WO2011017691A2 - Enhanced molecular detection of targeted intracellular mrna transcription using polynucleotide functionalized nanoparticles - Google Patents

Abstract

The present invention is directed to compositions and methods for enhanced molecular detection of targeted intracellular mRNA transcription using a polynucleotide functionalized nanoparticle (PN-NP).

Description

ENHANCED MOLECULAR DETECTION OF TARGETED INTRACELLULAR mRNA TRANSCRIPTION USING POLYNUCLEOTIDE FUNCTIONALIZED

NANOPARTICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit under 35 U.S.C. § U9(e) of U.S.

Provisional Application No. 61/232,302, filed August 7, 2009, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant Number EEC-0118025 awarded by the NSF (NSEC). The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention is directed to compositions and methods for enhanced molecular detection of targeted intracellular mRNA transcription using a polynucleotide functionalized nanoparticle (PN-NP).

BACKGROUND OF THE INVENTION

[0004] The detection of intracellular mRNA species in living cells has the potential to revolutionize a number of broad scientific fields from basic research in cell biology to highly translational work in the field of oncology [Santangelo et al., Annals of Biomedical Engineering. 34(1): 39-50 (2006)]. The current state-of-the art with regard to the detection of mRNA in live cells includes the use of fluorescent resonance energy transfer (FRET) probes (for example and without limitation, molecular beacons) which hybridize to targeted cytoplasmic mRNA targets whereupon detectable changes in intracellular fluorescence are demonstrative of target binding [Santangelo et al., Annals of Biomedical Engineering. 34(1): 39-50 (2006); Nitin et al., Nucleic Acids Res. 32(6): e58 (2004); Santangelo et al., Journal of Virology. 80(2): 682-8 (2006);

Santangelo et al., Journal of Biomedical Optics. 10(4): 44025 (2005)]. FRET techniques suffer from a number of drawbacks, most importantly: 1) Poor transfection efficiency of the FRET probes, thus, requiring the use of cytotoxic polymer and lipid transfection reagents, and 2) Intracellular FRET probe degradation which leads to non-specifically increasing background signal that cannot be distinguished from genuine target binding events. [0005] Recently, gold nanoparticle probes densely surfaee-fimctionalized with nucleic acids (PN-NP probes) have been used to detect intracellular mRNA targets using 'nano-flare' technology [Seferos et al, Journal of the American Chemical Society, 129(50): 15477-9 (2007)]. Importantly, the nano-flare technology addresses each of the drawbacks mentioned above for the competing technology.

[0006] In the case of intracellular FRET probes, a limiting drawback is the background fluorescence that is the result of incomplete FRET probe quenching and intracellular probe degradation. PN-NP nano-flares represent a significant advance with regard to increasing the signal-to-noise ratio with regard to bound versus un-bound target signaling in cells. This is due to the excellent quenching properties of the gold nanoparticles to which the flares are bound [Dubertret et al., Nat Biotechnol. 2001 19(4): 365-70 (2001)](6), and due to the intracellular stability of the conjugate structures [Rosi et al., Science. 312(5776): 1027-30 (2006)]. In the case of detecting intracellular mRNA targets in rather homogeneous cell populations, the sensitivity afforded by the current Au NP technology is adequate, and ensemble measurements can be seen as representative. However, detecting few cells (i.e. 1-10 target cells / 1 million total cells) expressing an intracellular target of choice from a high background of cells requires extremely low background signal and a significantly high target binding signal.

SUMMARY OF THE INVENTION

[0007] Described herein is a composition comprising a nanoparticle functionalized with a polynucleotide (PN-NP), and further comprising a transcriptional regulator, wherein the transcriptional regulator induces transcription of a target polynucleotide in a target cell. The compositions provided by the present disclosure are useful for the detection of targeted intracellular mRNA transcription.

[0008] In some embodiments, the transcriptional regulator induces transcription of the target polynucleotide to which the polynucleotide will hybridize. In other embodiments, the

transcriptional regulator decreases transcription of the target polynucleotide to which the polynucleotide will hybridize.

[0009] The disclosure contemplates compositions, in some aspects, wherein the transcriptional regulator is conjugated to the nanoparticle. Any substance that can regulate transcription is contemplated for use in the methods and compositions of the disclosure. In specific embodiments, the transcriptional regulator is selected from the group consisting of a polypeptide, a regulator polynucleotide, and an artificial transcription factor (ATF). In one specific aspect, the transcriptional regulator is a polypeptide, and in a further aspect the polypeptide is a hormone.

[0010] A polynucleotide or regulator polynucleotide of the composition comprises, in various aspects, RNA, DNA or a modified polynucleotide. In certain embodiments, the polynucleotide comprises about 5 nucleotides to about 100 nucleotides. In further embodiments, the

polynucleotide comprises about 10 nucleotides to about 50 nucleotides, and in still further embodiments the polynucleotide comprises about 15 nucleotides.

[0011] In some aspects the polynucleotide or regulatory polynucleotide comprises a detectable marker. The detectable marker is, in various embodiments, selected from the group consisting of a fluorophore, an isotope, a contrast agent, a redox active probe, a nanoparticle, a mass tag, a polypeptide, a peptide, a small molecule, an enzyme, a catalyst, an enzyme co-factor, a polynucleotide, a metal, and a quantum dot. In some embodiments, the detectable marker is quenched when the polynucleotide is not hybridized to the target polynucleotide.

[0012] The disclosure also contemplates that in some aspects, the polynucleotide or regulator polynucleotide is double stranded. In these aspects, it is further contemplated that the

polynucleotide comprises a first strand that is functionalized to the nanoparticle and a second strand that can hybridize to the first strand. In still further aspects, the second strand further comprises the detectable marker. In some embodiments, the first strand is sufficiently

complementary to the target polynucleotide to hybridize to the target polynucleotide, and in some aspects the hybridization dissociates the second strand from the first strand. The disclosure contemplates that in further aspects the dissociation results in a detectable change.

[0013] In various aspects, the disclosure also provides a composition wherein the

polynucleotide or the regulator polynucleotide is single stranded. In some of these aspects, the polynucleotide or regulator polynucleotide has secondary structure. In one aspect, the

polynucleotide or regulator polynucleotide forms a hairpin structure. In further aspects, hybridization of the polynucleotide to the target polynucleotide causes dissociation of the secondary structure and causes the detectable marker to move away from the nanoparticle resulting in a detectable change. [0014] In some aspects, the target polynucleotide is RNA or DNA.

[0015] The disclosure contemplates, in certain embodiments, that the target cell is selected from the group consisting of a cancer cell, a transformed cell, an in vivo target cell, a primary cell, a stem cell, a blood cell, a co-cultured cell, an immobilized cell, a suspended cell, a blood cell, a neuronal cell, and any cell derived from ectoderm, mesoderm, or endodermal lineage. In an embodiment, the target cell is a cancer cell, and in a specific embodiment the cancer cell is a prostate cancer cell.

[0016] In another embodiment, the target cell is a transformed cell, and in some aspects the transformed cell comprises an expression vector. The expression vector, in some embodiments, encodes a polypeptide fused in-frame to a detectable polypeptide. In further aspects, the detectable polypeptide is a fluorescent protein, and in one aspect the fluorescent protein is green fluorescent protein (GFP).

[0017] In further embodiments, it is contemplated that more than one target polynucleotide is detected in the target cell.

[0018] The disclosure also provides a method of detecting modulation of transcription of a target polynucleotide comprising administering a composition disclosed herein to a target cell and measuring a detectable change, wherein the administering yields an increased or decreased level of transcription of the target polynucleotide relative to a transcription level in the absence of the transcriptional regulator.

[0019] In another embodiment, the disclosure provides a method of detecting modulation of transcription of a target polynucleotide comprising administering a PN-NP and a transcriptional regulator and measuring a detectable change, wherein the transcriptional regulator increases or decreases transcription of the target polynucleotide in a target cell relative to a transcription level in the absence of the transcriptional regulator. In some aspects, the transcriptional regulator modulates transcription of the target polynucleotide to which the polynucleotide will hybridize.

[0020] In some aspects, the transcriptional regulator is conjugated to the nanoparticle.

[0021] Methods provided by the disclosure include those wherein the increase or decrease is from about 2-fold to about 100,000-fold. In some aspects, the increase or decrease is from about 100-fold to about 10,000-fold, and in a specific aspect the increase or decrease is about 2-fold. [0022] Methods of the disclosure also include those wherein a detectable change is measured. Accordingly, in some aspects, the measuring is done with a device that quantitates the detectable change. In further aspects, the device is a flow cytometer.

[0023] In further aspects of the methods, it is contemplated that the administration is performed in vivo.

[0024] Also provided in various embodiments are methods wherein the transcriptional regulator and the PN-NP are administered at different times. In some aspects, the transcriptional regulator is administered before the PN-NP, while in some aspects the transcriptional regulator is administered first, a biological sample is acquired second, and the PN-NP is administered third.

[0025] In various aspects of these methods, the time between the first and second step is from about 1 minute to about 21 days. In further aspects, the time between the first and second step is one day. In still further aspects, the time between the second and third step is from about 1 minute to about 21 days, and in further aspects the time between the second and third step is one day.

[0026] In further aspects of the methods, the administration is in vitro.

[0027] It is also contemplated by the disclosure that, in some aspects, a method is provided to identify a candidate transcriptional regulator as a transcriptional regulator. In some aspects, a library of candidate transcriptional regulators is screened for its ability to modulate the transcription of the target polynucleotide. In further aspects of these methods, an increase or decrease in the detectable change when the transcriptional regulator is administered relative to the detectable change when a different transcriptional regulator within the library is administered indicates that the candidate transcriptional regulator is a transcriptional regulator.

[0028] Another method provided by the disclosure is a method to identify the target polynucleotide. In some embodiments, a library of polynucleotides is screened for its ability to detect the increase or decrease in transcription of the target polynucleotide. In further

embodiments, each nanoparticle is functionalized with a polynucleotide of known sequence, and in still further embodiments, an increase or decrease in the detectable change when the transcriptional regulator is administered relative to the detectable change measured when a different nanopartiele functionalized with a polynucleotide within the library is administered is indicative of identifying the target polynucleotide.

[0029] The disclosure also, in various aspects, provides a kit comprising a composition as described herein.

DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 depicts the design of a nanoflare.

[0031] Figure 2 depicts the sensitivity of a nanoflare versus control, and in the presence of a transcriptional regulator.

[0032] Figure 3 depicts an experiment demonstrating a significantly increased PSA-specific flare signal in DHT-treated cells when compared to those cells grown under DHT-free experimental conditions.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure provides a composition comprising a nanopartiele

functionalized with a polynucleotide (PN-NP), and further comprising a transcriptional regulator, wherein the transcriptional regulator induces transcription of a target polynucleotide in a target cell. Accordingly, the disclosure contemplates a variety of nanoparticles for use in the compositions described herein.

[0034] As used herein, a "regulatory polynucleotide" will be understood to possess all of the characteristics and features of a "polynucleotide" as defined herein. A "regulatory

polynucleotide" differs from a "polynucleotide" in that it functions as a transcriptional regulator. A regulator polynucleotide can, in certain aspects, additionally function as a polynucleotide as described herein.

NANOPARTICLES

[0035] Compositions of the present disclosure comprise nanoparticles as described herein. Nanoparticles are provided which are functionalized to have a polynucleotide attached thereto. The size, shape and chemical composition of the nanoparticles contribute to the properties of the resulting PN-NP. These properties include for example, optical properties, optoelectronic properties, electrochemical properties, electronic properties, stability in various solutions, magnetic properties, and pore and channel size variation. Mixtures of nanoparticles having different sizes, shapes and/or chemical compositions, as well as the use of nanoparticles having uniform sizes, shapes and chemical composition, and therefore a mixture of properties are contemplated. Examples of suitable particles include, without limitation, aggregate particles, isotropic (such as spherical particles), anisotropic particles (such as non-spherical rods, tetrahedral, and/or prisms) and core-shell particles, such as those described in U.S. Patent No. 7,238,472 and International Publication No. WO 2003/08539, the disclosures of which are incorporated by reference in their entirety.

[0036] In one embodiment, the nanoparticle is metallic, and in various aspects, the nanoparticle is a colloidal metal. Thus, in various embodiments, nanoparticles of the invention include metal (including for example and without limitation, silver, gold, platinum, aluminum, palladium, copper, cobalt, indium, nickel, or any other metal amenable to nanoparticle formation), semiconductor (including for example and without limitation, CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (for example, ferromagnetite) colloidal materials.

[0037] Also, as described in U.S. Patent Publication No 2003/0147966, nanoparticles of the invention include those that are available commercially, as well as those that are synthesized, e.g., produced from progressive nucleation in solution {e.g., by colloid reaction) or by various physical and chemical vapor deposition processes, such as sputter deposition. See, e.g., HaVashi, Vac. Sci. Technol. A5(4) :1375-84 (1987); Hayashi, Physics Today, 44-60 (1987); MRS Bulletin, January 1990, 16-47. As further described in U.S. Patent Publication No 2003/0147966, nanoparticles contemplated are alternatively produced using HAuCU and a citrate-reducing agent, using methods known in the art. See, e.g. , Marinakos et al. , Adv. Mater. 11 :34-37(1999); Marinakos et al, Chem. Mater. 10: 1214-19(1998); Enustun & Turkevich, J. Am. Chem. Soc. 85: 3317(1963).

[0038] Nanoparticles can range in size from about 1 nm to about 250 nm in mean diameter, about 1 nm to about 240 nm in mean diameter, about 1 nm to about 230 nm in mean diameter, about 1 nm to about 220 nm in mean diameter, about 1 nm to about 210 nm in mean diameter, about 1 nm to about 200 nm in mean diameter, about 1 nm to about 190 nm in mean diameter, about 1 nm to about 180 ran in mean diameter, about 1 nm to about 170 nm in mean diameter, about 1 nm to about 160 nm in mean diameter, about 1 nm to about 150 nm in mean diameter, about 1 nm to about 140 nm in mean diameter, about 1 nm to about 130 nm in mean diameter, about 1 nm to about 120 nm in mean diameter, about 1 nm to about 110 run in mean diameter, about 1 nm to about 100 nm in mean diameter, about 1 nm to about 90 nm in mean diameter, about 1 nm to about 80 nm in mean diameter, about 1 nm to about 70 nm in mean diameter, about 1 nm to about 60 nm in mean diameter, about 1 nm to about 50 nm in mean diameter, about 1 nm to about 40 nm in mean diameter, about 1 nm to about 30 nm in mean diameter, or about 1 nm to about 20 nm in mean diameter, about 1 nm to about 10 nm in mean diameter. In other aspects, the size of the nanoparticles is from about 5 nm to about 150 nm (mean diameter), from about 5 to about 50 nm, from about 10 to about 30 nm, from about 10 to 150 nm, from about 10 to about 100 nm, or about 10 to about 50 nm. The size of the nanoparticles is from about 5 nm to about 150 nm (mean diameter), from about 30 to about 100 nm, from about 40 to about 80 nm. The size of the nanoparticles used in a method varies as required by their particular use or application. The variation of size is advantageously used to optimize certain physical characteristics of the nanoparticles, for example, optical properties or the amount of surface area that can be functionalized as described herein.

POLYNUCLEOTIDES

[0039] The terms "polynucleotide" and "nucleotide" or plural forms as used herein are interchangeable with modified forms as discussed herein and otherwise known in the art. In certain instances, the art uses the term "nucleobase" which embraces naturally-occurring nucleotides as well as modifications of nucleotides that can be polymerized. Thus, nucleotide or nucleobase means the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4- ethanocytosin, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C₃— C₆)-alkynyl- cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-rnethyl-4-tr- iazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et ah, U.S. Pat. No. 5,432,272 and Susan M. Freier and Karl-Heinz

Altmann, 1997, Nucleic Acids Research, vol. 25: pp 4429-4443. The term "nucleobase" also includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., 1991, Angewandte Chemie, International Edition, 30: 613-722 (see especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, 1990, pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of which are hereby incorporated by reference in their entirety). In various aspects, polynucleotides also include one or more "nucleosidic bases" or "base units" which include compounds such as heterocyclic compounds that can serve like nucleobases, including certain "universal bases" that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Universal bases include 3-nitropyrrole, optionally substituted indoles (e.g., 5- nitroindole), and optionally substituted hypoxanthine. Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.

[0040] Polynucleotides may also include modified nucleobases. A "modified base" is understood in the art to be one that can pair with a natural base (e.g. , adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring base. Exemplary modified bases are described in EP 1 072 679 and WO 97/12896, the disclosures of which are

incorporated herein by reference. Modified nucleobases include without limitation, 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 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-halo, 8-amino, 8-thiol, 8- thioalkyl, 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 bases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5 ,4-b][l,4]benzoxazin-2(3H)- one), phenothiazine cytidine (lH-pyrimido[5 ,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzox- azin-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 bases 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. Additional nucleobases include those disclosed in U.S. Pat. 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, 1991, Angewandte Chemie, International Edition, 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 bases are useful for increasing the binding affinity and 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° C and are, in certain aspects combined with 2'-O-methoxyethyl sugar modifications. See, U.S. Pat. Nos. 3,687,808, 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; 5,750,692 and 5,681,941, the disclosures of which are incorporated herein by reference.

[0041 ] Methods of making polynucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1 st Ed. (Oxford University Press, New York, 1991). Solid-phase synthesis methods are preferred for both polyribonucleotides and

polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well. See, e.g., U.S. Patent No. 7,223,833; Katz, J. Am. Chem. Soc, 74:2238 (1951); Yamane, et al, J. Am. Chem. Soc, 83:2599 (1961); Kosturko, et al, Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc, 76:6032 (1954); Zhang, et al, J. Am. Chem. Soc, 127:74-75 (2005); and Zimmermann, et al, J. Am. Chem. Soc, 124:13684-13685 (2002).

[0042] Nanoparticles provided that are functionalized with a polynucleotide, or modified form thereof, generally comprise a polynucleotide from about 5 nucleotides to about 100 nucleotides in length. More specifically, nanoparticles are functionalized with polynucleotide that are about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, and all polynucleotides intermediate in length of the sizes specifically disclosed to the extent that the polynucleotide is able to achieve the desired result. Accordingly, polynucleotides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length are contemplated.

[0043] Polynucleotides contemplated for attachment to a nanoparticle include those which modulate expression of a gene product expressed from a target polynucleotide. The

polynucleotides may, in various aspects, be comprised of DNA or RNA. Accordingly, antisense polynucleotides which hybridize to a target polynucleotide and inhibit translation, siRNA polynucleotides which hybridize to a target polynucleotide and initiate an RNAse activity (for example but not limited to RNAse H), triple helix forming polynucleotides which hybridize to double-stranded polynucleotides and inhibit transcription, and ribozymes which hybridize to a target polynucleotide and inhibit translation, are contemplated.

[0044] In some embodiments, the polynucleotide that is attached to the nanoparticle is an antagomiR. An antagomiR represents a novel class of chemically engineered polynucleotides. AntagomiRs are used to silence endogenous microRNA (miRNA) [Krutzfeldt et al., Nature 438 (7068): 685-9 (2005)]. AntagomiRs are, in some aspects, covalently modified with lipophoilic groups (for example and without limitation, cholesterol), or other agents specifically used to image the location of the antagomiR (for example and without limitation, a molecular

fluorophore).

[0045] In various aspects, if a specific mRNA is targeted, a single nanoparticle-binding agent composition has the ability to bind to multiple copies of the same transcript. In one aspect, a nanoparticle is provided that is functionalized with identical polynucleotides, i.e., each polynucleotide has the same length and the same sequence. In other aspects, the nanoparticle is functionalized with two or more polynucleotides which are not identical, i.e., at least one of the attached polynucleotides differ from at least one other attached polynucleotide in that it has a different length and/or a different sequence. In aspects wherein different polynucleotides are attached to the nanoparticle, these different polynucleotides bind to the same single target polynucleotide but at different locations, or substrate sites, or bind to different target

polynucleotides which encode different gene products. Accordingly, in various aspects, a single nanoparticle-binding agent composition target more than one gene product. Polynucleotides are thus target-specific polynucleotides, whether at one or more specific regions in the target polynucleotide, or over the entire length of the target polynucleotide as the need may be to effect a desired level of inhibition of gene expression.

MODIFIED POLYNUCLEOTIDES

[0046] Modified polynucleotides are contemplated for functionalizing nanoparticles wherein both one or more sugar and/or one or more internucleotide linkage of the nucleotide units in the polynucleotide is replaced with "non-naturally occurring" groups. In one aspect, this

embodiment contemplates a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of a polynucleotide is replaced with an amide containing backbone. See, for example US Patent Nos. 5,539,082; 5,714,331 ; and 5,719,262, and Nielsen et al. , Science, 1991, 254, 1497-1500, the disclosures of which are herein incorporated by reference.

[0047] Other linkages between nucleotides and unnatural nucleotides contemplated for the disclosed polynucleotides include those described in U.S. Patent 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; U.S. Patent Publication No. 20040219565; International Patent Publication Nos. WO 98/39352 and WO 99/14226; Mesmaeker et. al., Current Opinion in Structural Biology 5:343-355 (1995) and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 25:4429-4443 (1997), the disclosures of which are incorporated herein by reference.

[0048] Specific examples of polynucleotides include those containing modified backbones or non-natural internucleoside linkages. Polynucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified polynucleotides that do not have a phosphorus atom in their internucleoside backbone are considered to be within the meaning of "polynucleotide."

[0049] Modified polynucleotide backbones containing a phosphorus atom 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, thionoalkylphosphonates, 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 T to 2' linkage. Also contemplated are polynucleotides having inverted polarity comprising a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e. a single inverted nucleoside residue which may be abasic (the nucleotide is missing or has a hydroxy! group in place thereof). Salts, mixed salts and free acid forms are also contemplated.

[0050] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, 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; 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, the disclosures of which are incorporated by reference herein.

[0051] Modified polynucleotide backbones that do not include a phosphorus atom have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages; 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 methyl enehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. In still other embodiments, polynucleotides are provided with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and including -CH₂-NH-O-CH₂-, -CH₂-N(CH₃)- 0— CH₂- „— CH₂-O-N(CH₃)- CH₂-,— CH₂- N(CH₃)- N(CH₃)- CH₂- and ^0-N(CH₃)- CH₂-CH₂- described in US Patent Nos. 5,489,677, and 5,602,240. See, for example, U.S. Patent 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, the disclosures of which are incorporated herein by reference in their entireties.

[0052] In various forms, the linkage between two successive monomers in the oligo consists of 2 to 4, desirably 3, groups/atoms selected from— CH₂— ,— O— ,— S— ,— NRH— , >C=O, >C=NRH, >C=S,— Si(R")₂——SO—, -S(O)₂-,— P(O)₂-,— PO(BH₃)— ,— P(O,S)—— P(S)₂-,— PO(R">— , "-PO(OCH₃)— , and— PO(NHRH)-, where RH is selected from hydrogen and Cl-4-alkyl, and R" is selected from Cl-6-alkyl and phenyl. Illustrative examples of such linkages are— CH₂-CH₂-CH₂-, -CH₂-CO-CH₂-, -CH₂-CHOH-CH₂-,— O— CH2— O— , -O^ CH2— CH2— ,— O— CH2— CH=(including R5 when used as a linkage to a succeeding monomer), -CH₂-CH₂-O-, -NRH-CH₂-CH₂-, -CH₂-CH₂- NRH-, ^CH₂-NRH-CH₂- -,—O— CH₂-CH₂-NRH-, ^NRH-CO-O-,— NRH- CO— NRH-,— NRH- CS- NRH— ,— NRH— C(=NRH)— NRH- , -NRH-CO-CH₂- NRH- O— CO— O— ,— O— CO— CH₂-O-, ^0-CH₂-CO-O-, -CH₂-CO-NRH- ,— O- CO— NRH— , -NRH-CO-CH₂— ,—O— CH₂-CO-NRH-—O— CH₂-CH₂- NRH-, ^CH=N-O-, -CH₂-NRH-O-,— CH₂- O— N=(including R5 when used as a linkage to a succeeding monomer),— CH₂— O— NRH— ,— CO— NRH— CH₂— ,— CH₂- NRH-O-,— CH₂-NRH-CO-,— O— NRH— CH₂-,— O— NRH,— 0— CH₂-S-,— S— CH₂-O-,— CH₂- CH₂-S-,— O— CH₂- CH₂-S-,— S— CH₂- CH=(including R5 when used as a linkage to a succeeding monomer),— S— CH₂— CH₂— ,— S— CH₂— CH₂— O— ,— S— CH₂- CH₂-S-,— CH₂-S- CH₂-,— CH₂-SO- CH₂-,— CH₂- SO₂- CH₂-, ^0-SO-O-,—O— S(O)₂-O-,— O— S(O)₂- CH₂-,—O— S(O)₂- NRH-, -NRH-S(O)₂- CH₂-;—O— S(O)₂- CH₂-,— O- P(O)₂-O-,— O— P(O₅S)- 0—, ^0-P(S)₂-O-, ^S-P(O)₂-O-,— S— P(O₉S)- 0— , -S-P(S)₂-O-,— O—

P(O)₂-S-, ^O— P(O₅S)-S-, ^0-P(S)₂-S-, ^S-P(O)₂-S-, -S-P(O₅S)-S-, -S-P(S)₂-S-,— O— PO(R")- O— , ^O— PO(OCH₃)~^O^-, ^O— PO(O CH₂CH₃)- O— ,— O— PO(O CH₂CH₂S^R) -O— ,— 0^PO(BH₃ }— O— ,—0— PO(NHRN)-O-,— 0— P(O)₂-NRH H-, ^NRH-P(O)₂-O-,— O— P(0,NRH>— O— ,— CH₂-P(O)₂-O-,— 0-P(O)₂- CH₂-, and ^O— Si(R")₂— O— ; among which— CH₂-CO-NRH-,— CH₂- NRH-O-,— S— CH₂-O-, ^0-P(O)₂-O- O— P(- 0,S)-O-,— O— P(S)₂-O-,— NRH P(O)₂-O-,— O— P(0,NRH)— O— , ^O— PO(R")- O— ,— 0-PO(CH₃)- O— , and — O— PO(NHRN)— O— , where RH is selected form hydrogen and Cl-4-alkyl, and R" is selected from Cl-6-alkyl and phenyl, are contemplated. Further illustrative examples are given in Mesmaeker et. al., 1995, Current Opinion in Structural Biology, 5: 343-355 and Susan M. Freier and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol 25: pp 4429-4443.

[0053] Still other modified forms of polynucleotides are described in detail in U.S. Patent Application No. 20040219565, the disclosure of which is incorporated by reference herein in its entirety.

[0054] Modified polynucleotides may also contain one or more substituted sugar moieties. In certain aspects, polynucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; O-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C] to Ci₀ alkyl or C₂ to Cio alkenyl and alkynyl. Other embodiments include 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 polynucleotides comprise one of the following at the 2' position: Cl to ClO lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, 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 a polynucleotide, or a group for improving the pharmacodynamic properties of a polynucleotide, and other substituents having similar properties. In one aspect, a modification includes T- methoxyethoxy (2'-0-CH₂CH₂OCH₃, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995, HeIv. Chim. Acta, 78: 486-504) i.e., an alkoxyalkoxy group. Other modifications include 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-0^H₂- -O— CHr-N(CH₃)₂.

[0055] Still other modifications include 2'-methoxy (2'-O - -CH₃), 2'-aminopropoxy (T- OCH₂CH₂CH₂NH₂), 2'-allyl (2'-CH₂-CH=CH₂), 2'-0-allyl (2'-O^CH₂^CH=CH₂) and T- fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. In one aspect, a 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the polynucleotide, for example, at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked polynucleotides and the 5' position of 5' terminal nucleotide.

Polynucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for example, 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, the disclosures of which are incorporated by reference in their entireties herein.

[0056] In one aspect, a modification of the sugar 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 in certain aspects a methylene (— 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 WO 98/39352 and WO 99/14226, the disclosures of which are incorporated herein by reference.

METHODS OF ATTACHING POLYNUCLEOTIDES

[0057] Polynucleotides contemplated for use in the methods include those bound to the nanoparticle through any means. Regardless of the means by which the polynucleotide is attached to the nanoparticle, attachment in various aspects is effected through a 5' linkage, a 3' linkage, some type of internal linkage, or any combination of these attachments.

[0058] In one aspect, the nanoparticles, the polynucleotides or both are functionalized in order to attach the polynucleotides to the nanoparticles. Methods to functionalize nanoparticles and polynucleotides are known in the art. For instance, polynucleotides functionalized with alkanethiols at their 3'-termini or 5'-termini readily attach to gold nanoparticles. See Whitesides,

Proceedings of the Robert A. Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, Tex., pages 109-121 (1995). See also, Muck et al. [Chem. Commun. 555-557 (1996)] which describes a method of attaching 3' thiol DNA to flat gold surfaces. The alkanethiol method can also be used to attach polynucleotides to other metal, semiconductor and magnetic colloids and to the other types of nanoparticles described herein. Other functional groups for attaching polynucleotides to solid surfaces include phosphorothioate groups (see, for example, U.S. Pat. No. 5,472,881 for the binding of polynucleotide- phosphorothioates to gold surfaces), substituted alkylsiloxanes [(see, for example, Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci and Caruthers, J. Am. Chem. Soc, 103, 3185-3191 (1981) for binding of polynucleotides to silica and glass surfaces, and Grabar et al., [Anal. Chem., 67, 735-743] for binding of aminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes]. Polynucleotides with a 5' thionucleoside or a 3' thionucleoside may also be used for attaching polynucleotides to solid surfaces. The following references describe other methods which may be employed to attached polynucleotides to nanoparticles: Nuzzo et al., J. Am. Chem. Soc, 109, 2358 (1987) (disulfides on gold); Allara and Nuzzo, Langmuir, 1, 45 (1985) (carboxylic acids on aluminum); Allara and Tompkins, J. Colloid Interface Sci., 49, 410-421 (1974) (carboxylic acids on copper); Her, The Chemistry Of Silica, Chapter 6, (Wiley 1979) (carboxylic acids on silica); Timmons and Zisman, J. Phys. Chem., 69, 984-990 (1965) (carboxylic acids on platinum); Soriaga and Hubbard, J. Am. Chem. Soc, 104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Ace. Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides and other functionalized solvents on platinum); Hickman et al., J. Am. Chem. Soc, 111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv, Langmuir, 3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir, 3, 1034 (1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074 (1989) (silanes on silica); Eltekova and Eltekov, Langmuir, 3, 951 (1987) (aromatic carboxylic acids, aldehydes, alcohols and methoxy groups on titanium dioxide and silica); Lee et al., J. Phys. Chem., 92, 2597 (1988) (rigid phosphates on metals).

10059] U.S. patent application Ser. Nos. 09/760,500 and 09/820,279 and international application nos. PCT/USOl/01190 and PCT/US01/10071 describe polynucleotides functionalized with a cyclic disulfide. The cyclic disulfides in certain aspects have 5 or 6 atoms in their rings, including the two sulfur atoms. Suitable cyclic disulfides are available commercially or are synthesized by known procedures. Functionalization with the reduced forms of the cyclic disulfides is also contemplated. Functionalization with triple cyclic disulfide anchoring groups are described in PCT/US2008/63441, incorporated herein by reference in its entirety.

[0060] In certain aspects wherein cyclic disulfide functionalization is utilized, polynucleotides are attached to a nanoparticle through one or more linkers. In one embodiment, the linker comprises a hydrocarbon moiety attached to a cyclic disulfide. Suitable hydrocarbons are available commercially, and are attached to the cyclic disulfides. The hydrocarbon moiety is, in one aspect, a steroid residue. Polynucleotide-nanoparticle compositions prepared using linkers comprising a steroid residue attached to a cyclic disulfide are more stable compared to compositions prepared using alkanethiols or acyclic disulfides as the linker, and in certain instances, the polynucleotide-nanoparticle compositions have been found to be 300 times more stable. In certain embodiments the two sulfur atoms of the cyclic disulfide are close enough together so that both of the sulfur atoms attach simultaneously to the nanoparticle. In other aspects, the two sulfur atoms are adjacent each other. In aspects where utilized, the hydrocarbon moiety is large enough to present a hydrophobic surface screening the surfaces of the

nanoparticle.

[0061] In other aspects, a method for attaching polynucleotides onto a surface is based on an aging process described in U.S. application Ser. No. 09/344,667, filed Jun. 25, 1999; Ser. No. 09/603,830, filed Jun. 26, 2000; Ser. No. 09/760,500, filed Jan. 12, 2001 ; Ser. No. 09/820,279, filed Mar. 28, 2001; Ser. No. 09/927,777, filed Aug. 10, 2001 ; and in International application nos. PCT/US97/ 12783, filed JuI. 21, 1997; PCT/US00/ 17507, filed Jun. 26, 2000;

PCT/USOl/01190, filed Jan. 12, 2001; PCTVUSOl /10071, filed Mar. 28, 2001, the disclosures which are incorporated by reference in their entirety. The aging process provides nanoparticle- polynucleotide compositions with enhanced stability and selectivity. The process comprises providing polynucleotides, in one aspect, having covalently bound thereto a moiety comprising a functional group which can bind to the nanop articles. The moieties and functional groups are those that allow for binding (i.e., by chemisorption or covalent bonding) of the polynucleotides to nanoparticles. For example, polynucleotides having an alkanethiol, an alkanedisulfide or a cyclic disulfide covalently bound to their 5' or 3' ends bind the polynucleotides to a variety of nanoparticles, including gold nanoparticles. [0062] Compositions produced by use of the "aging" step have been found to be considerably more stable than those produced without the "aging" step. Increased density of the

polynucleotides on the surfaces of the nanoparticles is achieved by the "aging" step. The surface density achieved by the "aging" step will depend on the size and type of nanoparticles and on the length, sequence and concentration of the polynucleotides. A surface density adequate to make the nanoparticles stable and the conditions necessary to obtain it for a desired combination of nanoparticles and polynucleotides can be determined empirically. Generally, a surface density of at least 2 picomoles/cm² will be adequate to provide stable nanoparticle-polynucleotide compositions. Regardless, various polynucleotide densities are contemplated as disclosed herein.

(0063] An "aging" step is incorporated into production of functionalized nanoparticles following an initial binding or polynucleotides to a nanoparticle. In brief, the polynucleotides are contacted with the nanoparticles in water for a time sufficient to allow at least some of the polynucleotides to bind to the nanoparticles by means of the functional groups. Such times can be determined empirically. In one aspect, a time of about 12-24 hours is contemplated. Other suitable conditions for binding of the polynucleotides can also be determined empirically. For example, a concentration of about 10-20 nM nanoparticles and incubation at room temperature is contemplated.

[0064] Next, at least one salt is added to the water to form a salt solution. The salt is any water-soluble salt, including, for example and without limitation, sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, a combination of two or more of these salts, or one of these salts in phosphate buffer. The salt is added as a concentrated solution, or in the alternative as a solid. In various embodiments, the salt is added all at one time or the salt is added gradually over time. By "gradually over time" is meant that the salt is added in at least two portions at intervals spaced apart by a period of time. Suitable time intervals can be determined empirically.

[0065] The ionic strength of the salt solution must be sufficient to overcome at least partially the electrostatic repulsion of the polynucleotides from each other and, either the electrostatic attraction of the negatively-charged polynucleotides for positively-charged nanoparticles, or the electrostatic repulsion of the negatively-charged polynucleotides from negatively-charged nanoparticles. Gradually reducing the electrostatic attraction and repulsion by adding the salt gradually over time gives the highest surface density of polynucleotides on the nanoparticles. Suitable ionic strengths can be determined empirically for each salt or combination of salts. In one aspect, a final concentration of sodium chloride of from about 0.01 M to about 1.0 M in phosphate buffer is utilized , with the concentration of sodium chloride being increased gradually over time. In another aspect, a final concentration of sodium chloride of from about 0.01 M to about 0.5 M, or about 0.1 M to about 0.3 M is utilized, with the concentration of sodium chloride being increased gradually over time.

[0066] After adding the salt, the polynucleotides and nanoparticles are incubated in the salt solution for a period of time to allow additional polynucleotides to bind to the nanoparticles to produce the stable nanoparticle-polynucleotide compositions. An increased surface density of the polynucleotides on the nanoparticles stabilizes the compositions, as has been described herein. The time of this incubation can be determined empirically. By way of example, in one aspect a total incubation time of about 24-48, wherein the salt concentration is increased gradually over this total time, is contemplated. This second period of incubation in the salt solution is referred to herein as the "aging" step. Other suitable conditions for this "aging" step can also be determined empirically. By way of example, an aging step is carried out with incubation at room temperature and pH 7.0.

[0067J The compositions produced by use of the "aging" are in general more stable than those produced without the "aging" step. As noted above, this increased stability is due to the increased density of the polynucleotides on the surfaces of the nanoparticles which is achieved by the "aging" step. The surface density achieved by the "aging" step will depend on the size and type of nanoparticles and on the length, sequence and concentration of the polynucleotides.

[0068] As used herein, "stable" means that, for a period of at least six months after the compositions are made, a majority of the polynucleotides remain attached to the nanoparticles and the polynucleotides are able to hybridize with nucleic acid and polynucleotide targets under standard conditions encountered in methods of detecting nucleic acid and methods of

nano fabrication.

SURFACE DENSITY [0069] Nanoparticles as provided herein have a packing density of the polynucleotides on the surface of the nanoparticle that is, in various aspects, sufficient to result in cooperative behavior between nanoparticles and between polynucleotide strands on a single nanoparticle. In another aspect, the cooperative behavior between the nanoparticles increases the resistance of the polynucleotide to nuclease degradation. In yet another aspect, the uptake of nanoparticles by a cell is influenced by the density of polynucleotides associated with the nanoparticle. As described in PCT/US2008/65366, incorporated herein by reference in its entirety, a higher density of polynucleotides on the surface of a nanoparticle is associated with an increased uptake of nanoparticles by a cell.

[0070] A surface density adequate to make the nanoparticles stable and the conditions necessary to obtain it for a desired combination of nanoparticles and polynucleotides can be determined empirically. Generally, a surface density of at least 2 pmoles/cm² will be adequate to provide stable nanoparticle-polynucleotide compositions. In some aspects, the surface density is at least 15 pmoles/cm². Methods are also provided wherein the polynucleotide is bound to the nanoparticle at a surface density of at least 2 pmol/cm², at least 3 pmol/cm², at least 4 pmol/cm², at least 5 pmol/cm², at least 6 pmol/cm², at least 7 pmol/cm², at least 8 pmol/cm², at least 9 pmol/cm², at least 10 pmol/cm², at least about 15 pmol/cm², at least about 20 pmol/cm², at least about 25 pmol/cm², at least about 30 pmol/cm², at least about 35 pmol/cm², at least about 40 pmol/cm², at least about 45 pmol/cm², at least about 50 pmol/cm², at least about 55 pmol/cm², at least about 60 pmol/cm², at least about 65 pmol/cm², at least about 70 pmol/cm², at least about

7 9 9 9

75 pmol/cm , at least about 80 pmol/cm , at least about 85 pmol/cm^", at least about 90 pmol/cm , at least about 95 pmol/cm², at least about 100 pmol/cm², at least about 125 pmol/cm², at least about 150 pmol/cm², at least about 175 pmol/cm², at least about 200 pmol/cm², at least about 250 pmol/cm², at least about 300 pmol/cm , at least about 350 pmol/cm², at least about 400 pmol/cm², at least about 450 pmol/cm², at least about 500 pmol/cm², at least about 550 pmol/cm², at least about 600 pmol/cm², at least about 650 pmol/cm², at least about 700 pmol/cm², at least about 750 pmol/cm², at least about 800 pmol/cm², at least about 850 pmol/cm², at least about 900 pmol/cm², at least about 950 pmol/cm², at least about 1000 pmol/cm² or more. [0071] Density of polynucleotides on the surface of a nanoparticle has been shown to modulate specific polypeptide interactions with the polynucleotide on the surface and/or with the nanoparticle itself. Under various conditions, some polypeptides may be prohibited from interacting with polynucleotides associated with a nanoparticle based on steric hindrance caused by the density of polynucleotides. In aspects where interaction of polynucleotides with polypeptides that are otherwise precluded by steric hindrance is desirable, the density of polynucleotides on the nanoparticle surface is decreased to allow the polypeptide to interact with the polynucleotide.

[0072] Polynucleotide surface density has also been shown to modulate stability of the polynucleotide associated with the nanoparticle. In one embodiment, an RNA polynucleotide associated with a nanoparticle is provided wherein the RNA polynucleotide has a half-life that is at least substantially the same as the half-life of an identical RNA polynucleotide that is not associated with a nanoparticle. In other embodiments, the RNA polynucleotide associated with the nanoparticle has a half-life that is about 5% greater, about 10% greater, about 20% greater, about 30% greater, about 40% greater, about 50% greater, about 60% greater, about 70% greater, about 80% greater, about 90% greater, about 2-fold greater, about 3-fold greater, about 4-fold greater, about 5-fold greater, about 6-fold greater, about 7-fold greater, about 8-fold greater, about 9-fold greater, about 10-fold greater, about 20-fold greater, about 30-fold greater, about 40-fold greater, about 50-fold greater, about 60-fold greater, about 70-fold greater, about 80-fold greater, about 90-fold greater, about 100-fold greater, about 200-fold greater, about 300-fold greater, about 400-fold greater, about 500-fold greater, about 600-fold greater, about 700-fold greater, about 800-fold greater, about 900-fold greater, about 1000-fold greater, about 5000-fold greater, about 10,000- fold greater, about 50,000-fold greater, about 100,000-fold greater, about 200,000-fold greater, about 300,000-fold greater, about 400,000-fold greater, about 500,000-fold greater, about 600,000-fold greater, about 700,000-fold greater, about 800,000-fold greater, about 900,000-fold greater, about 1,000,000-fold greater or more than the half-life of an identical RNA polynucleotide that is not associated with a nanoparticle.

POLYNUCLEOTIDE FEATURES

[0073] The present disclosure provides, in various embodiments, PN-NP compositions that are useful for hybridizing to a target polynucleotide. In some aspects, the compositions are used to detect and/or quantify expression of the target polynucleotide. In some aspects, the compositions are used for gene regulation. In further aspects, the composition is useful both for quantitating a target polynucleotide and regulating the expression of the same or a different polynucleotide.

[0074] Gene regulatory activity is also, in various embodiments, achieved through the use of a regulatory polynucleotide which can modulate the transcription of a mRNA.

[0075] In some aspects, the PN-NP is functionalized with DNA. In some embodiments, the DNA is double stranded, and in further embodiments the DNA is single stranded. In further aspects, the PN-NP is functionalized with RNA, and in still further aspects the PN-NP is functionalized with double stranded RNA agents known as small interfering RNA (siRNA). The term "RNA" includes duplexes of two separate strands, as well as single stranded structures. Single stranded RNA also includes RNA with secondary structure. In one aspect, RNA having a hairpin loop in contemplated.

[0076] Polynucleotides that are contemplated for use in gene regulation and functionalized to a nanoparticle have complementarity to (i.e., are able to hybridize with) a portion of a target RNA (generally messenger RNA (mRNA)).

[0077] "Hybridization" means an interaction between two or three strands of nucleic acids by hydrogen bonds in accordance with the rules of Watson-Crick complementarity, Hoogstein binding, or other sequence-specific binding known in the art. Hybridization can be performed under different stringency conditions known in the art.

[0078] Generally, such complementarity is 100%, but can be less if desired, such as about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. For example, 19 bases out of 21 bases may be base-paired. Thus, it will be understood that a polynucleotide used in the methods need not be 100% complementary to a desired target nucleic acid to be

specifically hybridizable. Moreover, polynucleotides may hybridize to each other over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). Percent complementarity between any given polynucleotide can be determined routinely using BLAST programs (Basic Local Alignment Search Tools) and PowerBLAST programs known in the art (Altschul et ah, 1990, J. MoI. Biol., 215: 403-410; Zhang and Madden, 1997, Genome Res., 7: 649-656).

[0079] In some aspects, where selection between various allelic variants is desired, 100% complementarity to the target gene is required in order to effectively discern the target sequence from the other allelic sequence. When selecting between allelic targets, choice of length is also an important factor because it is the other factor involved in the percent complementary and the ability to differentiate between allelic differences.

TARGET POLYNUCLEOTIDE SEQUENCES AND HYBRIDIZATION

[0080] In some aspects, the disclosure provides methods of targeting a specific

polynucleotide. Any type of polynucleotide may be targeted, and the methods may be used, e.g., for therapeutic modulation of gene expression (See, e.g., PCT/US2006/022325, the disclosure of which is incorporated herein by reference).

[0081] In further embodiments, a polynucleotide is targeted for detection and quantitation of a relative amount of the target polynucleotide. In some embodiments, the nano flares as described herein are used to detect and quantitate an amount of the target polynucleotide in response to exposure to a transcriptional regulator as described by the disclosure.

[0082] Examples of polynucleotides that can be targeted by the methods of the invention include but are not limited to genes (e.g., a gene associated with a particular disease), viral RNA, niRNA, RNA, or single-stranded nucleic acids.

[0083] The target nucleic acid may be in cells, tissue samples, or biological fluids, as also known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995).

[0084] In some aspects, the target polynucleotide is in a target cell. In various embodiments, the target cell is selected from the group consisting of a cancer cell, a transformed cell, an in vivo target cell, a primary cell, a stem cell, a blood cell, a co-cultured cell, an immobilized cell, a suspended cell, a blood cell, a neuronal cell, and any cell derived from ectoderm, mesoderm, or endodermal lineage.

[0085] In those embodiments wherein the target cell is a cancer cell, the cancer cell is found in a human cancer. In further aspects, the cancer is selected from the group consisting of liver, pancreatic, stomach, colorectal, prostate, testicular, renal cell, breast, bladder, ureteral, brain, lung, connective tissue, hematological, cardiovascular, lymphatic, skin, bone, eye,

nasopharyngeal, laryngeal, esophagus, oral membrane, tongue, thyroid, parotid, mediastinum, ovary, uterus, adnexal, small bowel, appendix, carcinoid, gall bladder, pituitary, cancer arising from metastatic spread, and cancer arising from endodermal, mesodermal or ectodermally- derived tissues.

[0086] In some embodiments, the target cell is a transformed cell. In various aspects, the transformed cell comprises an expression vector, and in further aspects the expression vector encodes a polypeptide fused in-frame to a detectable polypeptide.

[0087] Any detectable polypeptide known in the art is useful in the methods of the disclosure, and in some aspects is a fluorescent protein. In some aspects, the fluorescent protein is selected from the list of proteins in Table 1, below.

Table 1. List of fluorescent polypeptides

[0088] In various embodiments the disclosure contemplates that more than one target polynucleotide is detected in the target cell.

[0089] The terms "start codon region" and "translation initiation codon region" refer to a portion of a mRNA or gene that encompasses contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and

"translation termination codon region" refer to a portion of such a mRNA or gene that encompasses contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions which may be targeted effectively with the polynucleotides on the functionalized nanoparticles.

[0090] Other target regions include the 5' untranslated region (5'UTR), the portion of an mRNA in the 5' direction from the translation initiation codon, including nucleotides between the 5' cap site and the translation initiation codon of a mRNA (or corresponding nucleotides on the gene), and the 3' untranslated region (3'UTR), the portion of a mRNA in the 3' direction from the translation termination codon, including nucleotides between the translation termination codon and 3' end of a mRNA (or corresponding nucleotides on the gene). The 5' cap site of a mRNA comprises an N7-methylated guanosine residue joined to the 5'~most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of a mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site.

[0091] For prokaryotic target nucleic acid, in various aspects, the nucleic acid is RNA transcribed from genomic DNA. For eukaryotic target nucleic acid, the nucleic acid is an animal nucleic acid, a plant nucleic acid, a fungal nucleic acid, including yeast nucleic acid. As above, the target nucleic acid is a RNA transcribed from a genomic DNA sequence. In certain aspects, the target nucleic acid is a mitochondrial nucleic acid. For viral target nucleic acid, the nucleic acid is viral genomic RNA, or RNA transcribed from viral genomic DNA.

[0092] In some embodiments of the disclosure, a target polynucleotide sequence is a microRNA. MicroRNAs (miRNAs) are 20-22 nucleotide (nt) molecules generated from longer 70-nt RNAs that include an imperfectly complementary hairpin segment [Jackson et al, Sci STKE 367: rel (2007); Mendell, Cell Cycle 4: 1 179-1184 (2005)]. The longer precursor molecules are cleaved by a group of proteins (Drosha and DCGR8) in the nucleus into smaller RNAs called pre-miRNA. Pre-miRNAs are then exported into the cytoplasm by exportin

[Virmani et al., J Vase Interv Radiol 19: 931-936 (2008)] proteins. The pre-miRNA in the cytoplasm is then cleaved into mature RNA by a complex of proteins called RNAi silencing complex or RISC. The resulting molecule has 19-bp double stranded RNA and 2 nt 3' overhangs on both strands. One of the two strands is then expelled from the complex and is degraded. The resulting single strand RNA-protein complex can then inhibit translation (either by repressing the actively translating ribosomes or by inhibiting initiation of translation) or enhance degradation of the mRNA it is attached to. There is, of course, a high degree of selectivity to this process, as the miRNA only binds to areas that are of high match to its sequence [Zamore et al., Science 309: 1519-1524 (2005)]. In one aspect, the target polynucleotide is microRNA-210.

[0093] Methods for inhibiting gene product expression provided include those wherein expression of the target gene product is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% compared to gene product expression in the absence of an polynucleotide- functionalized nanoparticle. In other words, methods provided embrace those which results in essentially any degree of inhibition of expression of a target gene product.

[0094] The degree of inhibition is determined in vivo from a body fluid sample or from a biopsy sample or by imaging techniques well known in the art. Alternatively, the degree of inhibition is determined in a cell culture assay, generally as a predictable measure of a degree of inhibition that can be expected in vivo resulting from use of a specific type of nanoparticle and a specific polynucleotide.

DETECTABLE MARKER

[0095] Methods are provided wherein presence of a polynucleotide is detected by an observable change. In one aspect, presence of the polynucleotide gives rise to a color change which is observed with a device capable of detecting a specific marker as disclosed herein. For example and without limitation, a fluorescence microscope can detect the presence of a fluorophore that is conjugated to a polynucleotide, which has been functionalized on a nanoparticle.

[0096] It will be understood that a marker contemplated will include any of the fluorophores described herein as well as other detectable markers known in the art. For example, markers also include, but are not limited to, a fluorophore, an isotope, a contrast agent, a redox active probe, a nanoparticle, a mass tag, a polypeptide, a peptide, a small molecule, an enzyme, a catalyst, an enzyme co-factor, a polynucleotide, a metal, and a quantum dot.

[0097] Suitable fluorescent molecules are also well known in the art and include without limitation 1,8-ANS (l-Anilinonaphthalene-8-sulfonic acid), l-Anilinonaphthalene-8-sulfonic acid (1 ,8-ANS), 5-(and-6)-Carboxy-2\ 7'-dichlorofluorescein pH 9.0, 5-FAM pH 9.0, 5-ROX (5- Carboxy-X-rhodamine, triethylammonium salt), 5-ROX pH 7.0, 5-TAMRA, 5-TAMRA pH 7.0, 5-T AMRA-MeOH, 6 JOE, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0, 6- Carboxyrhodamine 6G pH 7.0, 6-Carboxyrhodamine 6G, hydrochloride, 6-HEX, SE pH 9.0, 6- TET, SE pH 9.0, 7-Amino-4-rnethylcoumarin pH 7.0, 7-Hydroxy-4-methylcoumarin, 7- Hydroxy-4-methylcoumarin pH 9.0, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 532,

Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa 647, Alexa 660, Alexa 680, Alexa 700, Alexa Fluor 430 antibody conjugate pH 7.2, Alexa Fluor 488 antibody conjugate pH 8.0, Alexa Fluor 488 hydrazide-water, Alexa Fluor 532 antibody conjugate pH 7.2, Alexa Fluor 555 antibody conjugate pH 7.2, Alexa Fluor 568 antibody conjugate pH 7.2, Alexa Fluor 610 R- phycoerythrin streptavidin pH 7.2, Alexa Fluor 647 antibody conjugate pH 7.2, Alexa Fluor 647 R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 660 antibody conjugate pH 7.2, Alexa Fluor 680 antibody conjugate pH 7.2, Alexa Fluor 700 antibody conjugate pH 7.2, Allophycocyanin pH 7.5, AMCA conjugate, Amino Coumarin, APC (allophycocyanin) ,Atto 647, BCECF pH 5.5, BCECF pH 9.0, BFP (Blue Fluorescent Protein), BO-PRO-I-DNA, B0-PR0-3-DNA, BOBO-I- DNA, B0B0-3-DNA, BODIPY 650/665-X, MeOH, BODIPY FL conjugate, BODIPY FL, MeOH, Bodipy R6G SE, BODIPY R6G, MeOH, BODIPY TMR-X antibody conjugate pH 7.2, Bodipy TMR-X conjugate, BODIPY TMR-X, MeOH, BODIPY TMR-X, SE, BODIPY TR-X phallacidin pH 7.0, BODIPY TR-X, MeOH, BODIPY TR-X, SE, BOPRO-I, BOPRO-3, Calcein, Calcein pH 9.0, Calcium Crimson, Calcium Crimson Ca2+, Calcium Green, Calcium Green- 1 Ca2+, Calcium Orange, Calcium Orange Ca2+, Carboxynaphthofluorescein pH 10.0, Cascade Blue, Cascade Blue BSA pH 7.0, Cascade Yellow, Cascade Yellow antibody conjugate pH 8.0, CFDA, CFP (Cyan Fluorescent Protein), CI-NERF pH 2.5, CI-NERF pH 6.0, Citrine, Coumarin, Cy 2, Cy 3, Cy 3.5, Cy 5, Cy 5.5, CyQUANT GR-DNA, Dansyl Cadaverine, Dansyl Cadaverine, MeOH, DAPI, DAPI-DNA, Dapoxyl (2-aminoethyl) sulfonamide, DDAO pH 9.0, Di-8 ANEPPS, Di-8-ANEPPS-lipid, DiI, DiO, DM-NERF pH 4.0, DM-NERF pH 7.0, DsRed, DTAF, dTomato, eCFP (Enhanced Cyan Fluorescent Protein), eGFP (Enhanced Green

Fluorescent Protein), Eosin, Eosin antibody conjugate pH 8.0, Erythrosin-5-isothiocyanate pH 9.0, Ethidium Bromide, Ethidium homodimer, Ethidium homodimer-1-DNA, eYFP (Enhanced Yellow Fluorescent Protein), FDA, FITC, FITC antibody conjugate pH 8.0, FlAsH, Fluo-3, Fluo-3 Ca2+, Fluo-4, Fluor-Ruby, Fluorescein, Fluorescein 0.1 M NaOH, Fluorescein antibody conjugate pH 8.0, Fluorescein dextran pH 8.0, Fluorescein pH 9.0, Fluoro-Emerald, FM 1-43, FM 1-43 lipid, FM 4-64, FM 4-64, 2% CHAPS, Fura Red Ca2+, Fura Red, high Ca, Fura Red, low Ca, Fura-2 Ca2+, Fura-2, high Ca, Fura-2, no Ca, GFP (S65T), HcRed, Hoechst 33258, Hoechst 33258-DNA, Hoechst 33342, Indo-1 Ca2+, Indo-1, Ca free, Indo-1, Ca saturated, JC-I, JC-I pH 8.2, Lissamine rhodamine, LOLO-I-DNA, Lucifer Yellow, CH, LysoSensor Blue, LysoSensor Blue pH 5.0, LysoSensor Green, LysoSensor Green pH 5.0, LysoSensor Yellow pH 3.0, LysoSensor Yellow pH 9.0, LysoTracker Blue, LysoTracker Green, LysoTracker Red, Magnesium Green, Magnesium Green Mg2+, Magnesium Orange, Marina Blue, mBanana, mCherry, mHoneydew, MitoTracker Green, MitoTracker Green FM, MeOH, MitoTracker Orange, MitoTracker Orange, MeOH, MitoTracker Red, MitoTracker Red, MeOH, mOrange, mPlum, mRFP, mStrawberry, mTangerine, NBD-X, NBD-X, MeOH, NeuroTrace 500/525, green fluorescent Nissl stain-RNA, Nile Blue, EtOH, Nile Red, Nile Red-lipid, Nissl, Oregon Green 488, Oregon Green 488 antibody conjugate pH 8.0, Oregon Green 514, Oregon Green 514 antibody conjugate pH 8,0, Pacific Blue, Pacific Blue antibody conjugate pH 8.0, Phycoerythrin, PicoGreen dsDNA quantitation reagent, PO-PRO-I, PO-PRO-I-DNA, PO-PRO-3, PO-PRO-3- DNA, POPO-I, POPO-I-DNA, POPO-3, Propidium Iodide, Propidium Iodide-DNA, R- Phycoerythrin pH 7.5, ReAsH, Resomfin, Resorufin pH 9.0, Rhod-2, Rhod-2 Ca2+, Rhodamine, Rhodamine 110, Rhodamine 110 pH 7.0, Rhodamine 123, MeOH, Rhodamine Green,

Rhodamine phalloidin pH 7.0, Rhodamine Red-X antibody conjugate pH 8.0, Rhodaminen Green pH 7.0, Rhodol Green antibody conjugate pH 8.0, Sapphire, SBFI-Na+, Sodium Green Na+, Sulforhodamine 101, EtOH, SYBR Green I, SYPRO Ruby, SYTO 13-DNA, SYTO 45- DNA, SYTOX Blue-DNA, Tetramethylrhodamine antibody conjugate pH 8.0,

Tetramethylrhodamine dextran pH 7.0, Texas Red-X antibody conjugate pH 7.2, TO-PRO-I- DNA, TO-PRO-3-DNA, TOTO-I-DNA, TOTO-3-DNA, TRITC, X-Rhod-1 Ca2+, YO-PRO-I- DNA, Y0-PR0-3-DNA, YOYO-I-DNA, and Y0Y0-3-DNA.

[0098J In yet another embodiment, two types of fluorescent-labeled polynucleotides attached to two different particles can be used. This may be useful, for example and without limitation, to track two different cell populations. Suitable particles include polymeric particles (such as, without limitation, polystyrene particles, polyvinyl particles, acrylate and methacrylate particles), glass particles, latex particles, Sepharose beads and others like particles well known in the art. Methods of attaching polynucleotides to such particles are well known and routinely practiced in the art. See Chrisey et al, 1996, Nucleic Acids Research, 24: 3031-3039 (glass) and Charreyre et al, 1997 Langmiiir, 13: 3103-3110, Fahy et al, 1993, Nucleic Acids Research, 21: 1819-1826, Elaissari et al, 1998, J. Colloid Interface ScL, 202: 251-260, Kolarova et al, 1996,

Biotechniques, 20: 196-198 and Wolf et al , 1987 ^', Nucleic Acids Research, 15: 2911-2926 (polymer/latex). [0099] As used herein, a "contrast agent" is a compound or other substance introduced into a cell in order to create a difference in the apparent density of various organs and tissues, making it easier to see the delineate adjacent body tissues and organs. In some embodiments, the contrast agent is a paramagnetic compound, and in further embodiments the contrast agent is selected from the group consisting of gadolinium, xenon, iron oxide, and copper. Contrast agents may be detected using any device or procedure known in the art, including but not limited to magnetic resonance imaging (MRl).

[0100] The term "small molecule," as used herein, refers to a chemical compound, for instance a peptidometic or polynucleotide that may optionally be derivatized, or any other low molecular weight organic compound, either natural or synthetic. A small molecule may be a detectable marker or, in some aspects, is a therapeutic agent. Such small molecules may be a

therapeutically deliverable substance or may be further derivatized to facilitate delivery.

[0101] By "low molecular weight" is meant compounds having a molecular weight of less than 1000 Daltons, typically between 300 and 700 Daltons. Low molecular weight compounds, in various aspects, are about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 1000 or more Daltons.

[0102] Other labels besides fluorescent molecules can be used, such as chemiluminescent molecules, which will give a detectable signal or a change in detectable signal upon

hybridization.

[0103] Methods of labeling polynucleotides with fluorescent molecules and measuring fluorescence are well known in the art.

NANOFLARE TECHNOLOGY

[0104] Nano-flares take advantage of the unique optical properties of nanoparticles (NPs). NPs quench fluorescence with a greater efficiency [Dubertret et al, 2001, Nat. Biotechnol 19: 365-370] and over greater distances [Dulkeith et al, 2005, Nano Lett. 5: 585-589] than molecular quenchers. Likewise, all other types of nanoparticles described herein may be used as long as they are able to quench the detectable marker of an attached polynucleotide. Nano flares are described in detail in International Patent Application Number PCT/US2008/053603, the disclosure of which is incorporated by reference herein in its entirety.

[0105] Compositions and methods herein are useful in the practice of nano flare technology. In some embodiments, a composition is provided comprising a nanoparticle functionalized with a polynucleotide (PN-NP), and further comprising a transcriptional regulator, wherein the transcriptional regulator induces transcription of a target polynucleotide in a target cell.

[0106J In further embodiments, the polynucleotide comprises a detectable marker, wherein the detectable marker is quenched when the polynucleotide is not hybridized to a target

polynucleotide. In some aspects, the polynucleotide is double stranded, and the double stranded polynucleotide comprises a first strand that is functionalized to the nanoparticle and a second strand that can hybridize to the first strand. In further aspects, the second strand further comprises the detectable marker.

[0107] In certain aspects, the first strand is sufficiently complementary to the target polynucleotide to hybridize to the target polynucleotide, and the hybridization dissociates the second strand from the first strand. This dissociation results in a detectable change, that can be measured with a device as described herein.

[0108] In some embodiments, the polynucleotide that is attached to the nanoparticle is single stranded. In some of these aspects, the polynucleotide forms a hairpin structure, wherein hybridization of the polynucleotide to a target polynucleotide causes dissociation of the hairpin and causes the detectable marker to move away from the nanoparticle resulting in a detectable change. This detectable change is, in various aspects, measured with a device.

[0109] It is contemplated by the disclosure that the target polynucleotide is, in some aspects, RNA or DNA.

TRANSCRIPTIONAL REGULATORS

[0110] The present disclosure provides compositions comprising a nanoparticle functionalized with a polynucleotide (PN-NP), and further comprising a transcriptional regulator, wherein the transcriptional regulator induces transcription of a target polynucleotide in a target cell.

[0111] A transcriptional regulator as used herein is contemplated to be anything that induces a change in transcription of a niRNA. The change can, in various aspects, either be an increase or a decrease in transcription. In various embodiments, the transcriptional regulator is selected from the group consisting of a polypeptide, a polynucleotide, an artificial transcription factor (ATF) and any molecule known or suspected to regulate transcription.

Polypeptides

[0112] Compositions and methods of the disclosure include those wherein the transcriptional regulator is a polypeptide. Any polypeptide that acts to either increase or decrease transcription of a mRNA is contemplated for use herein. A peptide is also contemplated for use as a transcriptional regulator.

[0113] In some embodiments, the polypeptide is a transcription factor. In general, a transcription factor is modular in structure and contain the following domains.

[0114] DNA-binding domain (DBD), which attach to specific sequences of DNA (for example and without limitation, enhancer or promoter sequences) adjacent to regulated genes. DNA sequences that bind transcription factors are often referred to as response elements.

[0115] Trans-activating domain (TAD), which contain binding sites for other proteins such as transcription co-regulators. These binding sites are frequently referred to as activation functions (AFs) [Warnmark et al, MoI. Endocrinol. 17(10): 1901-9 (2003)].

[0116] An optional signal sensing domain (SSD) (for example and without limitation, a ligand binding domain), which senses external signals and, in response, transmits these signals to the rest of the transcription complex, resulting in up- or down-regulation of gene expression. Also, the DBD and signal-sensing domains may, in some aspects, reside on separate proteins that associate within the transcription complex to regulate gene expression.

[0117] Transcription factors are often classified based on the sequence similarity and hence the tertiary structure of their DNA-binding domains. The following Table (Table 2) of non- limiting examples of features and examples of transcription factors are contemplated for use in the compositions and methods of the disclosure.

Table 2. Transcription Factor Structural Features

• 1 Superclass: Basic Domains (Basic-helix-loop-helix) o 1.1 Class: Leucine zipper factors (bZIP) ■ 1.1.1 Family: AP-I (-like) components; includes (c-Fos/c-Jun)

■ 1.1.2 Family: CREB

• 1.1.3 Family: C/EBP-like factors

- 1.1.4 Family: bZIP / PAR

■ 1.1.5 Family: Plant G-box binding factors

■ 1.1.6 Family: ZIP only o 1.2 Class: Helix-loop-helix factors (WILH)

• 1.2.1 Family: Ubiquitous (class A) factors

■ 1.2.2 Family: Myogenic transcription factors (MyoD)

■ 1.2.3 Family: Achaete-Scute

■ 1.2.4 Family: Tal/Twist/Atonal/Hen o 1.3 Class: Helix-loop-helix / leucine zipper factors (bHLH-ZIP)

• 1.3.1 Family: Ubiquitous bHLH-ZIP factors; includes USF SREBP (SREBP)

■ 1.3.2 Family: Cell-cycle controlling factors; includes c-Myc o 1.4 Class: NF-I

- 1.4.1 Family: NF-I (A, B, C, X) o 1.5 Class: RF-X

■ 1.5.1 Family: RF-X (I, 2, 3, 4, 5, ANK) o 1.6 Class: bHSH

Superclass: Zinc -coordinating DNA-binding domains o 2.1 Class: Cys4 zinc finger of nuclear receptor type

■ 2.1.1 Family: Steroid hormone receptors

• 2.1.2 Family: Thyroid hormone receptor-like factors o 2.2 Class: diverse Cys4 zinc fingers

■ 2.2.1 Family: GATA-Factors

o 2.3 Class: Cys2His2 zinc finger domain

■ 2.3.1 Family: Ubiquitous factors, includes TFIlIA, SpI

■ 2.3.2 Family: Developmental / cell cycle regulators; includes Kruppel

■ 2.3.4 Family: Large factors with NF-6B-like binding properties o 2.4 Class: Cys6 cysteine-zinc cluster

o 2.5 Class: Zinc fingers of alternating composition

Superclass: Helix-turn-helix

o 3.1 Class: Homeo domain

■ 3.1.1 Family: Homeo domain only; includes Ubx

■ 3.1.2 Family: POU domain factors; includes Oct

■ 3.1.3 Family: Homeo domain with LIM region

■ 3.1.4 Family: homeo domain plus zinc finger motifs

o 3.2 Class: Paired box

■ 3.2.1 Family: Paired plus homeo domain

■ 3.2.2 Family: Paired domain only

o 3.3 Class: Fork head / winged helix

■ 3.3.1 Family: Developmental regulators; includes forkhead

■ 3.3.2 Family: Tissue-specific regulators

■ 3.3.3 Family: Cell-cycle controlling factors

■ 3.3.0 Family: Other regulators

o 3.4 Class: Heat Shock Factors ■ 3.4.1 Family: HSF o 3.5 Class: Tryptophan clusters

■ 3.5.1 Family. Myb

■ 3.5.2 Family: Ets-type

■ 3.5.3 Family: Interferon regulatory factors o 3.6 Class: TEA ( transcriptional enhancer factor) domain

■ 3.6.1 Family: TEA (TEADl, TEAD2, TEAD3, TEAD4) Superclass: beta-Scaffold Factors with Minor Groove Contacts o 4.1 Class: RHR (ReI homology region)

■ 4.1.1 Family: Rel/ankyrin; NF-kappaB

■ 4.1.2 Family: ankyrin only

- 4.1.3 Family: NFAT (Nuclear Factor of Activated T-cells) (NFATCl, NFATC2, NFATC3) o 4.2 Class: STAT

■ 4.2.1 Family: STAT o 4.3 Class: p53

- 4.3.1 Family: β53 o 4.4 Class: MADS box

• 4.4.1 Family: Regulators of differentiation; includes (Mef2)

• 4.4.2 Family: Responders to external signals, SRF (serum response

(SEB

o 4.5 Class: beta-Barrel alpha-helix transcription factors o 4.6 Class: TATA binding proteins

• 4.6.1 Family: TBP ■ 4.7.1 Family: SOX genes, SRY

« 4.7.2 Family: TCF-I (TCFl)

« 4.7.3 Family: HMG2-related, SSRPl

■ 4.7.5 Family: MATA

o 4.8 Class: Heteromeric CCAAT factors

■ 4.8.1 Family: Heteromeric CCAAT factors o 4.9 Class: Grainyhead

■ 4.9.1 Family: Grainyhead

o 4.10 Class: Cold-shock domain factors

■ 4.10.1 Family: csd

o 4.11 Class: Runt

* 4.11.1 Family: Runt

Superclass: Other Transcription Factors

o 0.1 Class: Copper fist proteins

o 0.2 Class: HMGI(Y) (HMGAl)

- 0.2.1 Family: HMGI(Y)

o 0.3 Class: Pocket domain

o 0.4 Class: El A-like factors

o 0.5 Class: AP2/EREBP-related factors

- 0.5.1 Family: AP2

■ 0.5.2 Family: EREBP

- 0.5.3 Superfamily: AP2/B3

■ 0.5.3.1 Family: ARF - 0.5.3.2 Family: ABI ■ 0.5.3.3 Family: RAV

Regulator Polynucleotides

[0118] In some embodiments, the transcription factor is a regulator polynucleotide. In certain aspects, the polynucleotide is RNA, and in further aspects the regulator polynucleotide is a noncoding RNA (ncRNA).

[0119] In some embodiments, the noncoding RNA interacts with the general transcription machinery, thereby inhibiting transcription [Goodrich et al., Nature Reviews MoI Cell Biol 7: 612-616 (2006)]. In general, RNAs that have such regulatory functions do not encode a protein and are therefore referred to as ncRNAs. Eukaryotic ncRNAs are transcribed from the genome by one of three nuclear, DNA-dependent RNA polymerases (Pol I, II or III). They then elicit their biological responses through one of three basic mechanisms: catalyzing biological reactions, binding to and modulating the activity of a protein, or base-pairing with a target nucleic acid.

[0120] ncRNAs have been shown to participate actively in many of the diverse biological reactions that encompass gene expression, such as splicing, mRNA turn over, gene silencing and translation [Storz, et al., Annu. Rev. Biochem. 74: 199-217 (2005)]. Notably, several studies have recently revealed that ncRNAs also actively regulate eukaryotic mRNA transcription, which is a key point for the control of gene expression.

[0121] In another embodiment, a regulatory polynucleotide is one that can associate with a transcription factor thereby titrating its amount. In some aspects, using increasing concentrations of the regulatory polynucleotide will occupy increasing amounts of the transcription factor, resulting in derepression of transcription of a mRNA.

[0122] In a further embodiment, a regulatory polynucleotide is an aptamer.

Artificial Transcription Factor

[0123] Many human diseases are characterized by altered gene expression patterns caused by malfunctioning transcriptional regulators. This has inspired increasing efforts in the chemical, biological, and medical community towards the design and synthesis of artificial transcription factors (ATFs), molecules that target specific genes and regulate their expression either positively or negatively [Ansari et al., Curr. Opin. Chem. Biol., 6: 765 (2002)]. Such factors would be powerful chemical tools for defining the maeromoleeular interactions that dictate gene expression patterns. Fully functional ATFs would have significant therapeutic potential as agents that could be used to restore normal patterns of gene expression in diseased cells.

[0124] Artificial transcription factors for use in the compositions and methods of the disclosure are generally described in Mapp [Org Biomol Chem, 1 : 2217-2220 (2003)], which is incorporated by reference herein in its entirety.

[0125] It is also contemplated by the disclosure that a transcription regulator is, in certain aspects, a stimulus encountered by a cell. For example and without limitation, the disclosure contemplates that stimuli that can function as a transcriptional regulator are temperature, radiation,and a compound that may be metabolized or not metabolized.

[0126] As used in this context, "temperature" can either be a temperature that induces transcription of, for example and without limitation, a heat shock gene, or can be a temperature that induces transcription of a cold response gene.

[0127] Radiation that can be used to regulate transcription is either ionizing radiation (for example and without limitation, ultraviolet (UV), X-ray and gamma ray) or non-ionizing radiation (for example and without limitation, neutron, electromagnetic, and thermal).

[0128] The term "compound," as used in the context of a transcriptional regulator, refers to a substance that may optionally be derivatized, or any other low molecular weight organic substance, either natural or synthetic.

METHODS

[0129] Methods provided by the disclosure include a method of detecting modulation of transcription of a target polynucleotide comprising administering a PN-NP and a transcriptional regulator and measuring a detectable change, wherein the transcriptional regulator increases or decreases transcription of the target polynucleotide in a target cell relative to a transcription level in the absence of the transcriptional regulator.

[0130] In various embodiments, transcription of the target polynucleotide is detected through the use of a composition of the disclosure. More specifically, the PN-NP in the composition detects the target polynucleotide through the nanoflare technology disclosed herein. In some aspects, a polynucleotide that is attached to a nanoparticle is double stranded, with one of the two strands further comprising a detectable marker. The detectable marker is quenched when it is in proximity to the nanoparticle. Hybridization of the PN-NP to a target polynucleotide causes, in some aspects, dissociation of the quenched strand of the polynucleotide. This dissociation results in a detectable change, and measurement of this change is related to the amount of target polynucleotide is present in a target cell. In various aspects the detectable change is a fluorescent signal. Accordingly, any device that can quantitate the fluorescent signal is contemplated for use. For example and without limitation, a flow cytometer may be used.

[0131] In some embodiments, the transcriptional regulator and the PN-NP are administered at different times, and in some aspects the transcriptional regulator is administered before the PN- NP. In further aspects, the transcriptional regulator is administered first, a biological sample is acquired second, and the PN-NP is administered third. In various aspects, the biological sample is selected from the group consisting of a tissue sample, a blood sample, a stool sample, a urine sample, saliva, tumor tissue, in vivo tumors, in vivo targeted organs, and in vivo targeted cells and tissues.

[0132] In some embodiments, the time between the first and second step is from about 1 minute to about 21 days. In a specific aspect, the time between the first and second step is one day. In various embodiments, the time between the first and second step is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours, about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours, about 22 hours, about 22.5 hours, about 23 hours, about 23.5 hours, about 24 hours, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, about 11 days, about 11.5 days, about 12 days, about 12.5 days, about 13 days, about 13.5 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 21 days, or more.

[0133] In further embodiments, the time between the second and third step is from about 1 minute to about 14 days. In a certain embodiment, the time between the second and third step is one day. In various embodiments, the time between the second and third step is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10,5 hours, about 1 1 hours, about 11.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours, about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours, about 22 hours, about 22.5 hours, about 23 hours, about 23.5 hours, about 24 hours, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, about 8 days, about 8.5 days, about 9 days, about 9.5 days, about 10 days, about 10.5 days, about 1 1 days, about 11.5 days, about 12 days, about 12.5 days, about 13 days, about 13.5 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 21 days, or more.

[0134] Methods as described herein are contemplated for in vivo or in vitro applications.

[0135] In various methods of the disclosure that are in vitro, it is contemplated that the method is to identify the transcriptional regulator as a candidate transcriptional regulator. In some of these methods, a library of transcriptional regulators is screened for its ability to modulate the transcription of the target polynucleotide. In various embodiments, the library is an expression library. In some aspects, the expression library is generated from a cDNA library. In further embodiments, the transcriptional regulator is a known regulator of transcription, and in some embodiments the transcriptional regulator is an unknown or hypothesized regulator of transcription.

[0136] Accordingly, in certain embodiments, a candidate transcriptional regulator is contacted with a target cell in vitro. In some aspects, the target cell is then contacted with a composition comprising a PN-NP as described herein. In a certain aspect, the polynucleotide that is attached to the nanoparticle is double stranded. In some aspects, the first strand of the double stranded polynucleotide is attached to the nanoparticle and is complementary to a target polynucleotide. In further aspects, a second strand of the double stranded polynucleotide further comprises a detectable marker that is quenched while the two strands of the polynucleotide remain hybridized to each other. In a specific aspect wherein the transcriptional regulator is capable of increasing transcription of a target polynucleotide, the first strand of the polynucleotide that is attached to the nanoparticle hybridizes to the target polynucleotide and causes the second strand to dissociate from the first strand. This dissociation results in a detectable change. The detectable change can be measured by a device as described herein, and if the detectable change is significantly different than the detectable change in a control cell that was contacted with the PN-NP but was either not contacted with a transcriptional regulator or was contacted with a different transcriptional regulator, then the candidate transcriptional regulator is identified as one that can increase transcription of the target polynucleotide. Thus, in some aspects, an increase or decrease in the detectable change when the transcriptional regulator is administered relative to the detectable change when a different transcriptional regulator within the library is administered is indicative of a candidate transcriptional regulator. These methods are useful, in certain aspects, for identifying a transcriptional regulator of a target polypeptide of interest.

[0137] In some aspects of the methods, a second transcriptional regulator is contacted with a target cell. In further aspects, the second transcriptional regulator is added concurrently with the first transcriptional regulator. In some aspects, the second transcriptional regulator is added after the first transcriptional regulator is added. It is therefore contemplated that in various aspects, the second transcriptional regulator is added about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes or more after the first transcriptional regulator is added. [0138] The disclosure also contemplates methods to identify the target polynucleotide. In some aspects of these methods, a library of polynucleotides is screened for its ability to detect the increase or decrease in transcription of the target polynucleotide. The library, in various aspects, is a polynucleotide library. In some aspects of these methods, a double stranded polynucleotide comprising a known sequence is functionalized to a nanoparticle, creating a first PN-NP. In some aspects, one strand of the double stranded polynucleotide further comprises a detectable marker that is quenched while the two strands of the polynucleotide remain hybridized to each other. The PN-NP is then contacted with a target cell concurrently with a transcriptional regulator. If the polynucleotide of known sequence that is functionalized on the nanoparticle hybridizes with the target polynucleotide, it results in a detectable change. The detectable change, in some aspects, is fluorescence. Observation of a detectable change that is significantly different from the detectable change observed by contacting the target cell with a second PN-NP in which the polynucleotide comprises a different sequence than the first PN-NP is indicative of identifying the target polynucleotide. Thus, in further aspects, each nanoparticle is

functionalized with a polynucleotide of known sequence, and in still further aspects, an increase or decrease in the detectable change when the transcriptional regulator is administered relative to the detectable change measured when a different nanoparticle functionalized with a

polynucleotide within the library is administered is indicative of identifying the target polynucleotide. Accordingly, in some aspects the methods provide for the identification of a mRNA that is regulated by a given transcriptional regulator. In various aspects, the mRNA is increased, and in some aspects the mRNA is decreased.

[0139] The present disclosure also provides methods for in vivo applications. In some embodiments, a composition of the disclosure is used to detect a target cell in a tissue. In some aspects, the tissue is superficial, and in further aspects the tissue is breast tissue. In further embodiments of these methods, the target cell is a cancer cell, and in some aspects the target cell is a breast cancer cell. Thus the present disclosure provides a method for delivering a

composition as described herein to a breast cancer cell. In some aspects of these methods, a composition is delivered locally to the breast cancer cell. Contact of the composition with the breast cancer cell results, in certain aspects, in a detectable change due to the properties of the PN-NP of the composition as discussed herein. The detectable change is then measured by a device capable of detecting the detectable change. [0140] In some embodiments, the disclosure provides a method to assess a temporal difference between transcription and translation of a detectable target polypeptide of interest. In some aspects of these methods, the target cell is a transformed cell. In various aspects, the transformed cell has been transformed with a vector. In further aspects, the vector encodes a target polypeptide. In still further aspects, the target polypeptide is a fusion protein, and in some aspects the target polypeptide is fused in-frame to a detectable polypeptide. The detectable polypeptide is selected, in various aspects, from the list of detectable polypeptides in Table 1. In a specific aspect, the detectable polypeptide is green fluorescent protein (GFP).

[0141] In various aspects the method contemplates contacting the target cell with a

composition comprising a PN-NP and a transcriptional regulator, wherein in one aspect the polynucleotide is double stranded, and in a further aspect a first strand is attached to the nanoparticle and is complementary to the mRNA that encodes the detectable polypeptide, and a second strand is hybridized to the first strand and further comprises a detectable marker that is quenched when the second strand is hybridized to the first strand. In a further aspect the transcriptional regulator increases transcription of the mRNA that encodes the detectable polypeptide. Thus, when the composition comprising the PN-NP and the transcriptional regulator contacts the target cell, transcription of the mRNA that encodes the detectable polypeptide is induced. In these aspects, a detectable change will occur when the first strand of the PN-NP hybridizes to the mRNA that encodes the detectable polypeptide and causes dissociation of the second strand from the first strand, thus distancing the second strand from the nanoparticle and resulting in a detectable change. This detectable change is measured by a device depending on the detectable marker that was used, and the difference between the time this detectable change is measured and the time at which the detectable polypeptide (for example and without limitation, GFP) is detected represents the temporal difference between transcription and translation of the detectable target polypeptide.

[0142] In a further aspect, methods are provided to detect cell membrane proteins that can be concomitantly labeled. Cell membrane proteins are labeled using conventional approaches, including without limitation, labeled antibodies, nanoparticles including quantum dots, aptamers, or other technologies. [0143] Local delivery of a composition comprising a PN-NP to a human is contemplated in some aspects of the disclosure. Local delivery involves the use of an embolic agent in combination with interventional radiology and a composition of the disclosure.

EMBOLIC AGENTS

[0144] The present disclosure employs the use of a composition comprising a nanoparticle functionalized with a polynucleotide (PN-NP), and further comprising a transcriptional regulator, wherein the transcriptional regulator induces transcription of a target polynucleotide in a target cell. In some aspects, the composition is administered with an embolic agent.

[0145] Embolic agents serve to increase localized drug concentration in target sites through selective occlusion of blood vessels by purposely introducing emboli, while decreasing drug washout by decreasing arterial inflow. In various aspects of the compositions and methods of the disclosure, the embolic agent is selected from the group consisting of a lipid emulsion (for example and without limitation, ethiodized oil or lipiodol), gelatin sponge, tris acetyl gelatin microspheres, embolization coils, ethanol, small molecule drags, biodegradable microspheres, non-biodegradable microspheres or polymers, and self-assemblying embolic material.

[0146] The present disclosure, in some aspects, provides methods for delivering a composition locally to a site of pathogenesis. This local delivery is termed "nanoembolization." In various embodiments, PN-NP particles are mixed with the embolic agent just prior to administration. The PN-NP/transcriptional regulator/embolic agent mixture may be used alone for

nanoembolization, or may be followed by administration of another embolic agent.

[0147] It has been shown that intraarterial (IA) delivery alone does not allow for dwell time at the desired site of therapy that is sufficient for efficient uptake of therapeutic PN-NPs. Thus the addition of embolic agent allows the therapy to block blood flow to a desired site increasing the dwell time of injected therapeutics which keeps the local concentration of therapeutic high and enhances delivery to tissue. Thus, using IA delivery of nanoparticles (NP) combined with an embolic agent greatly increases NP concentration in the vicinity of target cells and limits their distribution throughout the rest of the body, thereby greatly improving NP uptake in targeted cells of interest. [0148] Compositions of the present disclosure comprise ratios of PN-NPs and embolic agent. "Ratio," as used herein, can be a molar ratio, a volume to volume ratio or it can be the number of PN-NPs to the number of embolic agent molecules. One of ordinary skill in the art can determine the ratio to be used in the compositions of the present disclosure.

[0149] In some embodiments, the PN-NPs and the embolic agent are present in a ratio of about 1:1 to about 10:1. In further embodiments, the PN-NPs and the embolic agent are present in a ratio of about 2: 1 to about 5:1. In one aspect, the PN-NPs and the embolic agent are present in a ratio of about 3:1. The present disclosure contemplates, in various aspects, that

compositions of PN-NPs and the embolic agent are present in a ratio of about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1. about 42:1, about 43:1, about 44:1 , about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 55:1, about 60:1, about 65:1, about 70:1, about 75:1, about 80:1, about 85:1, about 90:1, about 95:1, about 100:1, about 150:1, about 200:1, about 250:1, about 300:1, about 350:1, about 400:1, about 450:1, about 500:1, about 550:1, about 600:1, about 650:1, about 700:1, about 750:1, about 800:1, about 850:1, about 900:1, about 950:1, about 1000:1, about 2000:1, about 5000:1, about 7000:1, about 10000:1 or greater.

[0150] In alternative aspects, compositions of PN-NPs and the embolic agent are present in a ratio of about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, about 1:25, about 1:26, about 1:27, about 1:28, about 1:29, about 1:30, about 1:31, about 1:32, about 1:33, about 1:34, about 1:35, about 1:36, about 1:37, about 1:38, about 1:39, about 1:40, about 1:41, about 1:42, about 1:43, about 1:44, about 1:45, about 1:46, about 1:47, about 1:48, about 1:49, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, about 1:95, about 1:100, about 1:150, about 1:200, about 1:250, about 1:300, about 1:350, about 1:400, about 1:450, about 1:500, about 1:550, about 1:600, about 1:700, about 1:750, about 1 :800, about 1 :850, about 1 :900, about 1:950, about 1 :1000, about 1 :2000, about 1 :5000, about 1 :10000 or greater.

[0151] In further embodiments, the PN-NPs are approximately 1 nanomolar (nM) to 10 micromolar (μM), while the embolic agent is in the μM to millimolar (mM) range. Accordingly, in some embodiments, this would yield PN-NP:embolic agent ratios of about 1 : 1, about 1 :10, about 1 :100, about 1:1000, about 1 :10,000 or higher.

THERAPEUTIC AGENTS

[0152] In some embodiments, a composition of the present disclosure further comprises a therapeutic agent. In some aspects, the therapeutic agent is associated with the nanoparticle. In other aspects, the therapeutic agent is co-administered with the PN-NP, but is separate from the PN-NP composition. In further aspects, the therapeutic agent is administered before the administration of the PN-NP composition, and in still further aspects, the therapeutic agent is administered after the administration of the PN-NP composition. One of ordinary skill in the art will understand that multiple therapeutic agents in multiple combinations can be administered at any time before, during or after administration of the PN-NP composition. In addition, repeated administration of a therapeutic agent is also contemplated.

[0153] In an embodiment of the invention, the therapeutic agent is selected from the group consisting of a protein, peptide, a chemotherapeutic agent, a small molecule, a radioactive material, and a polynucleotide.

[0154] Protein therapeutic agents include, without limitation peptides, enzymes, structural proteins, receptors and other cellular or circulating proteins as well as fragments and derivatives thereof, the aberrant expression of which gives rise to one or more disorders. Therapeutic agents also include, as one specific embodiment, chemotherapeutic agents. Still other therapeutic agents include polynucleotides, including without limitation, protein coding polynucleotides, polynucleotides encoding regulatory polynucleotides, and/or polynucleotides which are regulatory in themselves. Therapeutic agents also include, in various embodiments, a radioactive material.

[0155] In various aspects, protein therapeutic agents include cytokines or hematopoietic factors including without limitation IL-I alpha, IL-I beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor- 1 (CSF-I), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon- alpha (IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IL-7, IL-8, IL-9, IL-IO, IL-12, IL-13, IL-14, 1L-15, IL-16, IL-17, IL-18, thrombopoietin (TPO), angiopoietins, for example Ang-1, Ang-2, Ang-4, Ang-Y, the human angiopoietin-like polypeptide, vascular endothelial growth factor (VEGF), angiogenin, bone morphogenic protein- 1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein- 11, bone morphogenic protein- 12, bone morpho genie protein-13, bone morphogenic protein- 14, bone morphogenic protein- 15, bone morpho genie protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor, cytokine-induced neutrophil chemotactic factor 1 , cytokine-induced neutrophil, chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β. β endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial- derived neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor acidic, fibroblast growth factor basic, glial cell line-derived neutrophic factor receptor αl , glial cell line-derived neutrophic factor receptor α2, growth related protein, growth related protein α, growth related protein β, growth related protein γ, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor BB, platelet derived growth factor receptor α, platelet derived growth factor receptor β, pre-B cell growth stimulating factor, stem cell factor receptor, TNF, including TNFO, TNFl, TNF2, transforming growth factor α. transforming growth factor β, transforming growth factor βl, transforming growth factor βl .2, transforming growth factor β2, transforming growth factor β3. transforming growth factor β5, latent transforming growth factor βl, transforming growth factor β binding protein I, transforming growth factor β binding protein II, transforming growth factor β binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof.

[0156] In other aspects, chemotherapeutic agent include, without limitation, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); ethylenimines/methylmelamine such as thriethylenemelamine (TEM), Methylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5- fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5- azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6- thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate;

epipodophylotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinium coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists including

adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol

equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide.

[0157] Polynucleotide therapeutic agents include, in one aspect and without limitation, those which encode therapeutic proteins described herein and otherwise known in the art, as well as polynucleotides which have intrinsic regulatory functions. Polynucleotides that have regulatory functions have been described herein above and include without limitation RNAi , antisense, ribozymes, and triplex-forming polynucleotides, each of which have the ability to regulate gene expression. Methods for carrying out these regulatory functions have previously been described in the art (Dykxhoom D M, Novina C D and Sharp P A, Nature Review, 4: 457-467, 2003; Mittal V, Nature Reviews, 5: 355-365, 2004).

[0158] It will be appreciated that, in various aspects, a therapeutic agent as described herein is attached to the nanoparticle.

EXAMPLES

EXAMPLE 1

[0159] Cell Lines. LnCaP cells are a prostate cancer cell line collected as an aspirate from the lymph node of a 50 year old man with metastatic prostate cancer. The LnCaP line is unique in that it expresses a functional androgen receptor (AR). Accordingly, the LnCaP cell line is responsive to dihydrotestosterone (DHT) whereupon the AR binds DHT, translocates from the cell cytoplasm to the nucleus, and increases the transcription from androgen responsive genes. Prostate specific antigen (PSA), a marker for prostate cancer, is expressed by the LnCaP cell line, and is androgen responsive. Thus, stimulating LnCaP cells using DHT leads to an increased production of prostate specific antigen protein and PSA mRNA and is dose responsive [Lee et al., Endocrinology. 136(2): 796-803 (1995)].

[0160] LnCaP cells were propagated in RPMl- 1640 medium supplemented with 10% fetal bovine serum (FBS). For experiments, cells were sub-cultured in propagation media and allowed to adhere to the bottom of 12-well plates for a period of 24 hours. Following adhesion, the media in each well was changed to fresh experimental media whereupon the FBS in the propagation media was replaced with charcoal stripped serum (CSM, performed to remove endogenous steroid hormones). Experimental media without or supplemented with 10 nM DHT was used to induce differential PSA mRNA production. 10 nM DHT is well-known to induce PSA expression at the mRNA and protein level [Lee et al., Endocrinology. 136(2): 796-803 (1995)]. Cells grew in experimental media for period of approximately 12 hours prior to initiating experiments with and without gold nanoparticles. The time course of the experiment was chosen to capture the peak expression profiles of intracellular PSA mRNA following DHT induction (12-24 hours).

[0161] Co-Loaded Gold Nanoparticle Probes. As a means of measuring targeted versus non-specific intracellular mRNA levels and to assess probe degradation using DNA Au-NP immobilized nano-flares, a control (green)/target(far red) co-loaded DNA Au-NP probe design was used (Figure 1). On the surface of each DNA Au-NP were flares released upon PSA mRNA binding, and control flares of a sequence not complementary to any known mRNA sequence in humans.

[0162] In vitro experiments demonstrated approximate co-loading of the control and PSA- specific flares to the surface of the DNA Au-NPs (Figure 1). Further, in vitro experiments, where a mock PSA target was added to an aliquot of co-loaded DNA Au-NPs, demonstrated specific PSA-target flare removal following incubation at 37°C (Figure 2).

[0163] Flow Cytometry. In order to measure the ability of DHT to induce intracellular mRNA transcription in LnCaP cells we measured the median far red and green fluorescence from 10,000 LnCaP cells using a DakoCytomation flow cytometer. As demonstrated in Figure 3, very little fluorescence is observed in the control cells (no particles added) versus those treated with 100 pM of the co-loaded DNA-Au NPs. In addition, 10 nM DHT treated cells

demonstrated a significantly increased PSA-specific flare signal when compared to those cells grown under DHT-free experimental conditions (Figure 3).

[0164] The foregoing example demonstrated that a transcriptional regulator can be used to augment the target-specific flare signal generated from targeted cell types. This was a general and significant advance in the context of flow cytometry and the detection of live tumor cells, in this case prostate cancer cells. Furthermore, this work is of significant importance for in vitro live cell imaging, whereupon the non-toxic delivery of target mRNA-specific co-loaded nano- flare Au-NPs can be used to assess temporal differences between transcription and translation of labeled (for example and without limitation, GFP) protein targets of interest, or cell membrane proteins that are concomitantly labeled.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a nanoparticle functionalized with a polynucleotide (PN-NP), and further comprising a transcriptional regulator, wherein the transcriptional regulator induces transcription of a target polynucleotide in a target cell.

2. The composition of claim 1 wherein the transcriptional regulator induces transcription of the target polynucleotide to which the polynucleotide will hybridize.

3. The composition of claim 1 or claim 2 wherein the transcriptional regulator is conjugated to the nanoparticle.

4. The composition of any one of claim 1 through 3 wherein the transcriptional regulator is selected from the group consisting of a polypeptide, a regulator polynucleotide, and an artificial transcription factor (ATF).

5. The composition of any one of claims 1 through 4 wherein the transcriptional regulator is a polypeptide.

6. The composition of claim 4 wherein the polypeptide is a hormone.

7. The composition of any one of claims 1 through 6 wherein the polynucleotide is RNA, DNA or a modified polynucleotide.

8. The composition of any one of claims 1 through 7, wherein the polynucleotide comprises about 5 nucleotides to about 100 nucleotides.

9. The composition of any one of claims 1 through 8, wherein the polynucleotide comprises about 10 nucleotides to about 50 nucleotides.

10. The composition of any one of claims 1 through 9, wherein the polynucleotide comprises about 15 nucleotides.

11. The composition of any one of claims 1 through 10 wherein the polynucleotide comprises a detectable marker.

12. The composition of any one of claim 11 wherein the detectable marker is selected from the group consisting of a fluorophore, an isotope, a contrast agent, a redox active probe, a nanoparticle, a mass tag, a polypeptide, a peptide, a small molecule, an enzyme, a catalyst, an enzyme co-factor, a polynucleotide, a metal, and a quantum dot.

13. The composition of claim 11 or claim 12 wherein the detectable marker is quenched when the polynucleotide is not hybridized to the target polynucleotide

14. The composition of any one of claims 1 through 13 wherein the polynucleotide is double stranded.

15. The composition of claim 14 wherein the polynucleotide comprises a first strand that is functionalized to the nanoparticle and a second strand that can hybridize to the first strand.

16. The composition of claim 15 wherein the second strand further comprises the detectable marker.

17. The composition of claim 15 or claim 16 wherein the first strand is sufficiently complementary to the target polynucleotide to hybridize to the target polynucleotide.

18. The composition of claim 17 wherein the hybridization dissociates the second strand from the first strand.

19. The composition of claim 18 wherein the dissociation results in a detectable change.

20. The composition of any one of claims 1 through 13 wherein the polynucleotide is single stranded.

21. The composition of claim 20 wherein the polynucleotide has secondary structure.

22. The composition of claim 20 or claim 21 wherein hybridization of the

polynucleotide to the target polynucleotide causes dissociation of the secondary structure and causes the detectable marker to move away from the nanoparticle resulting in a detectable change.

23. The composition of any one of claims 1 through 22 wherein the target

polynucleotide is RNA or DNA.

24. The composition of any one of claims 1 through 23 wherein the target cell is selected from the group consisting of a cancer cell, a transformed cell, an in vivo target cell, a primary cell, a stem cell, a blood cell, a co-cultured cell, an immobilized cell, a suspended cell, a blood cell, a neuronal cell, and any cell derived from ectoderm, mesoderm, or endodermal lineage.

25. The composition of any one of claims 1 through 24 wherein the target cell is a cancer cell.

26. The composition of claim 25 wherein the cancer cell is a prostate cancer cell.

27. The composition of any one of claims 1 through 24 wherein the target cell is a transformed cell.

28. The composition of claim 27 wherein the transformed cell comprises an expression vector.

29. The composition of claim 28 wherein the expression vector encodes a polypeptide fused in-frame to a detectable polypeptide.

30. The composition of claim 29 wherein the detectable polypeptide is a fluorescent protein.

31. The composition of claim 30 wherein the fluorescent protein is green fluorescent protein (GFP).

32. The composition of any one of claims 1 through 31 wherein more than one target polynucleotide is detected in the target cell.

33. A method of detecting modulation of transcription of a target polynucleotide comprising administering the composition of any one of claims 1 through 32 to a target cell and measuring a detectable change, wherein the administering yields an increased or decreased level of transcription of the target polynucleotide relative to a transcription level in the absence of the transcriptional regulator.

34. A method of detecting modulation of transcription of a target polynucleotide comprising administering a PN-NP and a transcriptional regulator and measuring a detectable change, wherein the transcriptional regulator increases or decreases transcription of the target polynucleotide in a target cell relative to a transcription level in the absence of the transcriptional regulator.

35. The method of claim 34 wherein the transcriptional regulator modulates transcription of the target polynucleotide to which the polynucleotide will hybridize.

36. The method of claim 34 or claim 35 wherein the transcriptional regulator is conjugated to the nanoparticle.

37. The method of any one of claims 34 through 36 wherein the transcriptional regulator is selected from the group consisting of a polypeptide, a regulator polynucleotide, and an artificial transcription factor (ATF).

38. The method of any one of claims 34 through 37 wherein the transcriptional regulator is a polypeptide.

39. The composition of claim 38 wherein the polypeptide is a hormone.

40. The method of any one of claims 34 through 39 wherein the polynucleotide is RNA, DNA or a modified polynucleotide.

41. The method of any one of claims 34 through 40, wherein the polynucleotide comprises about 5 nucleotides to about 100 nucleotides.

42. The method of any one of claims 34 through 41 , wherein the polynucleotide comprises about 10 nucleotides to about 50 nucleotides.

43. The method of any one of claims 34 through 42, wherein the polynucleotide comprises about 15 nucleotides.

44. The method of any one of claims 34 through 43 wherein the polynucleotide comprises a detectable marker.

45. The method of any one of claim 44 wherein the detectable marker is selected from the group consisting of a fluorophore, an isotope, a contrast agent, a redox active probe, a nanoparticle, a mass tag, a polypeptide, a peptide, a small molecule, an enzyme, a catalyst, an enzyme co-factor, a polynucleotide, a metal, a quantum dot and a spectroscopically active molecule.

46. The composition of claim 44 or claim 45 wherein the detectable marker is quenched when the polynucleotide is not hybridized to the target polynucleotide

47. The method of any one of claims 34 through 46 wherein the polynucleotide is double stranded.

48. The method of claim 47 wherein the polynucleotide comprises a first strand that is functional ized to the nanoparticle and a second strand that can hybridize to the first strand.

49. The method of claim 48 wherein the second strand further comprises the detectable marker.

50. The method of claim 48 or claim 49 wherein the first strand is sufficiently complementary to the target polynucleotide to hybridize to the target polynucleotide.

51. The method of claim 50 wherein the hybridization dissociates the second strand from the first strand.

52. The method of claim 51 wherein the dissociation results in a detectable change.

53. The method of any one of claims 34 through 46 wherein the polynucleotide is single stranded.

54. The method of claim 53 wherein the polynucleotide has secondary structure.

55. The method of claim 20 or claim 21 wherein hybridization of the polynucleotide to the target polynucleotide causes dissociation of the secondary structure and causes the detectable marker to move away from the nanoparticle resulting in a detectable change.

56. The method of any one of claims 34 through 55 wherein the target polynucleotide is RNA or DNA.

57. The method of any one of claims 34 through 56 wherein the target cell is selected from the group consisting of a cancer cell, a transformed cell, an in vivo target cell, a primary cell, a stem cell, a blood cell, a co-cultured cell, an immobilized cell, a suspended cell, a blood cell, a neuronal cell, and any cell derived from ectoderm, mesoderm, or endodermal lineage.

58. The method of any one of claims 34 through 57 wherein the target cell is a cancer cell.

59. The method of claim 58 wherein the cancer cell is a prostate cancer cell.

60. The method of any one of claims 34 through 59 wherein the target cell is a transformed cell.

61. The method of claim 60 wherein the transformed cell comprises an expression vector.

62. The method of claim 61 wherein the expression vector encodes a polypeptide fused in-frame to a detectable polypeptide.

63. The method of claim 62 wherein the detectable polypeptide is a fluorescent protein.

64. The method of claim 63 wherein the fluorescent protein is green fluorescent protein (GFP).

65. The method of any one of claims 34 through 64 wherein more than one target polynucleotide is detected in the target cell.

66. The method of any one of claims 33 through 65 wherein the increase or decrease is from about 2-fold to about 100,000-fold.

67. The method of any one of claims 33 through 66 wherein the increase or decrease is from about 100-fold to about 10,000-fold.

68. The method of any one of claims 33 through 66 wherein the increase or decrease is about 2-fold.

69. The method of any one of claims 33 through 68 wherein the measuring is done with a device that quantitates the detectable change.

70. The method of any one of claims 33 through 69 wherein the device is a flow cytometer.

71. The method of any one of claims 33 through 70 wherein the administration is performed in vivo.

72. The method of any one of claims 34 through 71 wherein the transcriptional regulator and the PN-NP are administered at different times.

73. The method of any one of claims 34 through 72 wherein the transcriptional regulator is administered before the PN-NP.

74. The method of any one of claims 34 through 73 wherein the transcriptional regulator is administered first, a biological sample is acquired second, and the PN-NP is administered third.

75. The method of claim 74 wherein the time between the first and second step is from about 1 minute to about 21 days.

76. The method of claim 74 wherein the time between the first and second step is one day.

77. The method of any one of claims 74 through 76 wherein the time between the second and third step is from about 1 minute to about 21 days.

78. The method of any one of claims 74 through 76 wherein the time between the second and third step is one day.

79. The method of any one of claims 33 through 70 or claims 72 through 78 which is in vitro.

80. The method of claim 79 which is a method to identify the transcriptional regulator as a candidate transcriptional regulator.

81. The method of claim 80 wherein a library of transcriptional regulators is screened for its ability to modulate the transcription of the target polynucleotide.

82. The method of claim 81 wherein an increase or decrease in the detectable change when the transcriptional regulator is administered relative to the detectable change when a different transcriptional regulator within the library is administered is indicative of a candidate transcriptional regulator.

83. The method of claim 79 which is a method to identify the target polynucleotide.

84. The method of claim 83 wherein a library of polynucleotides is screened for its ability to detect the increase or decrease in transcription of the target polynucleotide.

85. The method of claim 83 or claim 84 wherein each nanoparticle is functionalized with a polynucleotide of known sequence.

86. The method of claim 84 or claim 85 wherein an increase or decrease in the detectable change when the transcriptional regulator is administered relative to the detectable change measured when a different nanoparticle functionalized with a polynucleotide within the library is administered is indicative of identifying the target polynucleotide.

87. A kit comprising the composition of any one of claims 1 through 32.