene is indicated by little to no detectable emissions from the nucleic acid reporter; whereas, increased gene expression is indicated by detectable emissions from the nucleic acid reporter which are greater from cells treated with the test compound compared to cells not treated with the test compound. This screening method is adaptable to high-throughput screening and imaging, for example using a fluorescence activated cell sorter, cell arrays, tissue arrays, or other microfluidic devices, which allow for high throughput detection and imaging. Combinatorial libraries can be used with this method to identify compounds that modulate gene expression in vivo. Another embodiment provides a method for detecting, localizing, or quantifying more than one target nucleic acid in living cell (multiplexing). In this embodiment, a first and a second nucleic acid reporter are independently operably linked to a PTD and optionally to a targeting signal. The first and second nucleic acid reporters each include different target recognition sequences. The target recognition sequences can be directed to transcripts of different forms or alleles of the same gene, or can be for transcripts of different genes. The first and second nucleic acid reporters typically provide different detectable signals, for example emit at different wavelengths. Another embodiment provides a method for determining the effectiveness of a therapeutic, for example a therapeutic that modulates gene expression of a host or suspected of modulating gene expression. The disclosed nucleic acid reporters can be used to obtain in vivo gene expression data from a individual or cell specific gene expression data of an individual or host. Such data can be used to determine whether the therapeutic is effective in the individual based on the level of specific gene expression in a host. One embodiment provides a method including the steps of administering a therapeutic to a host, obtaining cells from the host, contacting the cells with a nucleic acid reporter operably linked to a PTD and optionally to a targeting signal, wherein the nucleic acid reporter includes a target recognition sequence complementary to a predetermined target nucleic acid, irradiating TKHR DOCKET NO. 820701.1135

the cells with an exciting amount of radiation, and detecting electromagnetic emission from the cells in response to the exciting amount of radiation, and correlating the amount of detectable electromagnetic emission from the cells with amount of target nucleic acid in the cells. A therapeutic includes compounds, molecules, drugs or combinations thereof, administered to a host to treat, alleviated, mitigate, a pathology of the host or a symptom of a pathology. Alternatively, one or more cells from a host can be contacted in vitro with an agent, for example a therapeutic agent, suspected of modulating expression of a target nucleic acid. Typically, the cells can be incubated for a period of time to allow the therapeutic agent to have a biological effect. The cells can then be contacted with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon includes a target recognition sequence complementary to the target nucleic acid. The cells containing the nucleic acid reporter are irradiated with an exciting amount of radiation and electromagnetic emission from the cells emitted in response to the exciting mount of radiation can be detected. Emission from the cells exposed to the therapeutic agent and containing the molecular beacon can be compared with emissions from a control sample of cells containing the molecular beacon but which were not exposed to the therapeutic agent. A difference in emission between the cells exposed to the therapeutic agent and containing the molecular beacon compared with emissions from a control sample of cells containing the molecular beacon but which were not exposed to the therapeutic agent indicates that the therapeutic agent modulates expression of the target nucleic acid in the host. It will be appreciated by one of skill in the art that more than one nucleic acid reporter specific for more than one target nucleic acid can be used to establish a profile of gene expression modulation by a particular agent or compound for an individual host. Such data can provide information to a medical practitioner to assist in determining course of treatment and use of specific medicines personalized to one individual or host. Typically, the agent or compound will be an agent TKHR DOCKET NO. 820701.1135

believed to exert a therapeutic effect, i.e., treat or alleviate a pathology or symptom of a pathology. The data provided herein show that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. This opens new opportunities that become possible only with peptide-Iinked molecular beacons. For example, the fast delivery kinetics (~30 min) and high efficiency in probe internalization, combined with the high specificity and signal-to- background ratio of molecular beacon makes the peptide-Iinked molecular beacon probes a unique and novel system for studying gene expression in living cells in real time. When delivered through the endocytic pathway (e.g., liposome-based transfection), antisense oligonucleiotide probes tend to be trapped inside endocytic vesicles and degraded in the endosomes and lysosomes by nucleases (Dokka, S., Rojanasakul, Y. (2000) Adv. Drug Deliv. Rev. 44, 35-49). Peptide-based internalization has the potential to avoid the endocytic pathway, therefore reducing false-positive signals due to nuclease degradation. Consequently, ODN backbone modifications of the probe such as the use of 2'-O-methyl modified molecular beacons may not be necessary. As demonstrated in our study, combining fast probe delivery with prompt optical imaging, a high signal-to-background can be realized in live cell mRNA detection. Although the dual FRET molecular beacons approach (Tsourkas, A. et al. (2003) Analytic. Chemistry 75:3697-3703; Santangelo, P. J. et al. (2004) Nucleic Acids Res. 32©6)e57 can also significantly reduce the false- positive signal and give rise to enhanced sensitivity in mRNA detection in living cells, it requires simultaneous hybridization of two probes on the same target and typically takes much longer than 30 min. The peptide-Iinked molecular beacons approach, on the other hand, is faster (-30 min) and simpler, which may be more suitable for certain applications, including basic biological studies of mRNA expression level in living cells in response to drug molecules, toxin and external stimuli, the knock-down effect of RNAi, and disease detection and diagnosis. Using NIR fluorophores as the reporter, peptide-Iinked molecular beacons have the potential to become a powerful TKHR DOCKET NO. 820701.1135

tool for shallow tissue molecular imaging with high sensitivity and spatial resolution. Although the data demonstrated the high efficiency of peptide-based probe delivery, the underlying mechanism of cell membrane penetration remains largely unknown. Some studies suggest that cell entry is independent of the endosomal pathway (Simeoni, F. et al. (2003) Nucleic Acids Res. 31 :2717-2724) with the notion that the internalization is much faster than known endocytotic processes (Vives, E. et al. (2003) Curr. Protein Pept. Sci. 4:125-132) whereas others suggest that the CPP-induced cargo internalization is due to classical endocytosis (Lundberg, M. et al. (2001) Mol. Ther. 8:143-150; Console, S. et al. (2003) J. Bio. Chem. 278:35109-35114). One possibility is that the initial interaction between the peptide and cell surface is due to electrostatic forces, but subsequent cell membrane penetration is mediated by glycosaminoglycans (Ziegler, A. et al. (2003) Biochemistry 42:9185-9194). However, many questions still remain open. For example, for certain cell lines the TAT-1 and other peptides have the ability to deliver cargos into cell nuclei, suggesting a directed motion in the cytoplasm. It is still unclear whether different CPPs follow a common mechanism or have different pathways. More biochemical and biophysical studies of the CPP- based delivery mechanism are clearly warranted. There are many challenges in quantifying mRNA expression in living cells, including the need to create an internal control for fluorescence intensity of the reporter fluorophore in the intracellular environment, and determine the fraction of mRNA molecules hybridized with probes. The detection sensitivity is also affected by the properties of fluorophore, fluorescence detection method, the optical imaging instrumentation used and background signal. It has been suggested that the molecular beacon based approach could detect as low as 10 mRNA molecules in a single cell (Sokol, D.L. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11538-11543). Alternatively, the average mRNA expression over a large number of cells can be obtained using FACS (Fluorescence Activated Cell Sorter) to yield more reproducible and statistically more significant results. TKHR DOCKET NO. 820701.1135

6. Pharmaceutical Compositions Pharmaceutical compositions and dosage forms of the disclosure comprise a pharmaceutically acceptable salt of disclosed or a pharmaceutically acceptable polymorph, solvate, hydrate, dehydrate, co- crystal, anhydrous, or amorphous form thereof. Specific salts of disclosed compounds include, but are not limited to, sodium, lithium, potassium salts, and hydrates thereof. Pharmaceutical compositions and unit dosage forms of the disclosure typically also comprise one or more pharmaceutically acceptable excipients or diluents. Advantages provided by specific compounds of the disclosure, such as, but not limited to, increased solubility and/or enhanced flow, purity, or stability (e.g., hygroscopicity) characteristics can make them better suited for pharmaceutical formulation and/or administration to patients than the prior art. Pharmaceutical unit dosage forms of the compounds of this disclosure are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., intramuscular, subcutaneous, intravenous, intraarterial, or bolus injection), topical, or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient. The composition, shape, and type of dosage forms of the compositions of the disclosure will typically vary depending on their use. For example, a dosage form used in the detection of nucleic acids involved in a disease or disorder but expressed at a low level may contain larger amounts of the TKHR DOCKET NO. 820701.1135

nucleic acid reporter, for example the disclosed compounds or combinations thereof, than a dosage form used to detect nucleic acids that are overexpressed in a disease or disorder. Similarly, a parenteral dosage form may contain smaller amounts of the nucleic acid reporter than an oral dosage form used to detect the same disease or disorder. These and other ways in which specific dosage forms encompassed by this disclosure will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990). Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well known to those skilled in the art of pharmacy or pharmaceutics, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient. For example, oral dosage forms such as tablets or capsules may contain excipients not suited for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the specific active ingredients in the dosage form. For example, the decomposition of some active ingredients can be accelerated by some excipients such as lactose, or when exposed to water. Active ingredients that comprise primary or secondary amines are particularly susceptible to such accelerated decomposition. The disclosure further encompasses pharmaceutical compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers. In addition, pharmaceutical compositions or dosage forms of the disclosure may contain one or more solubility modulators, such as sodium chloride, sodium sulfate, sodium or potassium phosphate or organic acids. A specific solubility modulator is tartaric acid. TKHR DOCKET NO. 820701.1135

Like the amounts and types of excipients, the amounts and specific type of active ingredient in a dosage form may differ depending on factors such as, but not limited to, the route by which it is to be administered to patients. However, typical dosage forms of the compounds of the disclosure comprise a pharmaceutically acceptable salt, or a pharmaceutically acceptable polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous, or amorphous form thereof, in an amount sufficient to detect the target nucleic acid and include a range of from about 10 mg to about 1000 mg, preferably in an amount of from about 25 mg to about 750 mg, and more preferably in an amount of from 50 mg to 500 mg. Additionally, the compounds and/or compositions can be delivered using lipid- or polymer-based nanoparticles. For example, the nanoparticles can be designed to improve the pharmacological and therapeutic properties of drugs administered parenterally (Allen, T.M., Cullis, P.R. Drug delivery systems: entering the mainstream. Science. 303(5665): 1818-22 (2004)). 6.1. Parenteral Dosage Forms Parenteral dosage forms can be administered to patients by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, administration DUROS®-type dosage forms, and dose-dumping. Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; Water for Injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, Sodium TKHR DOCKET NO. 820701.1135

Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a disclosed composition herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms. 6.2. Topical, Transdermal And Mucosal Dosage Forms Topical dosage forms of the disclosure include, but are not limited to, creams, lotions, ointments, gels, shampoos, sprays, aerosols, solutions, emulsions, and other forms know to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, Pa. (1985). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon), or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990). TKHR DOCKET NO. 820701.1135

Transdermal and mucosal dosage forms of the compositions of the disclosure include, but are not limited to, ophthalmic solutions, patches, sprays, aerosols, creams, lotions, suppositories, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th Ed., Lea & Febiger, Philadelphia, Pa. (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes, as oral gels, or as buccal patches. Additional transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredient. Examples of transdermal dosage forms and methods of administration that can be used to administer the active ingredient(s) of the disclosure include, but are not limited to, those disclosed in U.S. Pat. Nos.: 4,624,665;

4,655,767 4,687,481 4,797,284; 4,810,499 4,834,978; 4,877,618 4,880,633 4,917,895 4,927,687; 4,956,171 5,035,894; 5,091 ,186 5,163,899 5,232,702 5,234,690; 5,273,755 5,273,756; 5,308,625 5,356,632 5,358,715 5,372,579; 5,421 ,816 5,466;465; 5,494,680 5,505,958 5,554,381 5,560,922; 5,585,111 5,656,285; 5,667,798 5,698,217 5,741 ,511 5,747,783; 5,770,219; 5,814,599; 5,817,332 5,833,647 5,879,322 and 5,906,830, each of which are incorporated herein by reference in their entirety. Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal and mucosal dosage forms encompassed by this disclosure are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue or organ to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1 ,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof, to form dosage forms that are non-toxic and pharmaceutically acceptable. TKHR DOCKET NO. 820701.1135

Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with pharmaceutically acceptable salts of the disclosed compostions. For example, penetration enhancers can be used to assist in delivering the active ingredients to or across the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, an tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as TWEEN 80 (polysorbate 80) and SPAN 60 (sorbitan monostearate). The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of the active ingredient(s). Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of the active ingredient(s) so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery- enhancing or penetration-enhancing agent. Different hydrates, dehydrates, co-crystals, solvates, polymorphs, anhydrous, or amorphous forms of the pharmaceutically acceptable salt of a disclosed composition can be used to further adjust the properties of the resulting composition.

TKHR DOCKET NO. 820701.1135

7. Kits This disclosure encompasses kits which include the discloses nucleic acid reporters and directions, for example written instructions, for their use. A typical kit comprises a unit of nucleic acid reporter having a region complementary to a predetermined target nucleic acid. The kit can further include appropriate buffers and reagents known in the art for administering the nucleic acids to cell cultures or hosts. The kits can also include a plurality ofthe disclosed nucleic acid reporters specific for more than one target nucleic acid. EXAMPLES

Example 1 : Design of peptide-Iinked molecular beacons Peptide-Iinked molecular beacons targeting the human GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and survivin mRNAs, as well as molecular beacons with a 'random' probe sequence were designed and synthesized. The specific design of these molecular beacons, and the sequence of the 11 amino-acid TAT-1 peptide used in the study are shown in Table 1. GAPDH is a glycolytic protein that has a diverse range of activities in mammalian cells including membrane fusion, microtubule bundling, nuclear RNA export, DNA replication and repair (Sirover, M.A. (1999) Biophys. Acta 1432:159-84). These activities suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Survivin is a member of the inhibitor of apoptosis protein (IAP) family and also regulates cell division Chiou, S.K. et al. (2003) Med. Sci. Monit. 9PI25-29). The GAPDH beacon is comprised of a 19-base probe domain targeting the Exon 6 region of GAPDH gene flanked by complementary 5-base sequences that hybridize to form the stem. The survivin beacon has a 16-base target sequence with a similar design of the stem. The 'random' beacon was designed as a negative control, with a 17- base probe sequence that does not have any match in the entire human genome. To ensure good target accessibility, molecular beacons complementary to different segments of the same mRNA molecule are TKHR DOCKET NO. 820701.1135 typically designed and tested to avoid targeting sequences that are occupied by RNA-binding proteins or where double stranded RNA is formed. Table 1. Design of peptide-Iinked molecular beacons Peptide TAT (N terminus) TyrGlyArgLysLysArgArgGlnArgArgArg (C terminus) (SEQ ID NO. 1) Unmodified Molecular Beacon

GAPDH 5'-Cv3-CGACGGAGTCCTTCCACGATACCACGTCG-BHQ2-3' (SEQ ID NO. 17 Modified Molecular Beacons

GAPDH 5'-Cv3-CGACGGAGTCCTTCCACGATACCACG/thiol-dT/CG-BHQ2-3' (SEQ ID NO. 18)

Survivin 5'-Cv3-CGACGGAGAAAGGGCTGCCACG/thiol-dT/CG-BHQ2-3' (SEQ ID NO. 19)

Random 5'-Cv3-CGACGCGACAAGCGCACCGATACG/thiol-dT/CG-BHQ2-3' (SEQ ID NO. 20) Three conjugation strategies were developed in attaching the delivery peptide to molecular beacons, as illustrated in Figure 1. In the first approach (Fig. 1 A), peptides were linked to a molecular beacon through a streptavidin- biotin bridge by introducing a modified oligonucleotide, biotin-dT, to the quencher arm of the stem through a carbon-12 linker. Exemplary peptide- linked molecular beacons included the biotin-modified molecular beacon, a streptavidin molecule, and biotin-modified TAT-1 peptides. Since each streptavidin molecule has four biotin-binding sites, biotin-modified molecular beacons and delivery peptides were able to be linked on the same streptavidin molecule. The stoichiometry was controlled so that the probability of having more than one molecular beacons linked to the same streptavidin is small. In the second design (Fig. 1 B), a thiol group was placed on the quencher-arm of the molecular beacon stem through a carbon linker; the thiol group then reacted with a maleimide group added to the C terminus of the peptide to form a thiol-maleimide linkage (Fig. 1 B). Both the streptavidin- biotin bridge and the thiol-maleimide linkage are stable in the cell cytoplasm. For certain cell types, CPPs including TAT-1 peptide tend to carry cargos into cell nucleus (Snyder, E.L. and Dowdy, S.F. (2001) Curr. Opin. TKHR DOCKET NO. 820701.1135

Mol. Ther. 3:147-152; Nori, A. et al. (2003) Bioconjug. Chem. 14:44-50). While this may be useful in detecting nuclear RNA targets such as polyadenylated nuclear RNA (PAN), in most applications however, the target mRNAs are in the cytoplasm, so should be the molecular beacons. Thus, as the third approach, the TAT-1 peptide was functionalized by adding a cysteine residue at the C terminus which forms a disulfide bridge with the thiol-modified molecular beacon as shown in Figure 1C. This cleavable design was based on the rationale that the reducing environment of the cytoplasm will cleave the disulfide bond once the construct enters the cell, thereby separating peptide from probe.

Example 2: Peptide conjugation In the design shown in Figure 1A, streptavidin was chosen as a linker molecule because of its high affinity for biotin and availability of multiple biotin- binding sites. The modified nucleotide (dT) at the third base from the 3' end on the quencher-arm of the stem was linked to biotin through a 12-carbon linker, and the biotin-modified peptides were conjugated to the biotin-modified molecular beacon through binding between biotin and streptavidin. The peptide-Iinked molecular beacon complex was dialyzed with PBS (1X) overnight using a Slide-A-Lyzer Dialysis Unit, 10K MWCO (Pierce Biotech Inc., Rockford, IL) to exchange the buffer and to remove the unconjugated peptide. The modified molecular beacons were synthesized at Integrated DNA Technologies, Inc (Coralville, IA) and MWG Biotech, Inc (High Point, NC), and the modified peptides were synthesized by Invitrogen Inc (Carlsbad, CA) and Synpep Corporation (Dublin, CA). In the direct linkage approaches shown in Figures 1B and 1C, the same position on the quencher-arm of the stem bore a modified nucleotide dT- amine group with a 6-carbon linker. To generate a direct, stable linkage between the peptide and molecular beacon, the peptide was modified with a maleimide group at its C terminus. Amine-modified molecular beacons were reacted in PBS buffer with a twofold molar excess of the heterobifunctional crosslinker SPDP, N-Succinimidyl 3-(2- pyridyldithio)propionate (Sigma-Aldrich) for 2 h, followed by reduction with a TKHR DOCKET NO. 820701.1135

twentyfold molar excess of TCEP, 3,3',3"-Phosphinidyne-tripropionic acid hydrochloride (Sigma-Aldrich), to create a free thiol (-SH) functional group. This thiolated beacon was then reacted with maleimide-modified peptide to form a stable chemical bridge between the beacon and the peptide. The peptide-Iinked molecular beacon complex was dialyzed overnight using Slide- A-Lyzer Dialysis Unit, 10K MWCO (Pierce Biotech Inc., Rockford, IL) to remove the unconjugated peptide. A similar approach was used in forming a cleavable disulfide bridge between the cysteine-modified peptide and the amine-modified molecular beacon. To reduce the probability of forming beacon-beacon conjugates, a higher (1.5x) concentration of peptides was used compared with that of beacons. Further, the positive charge of peptides helped prevent homodimerization of peptides. Example 3: Solution assays of hybridization kinetics and signal-to-background ratio Measurement of hybridization kinetics and signal-to-background ratio of peptide linked molecular beacons was carried out using a SAFIRE microplate monochromator reader (TECAN, Austria). For kinetic studies, 200 nM peptide-Iinked molecular beacons were mixed with 1 μM complementary oligonucleotide target at 37°C and the fluorescence intensity was recorded as a function of time for conventional molecular beacons, direct linked (stable and cleavable) peptide-molecular beacon complexes, and streptavidin-Iinked peptide-molecular beacon complex. To determine the signal-to-background ratios, 200 nM conventional and peptide-Iinked molecular beacons were mixed with 200 nM of complementary target respectively in the microplate reader, and the fluorescence intensity at equilibrium was recorded. The fluorescence signal of each molecular beacon type (conventional and peptide- Iinked) in the absence of target was recorded as the background signal. All solution assays were performed in 1x PBS buffer. Example 4: Cellular delivery of peptide-Iinked molecular beacons Primary human dermal fibroblast (HDF) cells (Cambrex, NJ) and a pancreatic cancer cell line MiaPaca-2 (ATCC, VA) were used. These cells TKHR DOCKET NO. 820701.1135

were cultured in an 8-well Nalge Nunc culture plate with a glass coverslip bottom in their respective cell culture media for 24 h prior to experiments. Delivery assays were performed by incubating cells at 37°C with the media containing peptide-Iinked molecular beacons. For molecular beacons targeting GAPDH, peptide-Iinked molecular beacons with three different concentrations (0.25 μM, 0.5 μM and 1.0 μM) were incubated with HDF cells for 30, 60 and 90 min. For molecular beacons targeting survivin, 0.5 μM of peptide-Iinked molecular beacons were incubated with HDF and MiaPaca-2 cells for 30 min. For all assays, the cells were washed twice with PBS to remove the incubation medium and placed in fresh medium for fluorescence imaging, which was carried out using a confocal microscope (Axiovert LSM- 100, Zeiss). Example 5: Fluorescence in-situ hybridization. Normal human dermal fibroblast cells were cultured in 8-well chambered coverslides for 24 hours in normal growth medium (FGM-2

Cambrex Co.) and then washing with 1x PBS (without Ca or Mg). The slide was fixed in 100% methanol at -20 °C for 10 minutes. After removing the methanol, the slides were allowed to air dry and stored overnight at -80 °C. In-situ hybridization assays were then performed by first washing the slides for 5 minutes in 1x PBS and hybridizing them overnight at 37 °C in 1x PBS (no Ca or Mg) containing 200 nM of unmodified GAPDH-targeting molecular beacons. After removing the hybridization solution with washing and adding 1x PBS, the cells were imaged using an Axiovert 100 epi-fluorescent microscope. Alternatively, normal HDF cells were fixed in 100% methanol at -20°C for 10 min, allowed to dry and kept at -80°C for 12 h. In-situ hybridization assays were performed as described above with 400 nM of fluorescently labeled linear probes targeting wild-type K-ras and GAPDH. The cells were imaged after removing the hybridization solution with washing and adding 1x PBS. TKHR DOCKET NO. 820701.1135

Example 6: Delivery of molecular beacons using commercial transfection reagents To compare the efficiency and functionality of different delivery methods, three commercially available transfection reagents were used:EFFECTINE® (Qiagen), SUPERFECT® (Qiagen), and

OLIGOFECTIMINE™ (Invitrogen). Transfection assays were carried out according to the procedure recommended by respective suppliers; both primary HDF cells and MiaPaca-2 pancreatic cancer cells were incubated with conventional (unmodified) molecular beacons for 0.5, 2 and 3.5 h. Example 7: Hybridization kinetics of peptide-Iinked molecular beacons To determine the effect of peptide conjugation on molecular beacon function, in-solution hybridization assays were carried out for the binding kinetics of peptide-Iinked molecular beacons with different conjugation methods. One concern was that, when the positively charged TAT-1 peptide is conjugated to a molecular beacon, it might interact with the negatively charged hairpin oligonucleotide, thus interfering with proper probe-target binding. Shown in Figure 2A are normalized fluorescence intensity verses time curves as a result of probe-target hybridization for unmodified GAPDH- targeting molecular beacons and peptide-Iinked molecular beacons with the streptavidin-biotin linkage, the stable thiol-maleimide linkage, and the cleavable disulfide bridge. Peptide-Iinked molecular beacons with the thiol- maleimide linkage had almost exactly the same probe-target hybridization kinetics as unmodified molecular beacons (black and green curves respectively in Fig. 2A), indicating that the conjugation of peptide using the thiol-maleimide linkage has essentially no effect on the functionality of molecular beacons. Molecular beacons with the cleavable disulfide bridge also behaved similarly to the unmodified ones. With streptavidin-biotin linkage, the hybridization kinetics of peptide-Iinked molecular beacons was slightly slower, but it did not affect the signal level, as can be seen from Figure 2A. It is possible that the streptavidin molecule, whose size is comparable to that of the molecule beacon, may have sterically hindered binding between the target and the hairpin probe, leading to a slightly reduced hybridization TKHR DOCKET NO. 820701.1135

kinetic rate. The effect of different conjugation methods on signal-to- background ratio (S/B) is relatively small (Fig. 2B), although the S/B for peptide-Iinked molecular beacons was slightly lower than that of the unmodified ones. Taken together, the results shown in Figure 2 indicate that conjugation of peptide to molecular beacons did not impair their normal function.

Example 8: Detection of GAPDH mRNA using peptide-Iinked molecular beacons To demonstrate the self-delivery and mRNA targeting functions of peptide-Iinked molecular beacons, detected mRNA of a housekeeping gene human GAPDH in normal human dermal fibroblast (HDF) cells was detected. After just 30 min of incubation with TAT-peptide conjugated GAPDH-targeting molecular beacons, clear and localized fluorescence signal in HDF cells as a result of molecular beacon-target mRNA hybridization for all three conjugation schemes, i.e., thiol-maleimide (Fig. 3A), disulfide bridge (Fig. 3B) and streptavidin-biotin (Fig. 3C) was observed. In contrast, peptide-Iinked random-sequence molecular beacons with streptavidin-biotin conjugation gave essentially no signal 30 min after delivery (Fig. 3D). Similar results were obtained using random-sequence molecular beacons with thiol-maleimide and disulfide linkages for peptide. This demonstrates that peptide-Iinked molecular beacons remained highly specific in living cells after internalization. Further, it was found that GAPDH mRNAs displayed a very intriguing filament-like localization pattern in HDF cells, with a clear tendency of surrounding the cell nucleus and following the cell morphology (Figs. 3A-C). Interestingly, molecular beacons with the cleavable (thiol-cysteine disulfide bridge) design seemed to give better localization patterns than those with the thiol-maleimide linkage, and the latter seemed to perform better than molecular beacons with the streptavidin-biotin linkage. Cleavage of the delivery peptide from the construct may have provided molecular beacons a better access to target mRNA molecules, although more studies of this phenomenon are required to validate this assumption. It is likely that a molecular beacon with a relatively bulky streptavidin molecule is TKHR DOCKET NO. 820701.1135

less able to penetrate into the secondary structure of the GAPDH mRNA, thus reducing its ability to seek out its targets. Almost all the HDF cells exposed to GAPDH peptide-Iinked molecular beacons showed strong fluorescence signal, implying a near 100% delivery efficiency (data not shown). Similar results were obtained after 60 min of incubation (Fig. 3E-H).

About the same level of fluorescence was observed for GAPDH-targeting molecular beacons with different linkages for peptide, whereas the random- sequence molecular beacons did not give much signal. Even after 90 min, there was essentially no increase in the signal level (data not shown), indicating that most of the peptide-Iinked molecular beacons entered the HDF cells within the first 30 min. Further, fluorescence signal levels and mRNA localization patterns in HDF cells were similar for experiments at three different nominal molecular beacon concentrations (0.25 μM, 0.5 μM and 1.0 μM). Due to the peptide conjugation process, it was estimated that, with 0.25 μM nominal molecular beacon concentration, the actual concentration of peptide-Iinked molecular beacons used in the assay was about 150-200 nM. Example 9: Comparison With in situ hybridization To correlate the results of present method with a traditional method, fluorescence in situ hybridization (FISH) assays targeting GAPDH mRNA in fixed HDF cells were performed. The probes used in the FISH assays were fluorescently labeled linear probes (5'-Cy5-GAGTCCTTCCACGATACCA-3') (SEQ ID NO. 21) that have the same probe sequence as the GAPDH- targeting molecular beacon. As demonstrated in Figure 4A, the fluorescence image obtained in FISH assays of GAPDH mRNA in HDF cells gave a filamentous localization pattern similar to that shown in Figure 3, confirming that the mRNA localization revealed in this study is not an artifact. However, the fluorescence signal as a result of FISH was not as sharp as that in living cell assays. Since the probes entered into both the cell cytoplasm and nucleus during FISH, a diffused fluorescence signal appeared in the fixed HDF cell nuclei (Fig. 4A). This is in contrast to the living cell images shown in Fig. 3 where very low signal can be observed in cell nucleus. This diffused signal may reflect the high abundance as well as a rather uniform distribution TKHR DOCKET NO. 820701.1135

of GAPDH mRNA in cell nucleus. As a negative control, we performed a FISH assay with fluorescently labeled linear Poly-A probes (5'-Cy5- AAAAAAAAAAAAAAAAAA-3') (SEQ ID NO. 22) and the resulting background signal was very low, as can be seen from Fig. 4B. This further confirmed that the fluorescence signal observed in our live cell and fixed cell studies of specific mRNA detection was truly due to probe/target hybridization. Example 10: Detection of survivin mRNA To demonstrate the ability of molecular beacons to determine gene transcription levels, expression of Survivin mRNA in live HDF and MiaPaca-2 cells was observed. Survivin expression level is very low in HDF cells, whereas in MiaPaCa-2 cells the level is relatively high. After 60 min of incubation with peptide-Iinked molecular beacons, the fluorescence signal in MiaPaca-2 cells was quite high, as shown in Figure 5A, but in HDF cells, only very low fluorescence signal can be observed (Fig. 5C). In addition, Survivin mRNAs shown an intriguing localization pattern, i.e., the Survivin mRNA molecules in MiaPaCa-2 cells seemed to be concentrated near one side of the cell nucleus (Fig. 5B). Although with very low expression level, Survivin mRNA localized in HDF in a similar fashion (Fig. 5C). Previous research suggested that the expression level and localization of Survivin may be an important indicator for cancer progression or prognosis (Okada, E. (2001) Cancer Lett. 163:109-116). Example 11 : Comparison with conventional transfection methods As mentioned above, cellular delivery of molecular beacons using conventional transfection methods, either liposome based or dendrimer based, typically requires 3-4 hours of incubation during which a high level of background signal is generated. To compare the efficiency of delivery and the stability of molecular beacons during internalization, we performed delivery studies of unmodified GAPDH-targeting molecular beacons with three commercially available transfection reagents: SUPERFECT® (dendrimer- based), OLIGOFECTAMINE™ (liposome-based), and EFFECTENE® (liposome based), and followed all the manufacturers' instructions. When fluorescence in HDF cells was observed after 30 min of GAPDH beacon TKHR DOCKET NO. 820701.1135

delivery using Superfect, OLIGOFECTAMINE™ or EFFECTENE®, there was essentially no signal (data not shown). It was found that, after 3.5 h of transfection with SUPERFECT®, the molecular beacons gave strong signal in HDF cells. However, the fluorescence image had different characteristics from that of peptide-Iinked molecular beacons. Specifically, the fluorescence signals were concentrated in random 'bright spots' in both cytoplasm and nucleus (Fig. 6A), suggesting that molecular beacons were trapped and degraded by endosomes, lysosomes and nucleus. Even more disturbing is that the random-sequence molecular beacons showed similar fluorescence signal levels and 'bright spots' in HDF cells (Fig. 6D), indicating that the signals in both cases were largely due to molecular beacon degradation. With OLIGOFECTAMINE™ transfection, the results after 3.5 h incubation were even worse: only highly concentrated bright spots were present in the HDF cells, with a similar signal level generated by GAPDH-targeting and random- sequence molecular beacons (Figs. 6B and 6E). Using EFFECTENE® for molecular beacon delivery gave fluorescence images (Figs. 6c and 5f with GAPDH-targeting and random-sequence molecular beacons, respectively) that resembled the corresponding images shown in Figures 6a and 6e. Clearly, caution must be taken in using these transfection methods in delivering molecular beacons for living cell gene detection assays. Example 12: Molecular beacons targeting K-ras The K-ras-targeting and 'random' sequence molecular beacon pairs adopted the shared-stem design (Tsourkas et al., 2003). The K-ras-targeting, GAPDH-targeting molecular beacons and Cy5-labeled random beacons were synthesized by Biosource International (Camarillo, CA) and MWG Biotech (High Point, NC). The Cy3-labeled random beacon and all of the synthetic targets were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The GAPDH-targeting molecular beacon (see Table 2) was designed such that the stem sequence is independent of the target sequence. The underlined bases in all the beacon designs shown in Table 2 indicate the bases added to form the stem of a molecular beacon. TKHR DOCKET NO. 820701.1135

Table 2. Target sequences and the design of molecular beacons

K-ras dual FRET molecular beacons Donor MB: 5'-/Cv3/CC 4CGCC CCΛGCrCCGTAGG/BHQ-2/-3' (SEQ ID NO. 23) Acceptor MB: 5'-/BHQ-3/AGTGCGCrGT \rCGTCA GGC/ACT/Cv5/-3' (SEQ ID NO. 24)

GAPDH molecular beacon '-Cv3-CGACGGAGTCCTTCCACGATACCACGΛhiol-dT/CG-BHQ2-3' (SEQ ID NO. 25) 'Random' sequence molecular beacons Donor MB: 5'-/Cv3/CACGTCGΛCA4GCGC/4CCGΛTACGTG/BHQ-2/-3' (SEQ ID NO. 26) Acceptor MB: 5WBHQ-3/ACGTGCG/AC/4 \GCGC/4CCG 7OACGT/Cv5/-3' (SEQ ID NO. 27).

Example 13: SLO delivery of molecular beacons and organelle labeling Molecular beacons targeting K-ras mRNA were delivered into cytoplasm of living cells using a reversible permeabilization method with SLO. The detailed protocol was described elsewhere (Santangelo el al. (2004) Nucleic Acids Res. 32(6): e57). Briefly, SLO was activated first by adding 5 mM of TCEP to 2 U/ml of SLO for 30 min at 37° C. Cells were grown in 24- well plates were incubated for 10 min in 200 μl of serum free medium contaiing 0.2 U/ml of activated SLO (0.5 U SLO per 10⁶ cells) and 5 μl of each molecular beacon. Cells were resealed by adding 0.5 mil of typical growth medium and incubated for 1 h at 37° C before performing fluorescence microscopy imaging. Molecular beacons targeting GAPDH mRNA was linked to the TAT peptide via a disulfide bridge as provided above. In order to image mitochondria and ER, MitoFluor Green (Molecular Probes) and ER-Tracker (Molecular Probes) were utilized respectively as per the protocols provided by Molecular Probes. Cells were incubated with each of the dyes for 30 min prior to the delivery of molecular beacons. Example 14: Dual FRET molecular beacons For dual FRET molecular beacons, Cy3 and Cy5 are selected as the donor and acceptor fluorophores, respectively, with excitation at 545 nm and emission detection at 670 nm for the FRET signal detection. MitoFlour Green was selected to label mitochondria to avoid possible spectral overlap with the TKHR DOCKET NO. 820701.1135

Cy3-Cy5 FRET pair. Specifically, the fluorescence signal of MitoFlour Green was detected using a bandpass filter of 510-530 nm under the excitation wavelength of 450 ± 25 nm. Since MitoFlour Green has essentially no excitation at 545 nm (Cy3 excitation) and no emission at 670 nm (Cy5 emission detection), as demonstrated in Fig. 8, the optics used for detecting the FRET signal due to molecular beacons should have no effect on the image of mitochondria shown in Fig. 7A. Likewise, there is very limited excitation of Cy3 at 488 nm and no fluorescence emission of Cy3 at 510-530 nm (Fig. 8). Therefore, there was no cross-talk between FRET detection of K- ras mRNA and the imaging of mitochondria labeled with MitoFluor Green. Further, in detecting the fluorescence signal of mitochondria labeled with MitoFlour Green and that of GAPDH mRNA using Cy3-labeled single beacons in the same cell, cross-talk in the confocal fluorescence imaging of the two was minimized since MitoFlour Green has no adsorption at 545 nm (Cy3 excitation) and Cy3 has little fluorescence emission at 520 nm (MitoFlour Green emission detection). ER-Tracker was chosen in ER labeling because it is specific to the ER (unlike other ER staining dyes) and has essentially no spectral overlap with MitoFluor Green. The fluorescence emission of ER-Tracker was detected at 460 nm under the excitation of 360 nm. This ensured that there was no cross-talk in the fluorescence imaging of mitochondrial and ER. Example 16: Fluorescence microscopy imaging Fluorescence imaging of live and fixed HDF cells was performed using a Zeiss Axiovert 100 TV epifluorescence microscope coupled to a Cooke Sensicam SVGA cooled CCD camera or a Zeiss Axiovert LSM-100 confocal microscope. An exposure time of 2 s was used for all imaging assays. For assays using dual FRET molecular beacons, excitation and emission detection were performed using 545 nm and 665 nm filters, respectively. Assays using peptide-Iinked, GAPDH-targeting molecular beacons were performed with excitation at 543 nm and emission detection using a 565-625 nm filter. TKHR DOCKET NO. 820701.1135

Example 17: Targeted Molecular Beacons Specific nuclear RNAs were detected and localized living cells using NLS-linked molecular beacons. Nuclear RNAs are non-coding RNA molecules that perform their functions in the cell nucleus including the splicing, processing, modification and biogenesis of mRNA, rRNA, tRNA and ribosomal complexes, which are transported to cell cytoplasm to produce proteins. To achieve effective and compartment-specific delivery of molecular beacons into live cell nucleus, a combination of reversible permeablization of cell membrane and NLS (nuclear localization signal) peptide was used.

Specifically, NLS peptides, which can deliver different cargos into nucleus via the nuclear import pathway, were linked to molecular beacons; the peptide- Iinked molecular beacons were delivered into the cytoplasm of living cells using Streptolysin O (SLO) (Cheung, CY. et al. (2001) Bioconjug. Chem. 12: 906-910). NLS-peptide linked molecular beacons were delivered into the nucleas of living cells of the whole cell population with near 100% efficiency, making this approach far more effective compared with microinjection. To demonstrate the specific detection and visualization of nuclear RNA in living cells, in this study, U3 small nucleolar RNA (U3 snoRNA), one of the best characterized nuclear RNAs, was selected as a representative target. Synthesized and actively retained in cell nucleus, U3 snoRNA is a class of nuclear RNAs that are associated with coiled bodies and transported to nucleolus for processing of rRNA, a critical step in biogenesis of ribosomes. U3-snoRNA-targeting molecular beacons were designed based on the secondary structure of U3 snoRNA and checked the target sequence using BLAST search to ensure high specificity For use in negative controls, 'random'-sequence molecular beacon ('random beacon') was designed whose specific target sequence does not match with any mammalian gene. The design of molecular beacons are provided in Table 3, with underlined bases represent based added to form the stem of a molecular beacon. Both the U3- snoRNA-targeting and 'random' sequence molecular beacons have Cy3 TKHR DOCKET NO. 820701.1135

(Cyanine 3) as the reporter and BHQ2 (black hole quencher-2) as the fluorescence quencher.

Table 3. The design of NLS peptide-Iinked molecular beacons

U3 snoRNA Molecular Beacon (5'-3') Cv3-CGACCGGCTTCACGCTCAGGGG(dT-C6-NH₇)CG-BHQ2 (SEQ ID NO. 28) Negative Control Molecular Beacon (5'-3') Cy3-CGACGCGACAAGCGCACCGATACG(dI-C6-NH₂)CG-BHQ2 (SEQ ID NO. 29) NLS Peptide CGGGPKKKRKVED (SEQ ID NO. 30)

To conjugate the NLS peptide to a molecular beacon, the stem domain of the molecular beacon was modified to introduce a functional amine group using dT-(C6)-NH₂ (Table 3). The specific NLS peptide sequence selected in this study was a segment of the SV40 large T antigen NLS which was shown to be able to deliver different cargos such as plasmids and nanoparticles to cell nucleus (Sebestyen, M.G. et al. (1998) Nat Biotechnol. 16: 80-5; Zanta, M.A. et al. (1999) Proc. Natl. Acad. Sci. U. S. A. 96: 91-6; Luo, D. and Saltzman, W.M. (2000) Nat. Biotechnol., 18, 33-7). As shown schematically in Fig. 1 D, the NLS sequence with a Cysteine

(Cys) at its N-terminus was conjugated to the modified molecular beacon through a stable linkage between the thiol and maleimide groups. This conjugation scheme allowed only one peptide to be linked to a molecular beacon. Both the U3-snoRNA-targeting and 'random' sequence peptide-Iinked molecular beacons were introduced into HeLa cells (ATCC, VA) using activated SLO. Conjugation of NLS to the probe allowed effective delivery of molecular beacons into cell nucleus within 2 h. Fig. 7 is a confocal fluorescence image of U3 snoRNAs in live HeLa cells indicating their localization in the nucleus. No significant fluorescence signal was observed in the cell cytoplasm, indicating the effective delivery of molecular beacons into cell nucleus using the NLS peptide, and high TKHR DOCKET NO. 820701.1135

specificity of molecular beacons in targeting nuclear RNA. A single NLS peptide on a molecular beacon was sufficient to deliver the probe into nucleus, in contrast to most nuclear import/transport studies in which multiple NLS peptides are conjugated to the same cargo. To demonstrate specific localization of U3 snoRNA in the cell nucleus, a higher magnification image is displayed in Fig. 7B indicating that some U3 snoRNAs are localized in the nucleolus of the cell, consistent with the suggestion that U3 snoRNA performs its major function in the nucleolus especially for biogenesis of ribosomes. Further, localization of U3 snoRNAs in other areas of nucleus (Fig. 7A) was observed, consistent with the observation that U3 snoRNAs are associated with coiled bodies involved in snRNP biogenesis. To further validate the results obtained in live cell detection assays, fluorescence in situ hybridization (FISH) in fixed cells was carried using hairpin probes (see detailed in Supporting Information). As demonstrated in Fig. 7C, the results of in situ hybridization show similar localization pattern of U3 snoRNA as visualized in live cells (Fig. 7A), confirming the specific detection of U3 snoRNA. The use of hairpin oligonucleotide probes in FISH studies resulted in a high signal to background ratio without extensive washing. As expected, fluorescently labeled linear oligonucleotide probes generated a much higher background signal in living cells compared with hairpin probes (result not shown), largely owing to its inability to distinguish between bound and unbound probes. As a negative control, the 'random'- sequence molecular beacons (Table 3) were delivered into the nucleus of HeLa cells and imaged the resulting fluorescence signal, which is due to possible beacon degradation, hairpin-binding proteins and non-specific interactions. Fig. 7D shows the background signal of the 'random' beacons. Evidently, the background signal is rather low without any localization pattern, further demonstrating the high specificity in detecting U3 snoRNA. All the fluorescence images shown in Fig. 7 were obtained using a confocal microscope under 545 nm excitation and 570 nm emission detection. TKHR DOCKET NO. 820701.1135

Example 18: Tat-linked molecular beacons for detecting RNA in cytoplasm and nucleus Tat-linked molecular beacons were used to detect both the cytoplasmic and nuclear population of RNA in live cells within 45 minutes (about half the time required for combination of SLO and NLS peptide as described above). This approach allows detection of both cytoplasmic and nuclear RNA simultaneously using a single probe and, since Tat peptide is used, it has the ability to translocate the probe across plasma as well as nuclear membranes, i.e., without the addition of a targeting signal. To demonstrate this ability, U1 snRNA was detected. U1 snRNA is transported from nucleus to cytoplasm where its 5' cap is processed, and then transported back to the nucleus. This RNA molecule is an essential component of the mRNA splicing machinery in cell nucleus. SnRNAs are transcribed by RNA polymerase II and, consequently, the resulting RNAs are capped. However, they are methylated differently from other mRNAs. The guanine base is methylated at position N7 as normal but, in addition, it is dimethylated at position 2. Thus most snRNAs have a characteristic 2,2,7-trimethyl-guanosine (m3G) cap. This structure is essential for assembly of the spliceosome and for transport of the spliceosome back into the nucleus. In this assay, Tat peptide to molecular beacons were used to detect both the cytoplasmic and nuclear population of U1 snRNA. The results of the assay are shown in Figure 8. In the cytoplasm, U1 snRNA was observed to be mainly in the perinuclear region, while in nucleus it is in a discrete, spotlike localization pattern in HDF cells. To visualize both the cytoplasmic and nuclear population of U1 snRNA, images were collected at different positions using z-stacks in confocal microscopy. Scans of different z -positions are shown in Figure 8. Using this approach, a single probe can be used to determine different expression levels and localization patterns of the target nucleic acid in the nucleus and cytoplasm respectively. Similar approaches may also be used to study the transport of RNA from nucleus to cytoplasm.

Claims

TKHR DOCKET NO. 820701.1135

We claim: 1. A molecular beacon operably linked to a protein transduction domain.

2. The molecular beacon of claim 1 having a stem-and-Ioop structure or a linear structure.

3. The molecular beacon of claim 1 , wherein the protein transduction domain is linked directly or indirectly to the molecular beacon.

4. The molecular beacon of claim 3, wherein the protein transduction domain is linked directly or indirectly to the molecular beacon by a disulfide, thiol-maleimide, or streptavidin-avidin linkage.

5. The molecular beacon of claim 2, wherein the protein transduction domain is linked to the molecular beacon by a linker.

6. The molecular beacon of claim 5, wherein the linker comprises a Cι-ι₂ alkyl group.

7. The molecular beacon of claim 5, wherein the protein transduction domain is linked to the linker by a disulfide, thiol-maleimide, or streptavidin-avidin linkage.

8. The molecular beacon of claim 5, wherein the linker is covalently attached to the molecular beacon.

9. The molecular beacon of claim 8, wherein the linker is covalently attached to a modified nucleotide of the molecular beacon.

10. The molecular beacon of claim 9, wherein the modified nucleotide is in a region of the molecular beacon that is not complementary to a target nucleic acid.

11. The molecular beacon of claim 9, wherein the modified nucleotide is in the stem of the molecular beacon.

12. The molecular beacon of claim 11 , wherein the modified nucleotide is within about 1 to about 6 nucleotides from a terminus of the molecular beacon.

13. The molecular beacon of claim 1 , wherein the molecular beacon is released from the protein transduction domain when exposed to intracellular conditions. TKHR DOCKET NO. 820701.1135

14. The molecular beacon of claim 1 , wherein the link is non- specifically cleaved by an enzyme.

15. The molecular beacon of claim 1 , further comprising a targeting sequence.

16. The molecular beacon of claim 15, wherein the targeting sequence is selected from the group consisting of a nuclear localization signal, a mitochondrial localization signal, a chloroplast localization signal, or membrane targeting signal.

17. The molecular beacon of claim 1.wherein the protein transduction domain comprises YGRKKRRQRRR, RKKRRQRRR, RRRRRRR (SEQ ID NO. 3); PTD-5 - RRQRRTSKLMKR (SEQ ID NO. 4); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO. 5); KALA - WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO. 6); and RQIKIWFQNRRMKWKK (SEQ ID NO. 7).

18. The molecular beacon of claim 1 , wherein the protein transduction domain is linked to the molecular beacon at a C-terminal cysteine residue of the protein transduction domain.

19. A pharmaceutical composition comprising a molecular beacon operably linked to a protein transduction domain and a pharmaceutically acceptable excipient.

20. A nucleic acid reporter comprising a molecular beacon operably linked to a targeting signal.

21. The nucleic acid reporter of claim 20, wherein the targeting signal comprises CGGGPKKKRKVED.

22. A protein transduction domain comprising a C-terminal cysteine residue.

23. The protein transduction domain of claim 22, comprising SEQ ID NO. 30.

24. A composition comprising: (a) a first and second end region, the first end region having a detectable label attached thereto and the second end region having a quencher attached thereto; TKHR DOCKET NO. 820701.1135

(b) a middle region between the first and second end regions complementary to a target nucleic acid; and (c) a protein transduction domain operably linked to the first or second end regions; wherein detectability of the detectable label is modulated upon binding of the middle region to the target nucleic acid.

25. A kit comprising: a molecular beacon operably linked to a protein transduction domain comprising a preselected target recognition sequence and instructions for using the molecular beacon to detect the preselected target recognition sequence.

26. A method for detecting cells expressing a target nucleic acid comprising: (a) contacting a cell suspected of expressing the target nucleic acid with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon comprises a region complementary to the target nucleic acid; (b) irradiating the molecular beacon with an exciting amount of electromagnetic radiation; and (c) detecting the electromagnetic radiation emitted by the molecular beacon, wherein a detectable amount of electromagnetic radiation emitted by the molecular beacon indicates that the cell expresses the target nucleic acid.

27. A method for sorting cells expressing a target nucleic acid comprising: (a) contacting a plurality of cells with at least one molecular beacon operably linked to a protein transduction domain, wherein the protein transduction domain facilitates translocation of the at least one molecular beacon to at least one of the plurality of cells' interior, and wherein the molecular beacon comprises a region complementary to the target nucleic acid; TKHR DOCKET NO. 820701.1135

(b) irradiating the plurality of cells with an exciting amount of electromagnetic energy; (c) detecting electromagnetic energy emitted in response to the exciting amount of electromagnetic radiation by the molecular beacon in the interior of at least of the plurality of cells; (d) separating cells comprising a molecular beacon emitting a detectable amount of electromagnetic radiation from cells which are not emitting a detectable amount of electromagnetic radiation.

28. A method for detecting a target nucleic acid in a host comprising: (a) administering to the host a molecular beacon operably linked to a protein transduction domain; (b) irradiating the molecular beacon with an exciting amount of electromagnetic radiation; and (c) detecting electromagnetic radiation emitted by the molecular beacon in response to the exciting amount of electromagnetic radiation.

29. A method for detecting expression of a target nucleic acid in a living cell comprising: (a) contacting the cell with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon is specific for the target nucleic acid; (b) irradiating the molecular beacon with an exciting amount of electromagnetic radiation; and (c) detecting the emission of electromagnetic radiation from the molecular beacon, wherein detectable emission from the molecular beacon is indicative of expression of the target nucleic acid in the cell.

30. A method for detecting expression of a target nucleic acid in a living cell comprising: (a) contacting the cell with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon is specific for the target nucleic acid; TKHR DOCKET NO. 820701.1135

(b) irradiating the molecular beacon with an exciting amount of electromagnetic radiation; and (c) detecting the emission of electromagnetic radiation from the molecular beacon, wherein detectable emission from the molecular beacon is indicative of expression of the target nucleic acid.

31. A method for identifying modulators of gene expression comprising: (a) contacting a cell with a test compound; (b) contacting the cell of step (a) with a nucleic acid reporter operably linked to a protein transduction domain, a targeting signal, or a combination thereof, wherein the nucleic acid reporter comprises a region complementary to a transcript of the gene; (c) irradiating the cell with an exciting amount of radiation; (d) detecting electromagnetic emissions from the cell; and (e) selecting the test compound that induces a change in the electromagnetic emissions of the cell contacted with the test compound compared to a control sample.

32. A method for determining effectiveness of an agent on a host comprising: (a) contacting a sample of the host's cells with an agent suspected of modulating expression of a target nucleic acid; (b) contacting the cells of step (a) with molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon comprises a target recognition sequence complementary to the target nucleic acid; (c) irradiating the cells of step (b) with an exciting amount of radiation; (d) detecting electromagnetic emission from the cells of step (c) emitted in response to the exciting amount of radiation; (e) comparing the emission from the cells of step (d) with emissions from a control sample, wherein a difference in emission of the cells TKHR DOCKET NO. 820701.1135

of step (d) compared to the control sample indicates that the agent modulates expression of the target nucleic acid in the host.

33. A method for delivering a molecular beacon to the interior of a cell comprising contacting a cell with a molecular beacon operably linked to a protein transduction domain and optionally linked to a targeting signal, wherein the protein transduction domain facilitates translocation of the molecular beacon to the interior of the cell.

34. The molecular beacon of claim 1 , wherein the molecular beacon comprises a peptide nucleic acid backbone (PNA), so that both the protein transduction domain and the PNA molecular beacon can be synthesized together in a single composition.

35. A method for detecting the transport and localization of a nucleic acid -protein complex in living cells comprising: (a) contacting a cell comprising a labeled target protein with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon is specific for a target nucleic acid; (b) irradiating the molecular beacon with an exciting amount of electromagnetic radiation; (c) irradiating the labeled target protein with an exciting amount of electromagnetic radiation; and (e) detecting the emission of electromagnetic radiation at a first wavelength from the molecular beacon and at a second wavelength from the labeled protein, wherein detectable emission from the molecular beacon is indicative of expression of the target nucleic acid in the cell, and detectable emission from the labeled protein at the same spatial location is indicative of the co-localization of the target protein with the target nucleic acid.

36. The method of claim 36, wherein the labeled protein is a fusion protein.

37. The method of claim 37, wherein the labeled protein is green fluorescence protein.