WO2011017691A2 - DÉTECTION MOLÉCULAIRE AMÉLIORÉE D'UNE TRANSCRIPTION D'ARNm INTRACELLULAIRE AU MOYEN DE NANOPARTICULES FONCTIONNALISÉES POLYNUCLÉOTIDIQUES - Google Patents
DÉTECTION MOLÉCULAIRE AMÉLIORÉE D'UNE TRANSCRIPTION D'ARNm INTRACELLULAIRE AU MOYEN DE NANOPARTICULES FONCTIONNALISÉES POLYNUCLÉOTIDIQUES Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
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- C12Q2600/00—Oligonucleotides characterized by their use
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- 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).
- PN-NP polynucleotide functionalized nanoparticle
- FRET fluorescent resonance energy transfer
- 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.
- PN-NP probes densely surfaee-fimctionalized with nucleic acids
- PN-NP probes gold nanoparticle probes densely surfaee-fimctionalized with nucleic acids
- 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)].
- 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.
- PN-NP polynucleotide
- the compositions provided by the present disclosure are useful for the detection of targeted intracellular mRNA transcription.
- the transcriptional regulator induces transcription of the target polynucleotide to which the polynucleotide will hybridize.
- the transcriptional regulator induces transcription of the target polynucleotide to which the polynucleotide will hybridize.
- transcriptional regulator decreases transcription of the target polynucleotide to which the polynucleotide will hybridize.
- 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.
- the transcriptional regulator is selected from the group consisting of a polypeptide, a regulator polynucleotide, and an artificial transcription factor (ATF).
- ATF artificial transcription factor
- the transcriptional regulator is a polypeptide, and in a further aspect the polypeptide is a hormone.
- a polynucleotide or regulator polynucleotide of the composition comprises, in various aspects, RNA, DNA or a modified polynucleotide.
- the polynucleotide comprises about 5 nucleotides to about 100 nucleotides.
- the polynucleotide comprises about 5 nucleotides to about 100 nucleotides.
- polynucleotide comprises about 10 nucleotides to about 50 nucleotides, and in still further embodiments the polynucleotide comprises about 15 nucleotides.
- 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.
- the detectable marker is quenched when the polynucleotide is not hybridized to the target polynucleotide.
- 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.
- the second strand further comprises the detectable marker.
- the first strand is sufficiently
- the disclosure contemplates that in further aspects the dissociation results in a detectable change.
- 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.
- 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.
- the target polynucleotide is RNA or DNA.
- 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.
- the target cell is a cancer cell, and in a specific embodiment the cancer cell is a prostate cancer cell.
- 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.
- the detectable polypeptide is a fluorescent protein, and in one aspect the fluorescent protein is green fluorescent protein (GFP).
- 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.
- 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.
- the transcriptional regulator modulates transcription of the target polynucleotide to which the polynucleotide will hybridize.
- the transcriptional regulator is conjugated to the nanoparticle.
- 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.
- 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.
- the administration is performed in vivo.
- transcriptional regulator and the PN-NP are administered at different times.
- 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.
- 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.
- the administration is in vitro.
- a method is provided to identify a candidate transcriptional regulator as a transcriptional regulator.
- a library of candidate transcriptional regulators is screened for its ability to modulate the transcription of the target polynucleotide.
- 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.
- Another method provided by the disclosure is a method to identify the target polynucleotide.
- a library of polynucleotides is screened for its ability to detect the increase or decrease in transcription of the target polynucleotide.
- 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.
- kits comprising a composition as described herein.
- Figure 1 depicts the design of a nanoflare.
- Figure 2 depicts the sensitivity of a nanoflare versus control, and in the presence of a transcriptional regulator.
- 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.
- composition comprising a nanopartiele
- PN-NP polynucleotide
- the disclosure contemplates a variety of nanoparticles for use in the compositions described herein.
- a "regulatory polynucleotide” will be understood to possess all of the characteristics and features of a “polynucleotide” as defined herein.
- 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.
- 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.
- 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.
- the nanoparticle is metallic, and in various aspects, the nanoparticle is a colloidal metal.
- 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.
- 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.
- 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).
- 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,
- 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.
- nucleotide and “nucleotide” or plural forms as used herein are interchangeable with modified forms as discussed herein and otherwise known in the art.
- nucleobase which embraces naturally-occurring nucleotides as well as modifications of nucleotides that can be polymerized.
- 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-(C 3 — C 6 )-alkynyl- cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-rnethyl-4-tr- iazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases
- 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.
- 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.
- 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
- 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- brom
- 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.
- 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.
- 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.
- 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.
- 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 in length
- Polynucleotides contemplated for attachment to a nanoparticle include those which modulate expression of a gene product expressed from a target polynucleotide.
- 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.
- 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
- 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
- a single nanoparticle-binding agent composition has the ability to bind to multiple copies of the same transcript.
- a nanoparticle is provided that is functionalized with identical polynucleotides, i.e., each polynucleotide has the same length and the same sequence.
- 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.
- 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 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
- PNA peptide nucleic acid
- the sugar- backbone of a polynucleotide is replaced with an amide containing backbone.
- 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 ;
- 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.”
- 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,
- 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.
- 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.
- polynucleotides are provided with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and including -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )- 0— CH 2 - consider— CH 2 -O-N(CH 3 )- CH 2 -,— CH 2 - N(CH 3 )- N(CH 3 )- CH 2 - and ⁇ 0-N(CH 3 )- CH 2 -CH 2 - described in US Patent Nos. 5,489,677, and 5,602,240. See, for example, U.S. Patent Nos.
- Modified polynucleotides may also contain one or more substituted sugar moieties.
- 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 Ci 0 alkyl or C 2 to Cio alkenyl and alkynyl.
- Other embodiments include O[(CH 2 ) n O] m CH 3 , O(CH2) n OCH 3 , O(CH 2 ) n NH 2 ,
- 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, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a poly
- a modification includes T- methoxyethoxy (2'-0-CH 2 CH 2 OCH 3 , 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.
- modifications include 2'-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 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 ⁇ H 2 - -O— CHr-N(CH 3 ) 2 .
- the 2'-modification may be in the arabino (up) position or ribo (down) position.
- a 2'-arabino modification is 2'-F.
- 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;
- 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 (— CH 2 — )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.
- 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.
- 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,
- 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.
- polynucleotides are attached to a nanoparticle through one or more linkers.
- 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.
- the two sulfur atoms of the cyclic disulfide are close enough together so that both of the sulfur atoms attach simultaneously to the nanoparticle.
- the two sulfur atoms are adjacent each other.
- the hydrocarbon moiety is large enough to present a hydrophobic surface screening the surfaces of the
- 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;
- 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.
- 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.
- 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/cm 2 will be adequate to provide stable nanoparticle-polynucleotide compositions. Regardless, various polynucleotide densities are contemplated as disclosed herein.
- An "aging” step is incorporated into production of functionalized nanoparticles following an initial binding or polynucleotides to a nanoparticle.
- 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.
- 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.
- the salt is added all at one time or the salt is added gradually over time.
- grade 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.
- 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.
- 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.
- 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.
- 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.
- an aging step is carried out with incubation at room temperature and pH 7.0.
- 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.
- 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
- 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.
- the cooperative behavior between the nanoparticles increases the resistance of the polynucleotide to nuclease degradation.
- 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.
- 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/cm 2 will be adequate to provide stable nanoparticle-polynucleotide compositions. In some aspects, the surface density is at least 15 pmoles/cm 2 .
- Methods are also provided wherein the polynucleotide is bound to the nanoparticle at a surface density of at least 2 pmol/cm 2 , at least 3 pmol/cm 2 , at least 4 pmol/cm 2 , at least 5 pmol/cm 2 , at least 6 pmol/cm 2 , at least 7 pmol/cm 2 , at least 8 pmol/cm 2 , at least 9 pmol/cm 2 , at least 10 pmol/cm 2 , at least about 15 pmol/cm 2 , at least about 20 pmol/cm 2 , at least about 25 pmol/cm 2 , at least about 30 pmol/cm 2 , at least about 35 pmol/cm 2 , at least about 40 pmol/cm 2 , at least about 45 pmol/cm 2 , at least about 50 pmol/cm 2 , at least about 55 pmol/cm 2 , at
- 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.
- RNA polynucleotide associated with a nanoparticle 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.
- 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
- PN-NP compositions that are useful for hybridizing to a target polynucleotide.
- the compositions are used to detect and/or quantify expression of the target polynucleotide.
- the compositions are used for gene regulation.
- the composition is useful both for quantitating a target polynucleotide and regulating the expression of the same or a different polynucleotide.
- Gene regulatory activity is also, in various embodiments, achieved through the use of a regulatory polynucleotide which can modulate the transcription of a mRNA.
- the PN-NP is functionalized with DNA.
- the DNA is double stranded, and in further embodiments the DNA is single stranded.
- 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).
- siRNA small interfering RNA
- 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.
- 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)).
- mRNA messenger RNA
- 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.
- 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%.
- 19 bases out of 21 bases may be base-paired.
- a polynucleotide used in the methods need not be 100% complementary to a desired target nucleic acid to be
- 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).
- BLAST programs Basic Local Alignment Search Tools
- PowerBLAST programs known in the art
- 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).
- a polynucleotide is targeted for detection and quantitation of a relative amount of the target polynucleotide.
- 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.
- 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).
- the target polynucleotide is in a target cell.
- 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.
- the cancer cell is found in a human cancer.
- 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.
- the target cell is a transformed cell.
- the transformed cell comprises an expression vector, and in further aspects the expression vector encodes a polypeptide fused in-frame to a detectable polypeptide.
- any detectable polypeptide known in the art is useful in the methods of the disclosure, and in some aspects is a fluorescent protein.
- the fluorescent protein is selected from the list of proteins in Table 1, below.
- the disclosure contemplates that more than one target polynucleotide is detected in the target cell.
- 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.
- stop codon region and
- translation termination codon region refers 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.
- 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).
- 5'UTR 5' untranslated region
- 3'UTR the 3' untranslated region
- 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.
- the nucleic acid is RNA transcribed from genomic DNA.
- the nucleic acid is an animal nucleic acid, a plant nucleic acid, a fungal nucleic acid, including yeast nucleic acid.
- the target nucleic acid is a RNA transcribed from a genomic DNA sequence.
- the target nucleic acid is a mitochondrial nucleic acid.
- the nucleic acid is viral genomic RNA, or RNA transcribed from viral genomic DNA.
- a target polynucleotide sequence is a microRNA.
- MicroRNAs 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
- RNAi silencing complex [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.
- RISC RNAi silencing complex
- 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.
- the target polynucleotide is microRNA-210.
- Methods for inhibiting gene product expression 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.
- methods provided embrace those which results in essentially any degree of inhibition of expression of a target gene product.
- 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.
- Methods are provided wherein presence of a polynucleotide is detected by an observable change.
- 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.
- a fluorescence microscope can detect the presence of a fluorophore that is conjugated to a polynucleotide, which has been functionalized on a nanoparticle.
- markers contemplated will include any of the fluorophores described herein as well as other detectable markers known in the art.
- 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.
- 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-Hyd
- 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 332
- 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,
- 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.
- 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.
- 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).
- small molecule 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.
- 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 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.
- 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.
- compositions and methods herein are useful in the practice of nano flare technology.
- 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.
- PN-NP polynucleotide
- the polynucleotide comprises a detectable marker, wherein the detectable marker is quenched when the polynucleotide is not hybridized to a target
- 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.
- the second strand further comprises the detectable marker.
- 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.
- the polynucleotide that is attached to the nanoparticle is single stranded.
- 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.
- the target polynucleotide is, in some aspects, RNA or DNA.
- 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.
- PN-NP polynucleotide
- 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.
- 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.
- 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.
- the polypeptide is a transcription factor.
- a transcription factor is modular in structure and contain the following domains.
- DNA-binding domain 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.
- TAD Trans-activating domain
- AFs activation functions
- 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.
- the DBD and signal-sensing domains may, in some aspects, reside on separate proteins that associate within the transcription complex to regulate gene expression.
- Transcription factors are often classified based on the sequence similarity and hence the tertiary structure of their DNA-binding domains.
- Table 2 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.
- MyoD Myogenic transcription factors
- Ubiquitous factors includes TFIlIA, SpI
- ⁇ 3.1.1 Family Homeo domain only; includes Ubx
- TEA TEADl, TEAD2, TEAD3, TEAD4
- RHR ReI homology region
- NFAT Nuclear Factor of Activated T-cells
- the transcription factor is a regulator polynucleotide.
- the polynucleotide is RNA, and in further aspects the regulator polynucleotide is a noncoding RNA (ncRNA).
- the noncoding RNA interacts with the general transcription machinery, thereby inhibiting transcription [Goodrich et al., Nature Reviews MoI Cell Biol 7: 612-616 (2006)].
- 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.
- 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.
- 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.
- a regulatory polynucleotide is an aptamer.
- ATFs artificial transcription factors
- 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.
- a transcription regulator is, in certain aspects, a stimulus encountered by a cell.
- stimuli that can function as a transcriptional regulator are temperature, radiation,and a compound that may be metabolized or not metabolized.
- 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.
- 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).
- ionizing radiation for example and without limitation, ultraviolet (UV), X-ray and gamma ray
- non-ionizing radiation for example and without limitation, neutron, electromagnetic, and thermal.
- compound refers to a substance that may optionally be derivatized, or any other low molecular weight organic substance, either natural or synthetic.
- 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.
- 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.
- 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.
- the detectable change is a fluorescent signal.
- any device that can quantitate the fluorescent signal is contemplated for use.
- a flow cytometer may be used.
- 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.
- the transcriptional regulator is administered first, a biological sample is acquired second, and the PN-NP is administered third.
- 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.
- 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,
- 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,
- the method is to identify the transcriptional regulator as a candidate transcriptional regulator.
- a library of transcriptional regulators is screened for its ability to modulate the transcription of the target polynucleotide.
- the library is an expression library.
- the expression library is generated from a cDNA library.
- the transcriptional regulator is a known regulator of transcription, and in some embodiments the transcriptional regulator is an unknown or hypothesized regulator of transcription.
- a candidate transcriptional regulator is contacted with a target cell in vitro.
- the target cell is then contacted with a composition comprising a PN-NP as described herein.
- the polynucleotide that is attached to the nanoparticle is double stranded.
- the first strand of the double stranded polynucleotide is attached to the nanoparticle and is complementary to a target polynucleotide.
- 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.
- 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.
- 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.
- a second transcriptional regulator is contacted with a target cell.
- the second transcriptional regulator is added concurrently with the first transcriptional regulator.
- the second transcriptional regulator is added after the first transcriptional regulator is added.
- 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
- the disclosure also contemplates methods to identify the target polynucleotide.
- 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.
- a double stranded polynucleotide comprising a known sequence is functionalized to a nanoparticle, creating a first PN-NP.
- 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.
- each nanoparticle is fluorescence.
- the methods provide for the identification of a mRNA that is regulated by a given transcriptional regulator.
- the mRNA is increased, and in some aspects the mRNA is decreased.
- the present disclosure also provides methods for in vivo applications.
- a composition of the disclosure is used to detect a target cell in a tissue.
- the tissue is superficial, and in further aspects the tissue is breast tissue.
- the target cell is a cancer cell, and in some aspects the target cell is a breast cancer cell.
- the disclosure provides a method to assess a temporal difference between transcription and translation of a detectable target polypeptide of interest.
- the target cell is a transformed cell.
- the transformed cell has been transformed with a vector.
- the vector encodes a target polypeptide.
- 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).
- 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.
- the transcriptional regulator increases transcription of the mRNA that encodes the detectable polypeptide.
- 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.
- 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.
- 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.
- the composition is administered with an embolic agent.
- 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.
- 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.
- 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.
- compositions of the present disclosure comprise ratios of PN-NPs and embolic agent.
- Ratio 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.
- ratio can be determined the ratio to be used in the compositions of the present disclosure.
- 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.
- 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,
- the PN-NPs are approximately 1 nanomolar (nM) to 10 micromolar ( ⁇ M), while the embolic agent is in the ⁇ M 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.
- a composition of the present disclosure further comprises a therapeutic agent.
- the therapeutic agent is associated with the nanoparticle.
- the therapeutic agent is co-administered with the PN-NP, but is separate from the PN-NP composition.
- 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.
- multiple therapeutic agents in multiple combinations can be administered at any time before, during or after administration of the PN-NP composition.
- repeated administration of a therapeutic agent is also contemplated.
- 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.
- 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.
- 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
- 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 ⁇ l , glial cell line-derived neutrophic factor receptor ⁇ 2, growth related protein, growth related protein ⁇ , growth related protein ⁇ , growth related protein ⁇ , growth related protein ⁇ , 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 ⁇ , nerve growth factor nerve growth factor receptor, neuro
- transforming growth factor ⁇ transforming growth factor ⁇ , transforming growth factor ⁇ l, transforming growth factor ⁇ l .2, transforming growth factor ⁇ 2, transforming growth factor ⁇ 3.
- transforming growth factor ⁇ 5 latent transforming growth factor ⁇ l, transforming growth factor ⁇ binding protein I, transforming growth factor ⁇ binding protein II, transforming growth factor ⁇ 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.
- 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, cylating agents, such as 5-
- 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
- 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
- antiestrogen such as tamoxifen
- androgens including testosterone propionate and fluoxymesterone/equivalents
- antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide
- non-steroidal antiandrogens such as flutamide
- 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).
- a therapeutic agent as described herein is attached to the nanoparticle.
- 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).
- AR functional androgen receptor
- 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.
- DHT dihydrotestosterone
- PSA Prostate specific antigen
- 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)].
- LnCaP cells were propagated in RPMl- 1640 medium supplemented with 10% fetal bovine serum (FBS).
- FBS fetal bovine serum
- 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).
- CSM charcoal stripped serum
- 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.
- 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.
- a transcriptional regulator can be used to augment the target-specific flare signal generated from targeted cell types.
- 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.
- labeled for example and without limitation, GFP
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Abstract
La présente invention concerne des compositions et des procédés permettant une meilleure détection moléculaire de la transcription de ARNm intracellulaire ciblée au moyen d'une nanoparticule fonctionnalisée polynucléotidique (PN-NP).
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US10973927B2 (en) | 2017-08-28 | 2021-04-13 | The Chinese University Of Hong Kong | Materials and methods for effective in vivo delivery of DNA nanostructures to atherosclerotic plaques |
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BAPTISTA, P. ET AL.: 'Colorimetric detection of eukaryotic gene expression with DNA-derivatized gold nanoparticles.' J. BIOTECHNOL. vol. 119, 2005, pages 111 - 117 * |
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US10973927B2 (en) | 2017-08-28 | 2021-04-13 | The Chinese University Of Hong Kong | Materials and methods for effective in vivo delivery of DNA nanostructures to atherosclerotic plaques |
CN111551724A (zh) * | 2020-04-03 | 2020-08-18 | 西北农林科技大学 | 一种荧光探针、检测四环素的方法及应用 |
CN111551724B (zh) * | 2020-04-03 | 2023-06-09 | 西北农林科技大学 | 一种荧光探针、检测四环素的方法及应用 |
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