WO2005021712A2 - Modified molecular beacons - Google Patents
Modified molecular beacons Download PDFInfo
- Publication number
- WO2005021712A2 WO2005021712A2 PCT/US2004/020232 US2004020232W WO2005021712A2 WO 2005021712 A2 WO2005021712 A2 WO 2005021712A2 US 2004020232 W US2004020232 W US 2004020232W WO 2005021712 A2 WO2005021712 A2 WO 2005021712A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- molecular beacon
- nucleic acid
- cells
- cell
- transduction domain
- Prior art date
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- QOFZZTBWWJNFCA-UHFFFAOYSA-N texas red-X Chemical compound [O-]S(=O)(=O)C1=CC(S(=O)(=O)NCCCCCC(=O)O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 QOFZZTBWWJNFCA-UHFFFAOYSA-N 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- JADVWWSKYZXRGX-UHFFFAOYSA-M thioflavine T Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C1=[N+](C)C2=CC=C(C)C=C2S1 JADVWWSKYZXRGX-UHFFFAOYSA-M 0.000 description 1
- 238000006177 thiolation reaction Methods 0.000 description 1
- NJRXVEJTAYWCQJ-UHFFFAOYSA-N thiomalic acid Chemical compound OC(=O)CC(S)C(O)=O NJRXVEJTAYWCQJ-UHFFFAOYSA-N 0.000 description 1
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 210000003956 transport vesicle Anatomy 0.000 description 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 230000009447 viral pathogenesis Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 239000008136 water-miscible vehicle Substances 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/006—Biological staining of tissues in vivo, e.g. methylene blue or toluidine blue O administered in the buccal area to detect epithelial cancer cells, dyes used for delineating tissues during surgery
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
Definitions
- the disclosure is generally related to molecular beacons, for example, dual-labeled oligonucleotide probes with a fluorophore at one end and a quencher at the other end, more particularly to molecular beacons modified to translocate across membranes.
- Probes must be able to recognize the target with high specificity, convert target recognition directly into a measurable signal with high signal-to-background ratio, and allow for differentiation between true and false-positive events (Molenaar, C. et al. (2001 j Nucleic Acids Res. 29, E89-9). Probes must also be delivered into living cells with high efficiencies. Among the technologies currently under development for living cell gene detection and quantification, the most promising one is perhaps molecular beacons. Molecular beacons are dual-labeled antisense oligonucleotide (ODN) probes with a fluorophore at one end and a quencher at the other end (Tyagi, S., Kramer, F.R. (1996) Nat.
- ODN dual-labeled antisense oligonucleotide
- molecular beacons are designed to form a stem- loop (hairpin) structure in the absence of complementary target so that fluorescence of the fluorophore is quenched. Hybridization with target mRNA opens the hairpin and physically separates the reporter form the quencher, allowing a fluorescence signal to be emitted upon excitation.
- molecular beacons enable a homogenous assay format where background is low without the need to wash away free probes.
- microinjection Sokol, D.L. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11538-11543) and electroporation (Yin, D., Tang, J.G. (2001) FEBS Lett. 495, 16-20) are invasive and may cause severe damage to cells. Further, microinjection is labor-intensive and only practical for studying a small number of cells.
- compositions and methods for detecting, real time imaging and/or quantifying a target nucleic acid with high signal to background ratio provide compositions that can non-invasively detect or quantify a target nucleic acid in a living cell.
- nucleic acid reporters can be modified to translocate across membranes and thereby avoid invasive delivery techniques such as microinjection or poration of membranes.
- the present disclosure provides modified nucleic acid reporters, including but not limited to, molecular beacons operably linked to a protein transduction domain.
- the nucleic acid reporters linked to a protein transduction domain can be further modified to be operably linked to a targeting signal, including for example, a nuclear localization signal.
- compositions can quickly and efficiently enter living cells without the need of any other delivery reagent, and enter specific membrane bound organelles or localize to specific intracellular regions.
- a particular aspect provides molecular beacons conjugated to a protein transduction domain (PTD) peptide TAT-1 providing a multifunctional probe that can enter into living cells with nearly 100% efficiencies, fast (-30 min) delivery kinetics, and the ability to localize in cell cytoplasm.
- PTD protein transduction domain
- the disclosed nucleic acid reporters function well (with much better signal to background ratio) for mRNA detection in living cells as compared with the results of in situ hybridization. This nucleic acid reporter design provides a TKHR DOCKET NO. 820701.1135
- nucleic acid reporter having a streptavidin- biotin linkage.
- the nucleic acid reporter includes a modified nucleotide, for example biotin-dT, in the quencher arm of the stem.
- the biotin moiety can be linked to the modified nucleotide via a linker, for example an alkyl linker.
- a biotin-modified PTD is linked to the biotin modified nucleic acid reporter through a streptavidin molecule, which has four biotin-binding sites.
- the disclosed nucleic acid reporter includes a thiol- maleimide linkage in which the nucleic acid reporter is modified by adding a thiol group which can react with a maleimide group placed to the C terminus of the PTD to form a direct, stable linkage.
- a nucleic acid reporter linked to a PTD or targeting signal with a cleavable disulfide bridge can be modified by adding a cysteine residue at the C terminus which forms a disulfide bridge with the thiol-modified nucleic acid reporter. This disulfide bridge design allows the PTD to be cleaved from the nucleic acid reporter by the reducing environment of the cytoplasm.
- the delivery peptide sequence may be synthesized along with the nucleic acid reporter, for example, in the case of a PNA probe, where both the delivery peptide and the nucleic acid probe sequence can be generated using a single peptide.
- the linkage between the delivery peptide and the nucleic acid probe can be tailored to allow for specific cleavage, for example by using a reducing disulfide bridge or using enzymatic cleavage sites in the linkage.
- nucleic acid reporter with a delivery peptide
- the delivery vehicle itself may be dendrimer-based or lipid-based such as liposomes/polymeric or any combination of the above, in which nucleic acid probes are packaged inside the delivery vehicle, with delivery peptides attached to the surface of such construct.
- the attached delivery peptides may allow TKHR DOCKET NO. 820701.1135
- compositions and methods to detect a target nucleic acid for example nuclear RNA
- a target nucleic acid for example nuclear RNA
- This approach combines the ability of site directed delivery, for example, nucleus-specific delivery of probes using a toxin-based reversible permeablization of cells and a targeting signal, for example NLS peptide, and the sensitive detection of target nuclear RNA using molecular beacons.
- nucleic acid reporters having both a targeting signal and cell permeating peptides (e.g., Tat, poly- Arginine) for faster and more efficient delivery of probes into specific regions of a cell, for example the cell nucleus.
- Still another aspect provides a method for determining the effects of an agent on gene expression in a host by contacting one or more cells of the host with an agent, contacting the cells with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon is specific for a target nucleic acid, irradiating the cells with an exciting amount of radiation, detecting the electromagnetic emissions in response to the exciting amount of radiation, and comparing the emissions in cells treated with the agent to emissions from a control sample.
- a targeting signal e.g., Tat, poly- Arginine
- Figures 1A-D are schematic illustrations of three exemplary embodiments for linking the delivery peptide to molecular beacons.
- Figure 2A is a line graph of normalized fluorescence intensity as a function of time for unmodified molecular beacons, and for the three types of peptide-Iinked molecular beacons.
- Figure 2B is a bar graph of signal-to-background ratios of probe-target hybridization for exemplary peptide-Iinked molecular beacons linked by different conjugation methods.
- Figures 3A-G are fluorescence micrographs of HDF cells incubated with exemplary molecular beacons. TKHR DOCKET NO. 820701.1135
- Figures 4A and B are fluorescence in situ hybridization micrographs of HDF cells showing GAPDH mRNA.
- Figures 5A and B are fluorescence micrographs showing the detection of survivin mRNA in live HDF and MiaPaca-2 cells.
- Figures 6A-F are fluorescence micrographs of HDF cells comparing cellular delivery of molecular beacons using conventional techniques.
- Figures 7A-D are fluorescence micrographs of HDF cells showing nuclear delivery of an exemplary embodiment of the disclosed nucleic acid reporters.
- Figure 8 is a panel of fluorescence micrographs of HDF cells showing nuclear and cytoplasmic localization of U1 snRNA using a U1 snRNA targeted molecular beacon linked with Tat-1 peptide.
- amino acid residue sequences are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus.
- amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y),
- Variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties.
- a TKHR DOCKET NO. 820701.1135 typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
- a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties. In making such changes, the hydropathic index of amino acids can be considered.
- hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
- Those indices are: isoleucine (+4.5); valine (+4.2); leucine • (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn TKHR DOCKET NO. 820701.1135
- polypeptide defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred. Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments.
- hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ⁇ 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
- amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide.
- substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.
- amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (lie: Leu, Val), (Leu: lie, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu).
- Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set TKHR DOCKET NO. 820701.1135
- embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.
- Identity is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. "Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
- the percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST).
- analysis software i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.
- Needelman and Wunsch J. Mol. Biol., 48: 443-453, 1970
- NBLAST e.g., NBLAST, and XBLAST
- the default parameters are used to determine the identity for the polypeptides of the present invention.
- a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%.
- Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-
- the term "purified" and like terms relate to the isolation of a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components normally associated with the molecule or compound in a native environment.
- the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
- the term “treating” includes alleviating the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
- "Operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence, and an organelle localization sequence operably linked to protein will direct the linked protein to be localized at the specific organelle.
- “Localization Signal or Sequence or Domain” or “Targeting Signal or Sequence or Domain” are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, or intracellular region.
- the signal can be polynucleotide, polypeptide, or carbohydrate moiety or can be an organic or inorganic compound sufficient to direct an attached molecule TKHR DOCKET NO. 820701.1135
- organelle localization signals include nuclear localization signals known in the art and other organelle localization signals known in the art such as those provided in Tables 1 and 2 and described in Emanuelson et al., Predicting Subcellular Localization of Proteins Based on Their N-terminal Amino Acid Sequence. Journal of Molecular Biology.
- Organelle localization signals of the present invention can have 80 to 100% homology to the sequences in Tables 1 and 2.
- the organelle localization signals include signals having or conferring a net charge, for example a positive charge.
- PTD Protein Transduction Domain
- a PTD attached to another molecule facilitates the molecule traversing membranes, for example going from extracellular space to intracellular space, or cytosol to within an organelle.
- Exemplary PTDs include but are not limited to HIV TAT YGRKKRRQRRR (SEQ ID NO. 1) or RKKRRQRRR (SEQ ID NO.
- exogenous DNA or “exogenous nucleic acid sequence” or “exogenous polynucleotide” refers to a nucleic acid sequence TKHR DOCKET NO. 820701.1135
- the introduced exogenous sequence is a recombinant sequence.
- the term "transfection” refers to the introduction of a nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus or chloroplast.
- the nucleic acid may be in the form of naked DNA or RNA, associated with various proteins or the nucleic acid may be incorporated into a vector.
- vector is used in reference to a vehicle used to introduce a nucleic acid sequence into a cell.
- a viral vector is virus that has been modified to allow recombinant DNA sequences to be introduced into host cells or cell organelles.
- organelle refers to cellular membrane bound structures such as the chloroplast, mitochondrion, and nucleus.
- organelle includes natural and synthetic organelles.
- non-nuclear organelle refers to any cellular membrane bound structure present in a cell, except the nucleus.
- polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
- the terms "nucleic acid,” “nucleic acid sequence,” or “oligonucleotide” also encompasses a polynucleotide as defined above.
- polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the strands in such regions may be from the same molecule or from different molecules.
- the regions may include all of one or more of the molecules, but more typically TKHR DOCKET NO. 820701.1135
- polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
- DNAs or RNAs with backbones modified for stability or for other reasons are examples of DNAs or RNAs with backbones modified for stability or for other reasons.
- polynucleotides as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
- polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
- Oxygenucleotide(s) refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and double- stranded DNAs, among others.
- Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength.
- fluorophores include, but are not limited to, 1 ,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofIuorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5- FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5- Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7- Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4- methylcoumarin; 9-Amino-6-chloro-2-methoxyacrid
- AFPs - AutoFluorescent Protein - see sgGFP, sgBFP; Alexa Fluor 350TM; Alexa Fluor 430TM; Alexa Fluor 488TM; Alexa Fluor 532TM; Alexa Fluor 546TM; Alexa Fluor 568TM; Alexa Fluor 594TM; Alexa Fluor 633TM; Alexa Fluor 647TM; Alexa Fluor 660TM; Alexa Fluor 680TM; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B;
- Phorwite AR Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R;
- PKH67 PMIA; Pontochrome Blue Black; POPO-1 ; POPO-3; PO-PRO-1 ; PO- PRO-3; Primuline; Procion Yellow; Propidium lodid (PI); PyMPO; Pyrene;
- Rhodamine 110 Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G;
- Rhodamine B Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine
- Rhodamine Red Rhodamine WT; Rose Bengal; R-phycocyanine;
- PE R-phycoerythrin
- rsGFP R-phycoerythrin
- S65A S65C
- S65L S65T
- Sapphire GFP R-phycoerythrin
- SITS Tin Isothiosulphonic Acid
- SNARF calcein; SNARF1 ; Sodium Green; SpectrumAqua; SpectrumGreen;
- SpectrumOrange Spectrum Red; SPQ (6-methoxy-N-(3- sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 11 ; SYTO 12; SYTO 13; SYTO 14; SYTO 15;
- nucleic acid reporter means a compound, molecule, or polymer that specifically detects or can be used to detect a specific nucleic acid sequence.
- exemplary nucleic acid reporters include, but are not limited to, labeled polynucleotides, for example labeled antisense polynucleotides.
- the nucleic acid reporters can be single or multistranded.
- Exemplary nucleic acid reporters also include molecular beacons.
- molecular beacon generally means dual-labeled antisense oligonucleotide (ODN) probes.
- Exemplary backbones include, but are not limited to, unmodified oligodeoxyribonucleotides, 2'-O-methyl oligoribonucleotides, phosphorothioate oligodeoxynucleotides, and oligodeoxynucleotides containing methylphosphonate, phosphoramidate, methythiophosphonate, or methythiophosphotriester.
- Exemplary labels include, but are not limited to, a fluorophore at one end and a quencher at the other end.
- a quencher can be any molecule or substance that reduces or eliminates the detectable single from the other label.
- Exemplary quenchers include but are not limited to, metal particles less than about 100 nm in diameter, typically less than about 10 nm in diameter, such as gold or silver or other dyes including but not limited to DABCYL (4- ⁇ [4- (dimethylamino)phenyl]diazenyl ⁇ benzoyl, BHQ-1, and BHQ-2.
- the quenchers can have no fluorescence themselves. Such quenchers are typically referred to as dark quenchers. Alternatively, the quenchers can be fluorescent themselves.
- nucleic acid reporter constructs that can non-invasively report the presence of a target nucleic acid either in vivo as well as in vitro.
- Non-invasive delivery refers to delivery without significant physical damage to a cell or tissue using for example, a mechanical device such as needle or other mechanical or physical means such as poration that may cause significant cellular or tissue damage.
- Embodiments of the disclosure provide nucleic acid reporters modified with a protein transduction domain to facilitate translocation of the nucleic acid reporter from the extracellular space to intracellular space.
- the nucleic acid reporter can translocate to any region of the interior of a cell including the interior of membrane bound organelles such as the nucleus, mitochondrion, or chloroplast.
- any membrane organelle is included within the scope of the disclosure. It will be further appreciated that any cell having a membrane is within the scope of this disclosure including, but not limited to animal cells such as human cells, or plant cells.
- Other embodiments provide nucleic acid reporters that are further modified to include targeting signals such as intracellular targeting signals, organelle targeting signals, cellular targeting signals, tissue targeting signals, or organ targeting signals. Generally, such targeting signals are known in the art. Targeting signals include, but are not limited to, amino acid or nucleic acid sequences, as will as lipids or carbohydrates that target the nucleic acid reporter to a specific cell, tissue, organ or intracellular region of a cell.
- targeting can be accomplished through receptor: ligand interactions or by using a targeting signal that modifies the polarity, hydrophobicity, hydrophilicity, or any combination thereof, of the nucleic acid reporter.
- the targeting signal can confer a positive or negative charge to the nucleic acid reporter as needed.
- Exemplary targeting signals include, but are not limited to, growth factors, growth factor receptors, antibodies or fragments thereof specific for extracellular eptiopes, carbohydrates, lipids, peptides, TKHR DOCKET NO. 820701.1135
- nucleic acid reporters can include a PTD, a targeting signal, or a combination thereof.
- the PTD, the targeting signal, or both can be releaseably linked to the nucleic acid reporter, for example through cleavable bonds, so that the nucleic acid reporter is released from the PTD or targeting signal when the nucleic acid reporter arrives at a desired location.
- the PTD is cleaved when the nucleic acid reporter enters the cytosol and the targeting signal remains linked to the nucleic acid reporter.
- the targeting signal is removed from the nucleic acid reporter when the nucleic acid reporter reaches the desired location.
- the disclosed nucleic acid reporters include, but are not limited to, labeled oligonucleotides such as molecular beacons.
- Molecular beacons are polynucleotides generally having a pair of labels, for example a label at each end of the molecule.
- the polynucleotides include a sequence that is complementary to a target nucleic acid (target recognition sequence). The degree of complementarity is sufficient to enable sequence specific interactions between the nucleic acid reporter and the target nucleic acid.
- Some embodiments can detect single base differences or single nucleotide polymorphisms in a target nucleic acid.
- the disclosed nucleic acid reporters may have target recognition sequences 7-140 nucleotides, but it will be appreciated that the target recognition sequence can be of any length that permits sequence specific association with the target nucleic acid.
- the sequences flanking the target recognition sequences form a stem hybrid, or "stem duplex" 3-25 nucleotides in length.
- Modified nucleotides and modified nucleotide linkages may be used to produce the disclosed nucleic acid reporters and are described more fully below. Such modifications are known in the art and include modifications to increase resistance to the enzymatic degradation.
- labile phosphodiester or phosphoester linkages may be replaced with more stable TKHR DOCKET NO. 820701.1135
- nucleic acid reporters may include, for example, peptide nucleic acid (“PNA") linkages.
- PNA peptide nucleic acid
- the disclosed compositions and target nucleic acids can be DNA, RNA including rRNA, nuclear RNA, mRNA, cDNA, genomic DNA, or combinations thereof.
- Modified Nucleotide Linkages Some embodiments provide nucleic acid reporters including a plurality of nucleic acids or oligonucleotides containing modified backbones or non- natural intemucleoside linkages. Exemplary modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
- modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonat.es, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
- Some oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
- Various salts, mixed salts and free acid forms are also included.
- Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.
- oligonucleotide backbones do not include a phosphorus atom therein and have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
- morpholino linkages formed in part from the sugar portion of a nucleoside
- siloxane backbones sulfide, sulfoxide and sulfone backbones
- formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
- riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methytenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
- nucleic acid reporters containing oligonucleotide mimetics in which both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
- the base units are maintained for hybridization with an appropriate nucleic acid target compound.
- an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
- PNA peptide nucleic acid
- the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
- the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
- PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al. (1991) Science 254:1497-1500.
- nucleic acid reporters having oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH 2 -NH-O-CH 2 -, -CH 2 -N(CH 3 )- O-CH 2 - [known as a methylene (methylimino) or MMI backbone], -CH 2 -O- N(CH 3 )-CH 2 - -CH 2 -N(CH 3 )-N(CH 3 )-CH2- and -O-N(CH 3 )-CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as -O-P-O- CH 2 -] of the above referenced U.S.
- the nucleic acid reporters may comprise modified oligonucleotides containing one or more substituted sugar moieties.
- modified oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to C 10 alkyl or C 2 to C-io alkenyl and alkynyl.
- oligonucleotides comprise one of the following at the 2' position: Ci to C-io lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, 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 the nucleic acid reporter and other substituents having similar properties.
- Another modification includes 2'-methoxyethoxy (2'-O- CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al. (1995) Helv. Chim. Acta, , 78, 486-504) i.e., an alkoxyalkoxy group.
- a further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, and I'll TKHR DOCKET NO. 820701.1135
- dimethylaminoethoxyethoxy also known in the art as 2'-O-dimethyl-amino- ethoxy-ethyl or 2'-DMAEOE
- the 2'-modification may be in the arabino (up) position or ribo (down) position.
- An exemplary 2'-arabino modification is 2'-F.
- Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
- Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
- a further modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
- the linkage is preferably a methelyne (-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 U.S. Patent No.
- Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
- nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
- Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and TKHR DOCKET NO. 820701.1135
- nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1 H-pyrimido[5,4-b][1 ,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1 H-pyrimido[5,4-b][1 ,4]benzothiazin-2(3H)-one), G- clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)- H-pyrimido[5,4-b][1 ,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
- Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone.
- Further nucleobases include those disclosed in U.S. Pa No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613, and those disclosed by Sanghvi, Y.
- nucleobases may be particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure.
- nucleobases include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, TKHR DOCKET NO. 820701.1135
- compositions that contain a reporter moiety whose reporting ability changes depending on whether the nucleic acid reporter is bound to its complement or target nucleic acid.
- some nucleic acid reporters are designed to take advantage of quenching by fluorescence resonance energy transfer (FRET) or LRET (luminescence resonance energy transfer) to detect and report binding to target molecules.
- FRET fluorescence resonance energy transfer
- LRET luminescence resonance energy transfer
- FRET is a highly distance-dependent interaction between a fluorescent reporter dye in an excited state and a quencher in its ground state. Energy is transferred from one molecule (the fluorophore) to the other (the quencher) without the emission of a photon. Additional examples of reporting abilities may also include the use of interchelating dyes conjugated to nucleic acid probes, such that there is significant increase/decrease in signal upon binding to target nucleic acid. Further examples may include the use of reporters which change the polarization state of emission energy upon binding to target nucleic acid. As noted above, some embodiments of the disclosed nucleic acid reporters include a pair of labels.
- pairs of labels include, but are not limited to, at least one donor/quencher pair, for example a dye pair, or a dye and a non-dye quencher.
- the pair of labels typically includes a fluorescent donor dye and a quencher for the donor fluorophore.
- TKHR DOCKET NO. 820701.1135
- the labels are linked to a sequence or structure in the nucleic acid reporter which does not hybridize directly to the target sequence.
- the disclosed nucleic acid reporters include any nucleic acid sequence or structure which can be labeled such that the presence of its complement or target nucleic acid indicates the presence of the target sequence.
- the nucleic acid reporter moiety is labeled with a donor/quencher dye pair such that donor fluorescence is quenched prior to the sequence specific binding of the nucleic acid reporter to the target nucleic acid, and such that quenching of donor fluorescence is reduced as an indication of the presence of the target.
- the nucleic acid reporter may have a secondary structure such as a stem-loop (or hairpin) as described in U.S. Pat. No.
- the secondary structure is labeled such that the donor and quencher are in close proximity when the secondary structure is folded, resulting in quenching of donor fluorescence.
- the secondary structure In the presence of a target, the secondary structure is unfolded in a target-dependent reaction so that the distance between the donor and quencher is increased. This decreases quenching and produces an increase in donor fluorescence which can be detected as an indication of the presence of the target sequence.
- the fluorophore and quencher molecules are typically less than about 100 A apart.
- the absorption spectrum of the quencher overlaps with the emission spectrum of the fluorophore.
- donor/quencher dye pairs known in the art are useful in some embodiments of the present invention. These include, for example, fluorescein isothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TRITC), FITC/Texas RedTM (Molecular Probes), FITC/N- hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS)/FITC, FITC/Rhodamine X, FITC/tetramethylrhodamine (TAMRA), and others.
- FITC fluorescein isothiocyanate
- TRITC FITC/tetramethylrhodamine isothiocyanate
- donor/quencher pairs can be selected so that the emission wavelengths of the donor fluorophore overlap the excitation wavelengths of the quencher, i.e., there must be TKHR DOCKET NO. 820701.1135
- DABYL P-(dimethyl aminophenylazo) benzoic acid
- EDANS 5-(2'-aminoethyl) aminonaphthalene
- Any dye pair which produces fluorescence quenching in the disclosed nucleic acid reporters are suitable for use in the disclosed methods, regardless of the mechanism by which quenching occurs.
- Terminal and internal labeling methods are also known in the art and may be routinely used to link the donor and quencher dyes at their respective sites in the nucleic acid reporter.
- Embodiments of the present disclosure also include nucleic acid reporters operably linked to a protein transduction domain.
- PTDs protein transduction domains
- CPPs cell penetrating peptides
- PTDs Although several of PTDs have been documented, the two most commonly employed PTDs are derived from TAT protein of HIV (Frankel and Pabo (1988) Cell 55(6): 1189-93) and Antennapedia transcription factor from Drosophila, whose PTD is known as Penetratin (Derossi et al.(1994) J Biol Chem. 271 (30):18188-93).
- the Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices.
- Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia.(Fenton et al. (1998) J Immunol Methods 212(1):41-8).
- TAT protein consists of 86 amino acids and is involved in the replication of HIV-1.
- the TAT PTD consists of an 11 amino acid sequence domain (residues 47 to 57; YGRKKRRQRRR (SEQ ID NO. 1) of the parent protein that appears to be critical for uptake (Vives et al. (1997) J Biol Chem. 272(25): 16010-7).
- TKHR DOCKET NO. 820701.1135
- the basic domain Tat(49-57) or RKKRRQRRR (SEQ ID NO. 2) (Wender et al. (2000) Proc Natl Acad Sci U S A. 97(24): 13003-8) has been shown to be a PTD.
- TAT has been favored for fusion to proteins of interest for cellular import.
- modifications to TAT including substitutions of Glutatmine to Alanine, i.e., Q to A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al. (2000) Proc Natl Acad Sci U S A. 97(24): 13003-8) to up to 33 fold in mammalian cells. (Ho et al. (2001) Cancer Res.
- PTDs that are cationic or amphipathic.
- exemplary PTDs include but are not limited to poly-Arg - RRRRRRR (SEQ ID NO. 3); PTD-5 - RRQRRTSKLMKR (SEQ ID NO. 4); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO. 5); KALA - WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO.
- the disclosed nucleic acid reporters can have the protein transduction domain linked directly or indirectly to the reporter composition.
- One embodiment provides a nucleic acid reporter having a modified monomer, such as a nucleotide, to facilitate linking chemistries.
- the monomer can be modified with a reactive group such as an amine, carbonyl, carboxyl, and thiol or have a biotin group attached to the nucleotide.
- modified monomers are known in the art, and are commercially available and include modified nucleotides such as dT (Link Technologies, Scotland, UK).
- the protein transduction domain can be directly linked using conventional linking chemistry to the modified nucleotide or can be indirectly linked to the amine modified nucleotide using a linker or spacer group.
- the linker group can be linked to the modified nucleotide at one end, and linked to the PTD on the other end.
- the protein transduction domain is operably linked to the nucleic acid reporter using a streptavidin-biotin linkage.
- the nucleic acid reporter includes a modified monomer, for example biotin-dT, in the region of the nucleic acid reporter that is not complementary to the target nucleic acid, for example to the quencher arm of the stem.
- the linkage can be through a carbon linker.
- Protein transduction domains can be modified to include a biotin moiety.
- the modified PTD and the modified nucleic acid reporter can then be linked through a streptavidin molecule.
- a thiol-maleimide linkage is used to link the PTD to the nucleic acid reporter.
- the non-complementary region of the nucleic acid reporter is modified by adding a thiol group to a monomer of the nucleic acid reporter.
- the thiol group can react with a maleimide group placed at the C terminus of the PTD or targeting signal to form a linkage, in particular a direct and stable linkage.
- Another embodiment incorporates a cleavable disulfide bridge in which the PTD or targeting signal is modified by adding a cysteine residue at the C terminus which forms a disulfide bridge with the thiol-modified nucleic acid reporter.
- This disulfide bridge design allows the PTD or targeting signal to be cleaved from the nucleic acid reporter by the reducing environment of the cytoplasm.
- Another embodiment allows for combined synthesis of the delivery peptide sequence along with the nucleic acid reporter, for example, in the case of a PNA probe, where both the delivery peptide and the nucleic acid probe sequence can be generated using a single peptide.
- the linkage between the delivery peptide and the nucleic acid probe can be tailored to allow for specific cleavage using reducing disulfide bridge or using enzymatic cleavage sites in the linkage.
- nucleic acid reporter with delivery peptide
- the delivery vehicle itself may be dendrimer-based or lipid-based such as liposomes/polymeric or any combination of the above, in which nucleic acid probes are packaged inside TKHR DOCKET NO. 820701.1135
- the disclosed nucleic acid reporters can be linked to a protein transduction domain or targeting signal via a linker or spacer.
- the linker can be one or more monomer units including atoms, amino acids, nucleic acids, sugars, or natural or synthetic polymer monomers.
- the linker is composed of monomers that are substantially inert or do not otherwise chemically react once coupled to the nucleic acid reporter.
- Representative linkers include alkyl linkers of 1 to 12 carbons, typically about 6 carbons.
- the alkyl groups of the linker can be substituted, for example with alkyl or aryl groups, heterocycles, halogens, and the like.
- the number of monomers of the linker can vary, however, the linker should have enough monomers to prevent the protein transduction domain or targeting signal from sterically interfering with the binding of the nucleic acid reporter to its target nucleic acid.
- the linker is modified to link the protein transduction domain or target signal via conventional linking chemistry.
- the linkers can be linked with or contain cleavable bonds, for example photo cleavable, thermally cleavable, or enzymatically cleavable bonds. Such bonds are known in the art.
- the cleavable bond Upon entry into the cell, the cleavable bond can be cleaved. The cleavage of the bond can result in the separation of the PTD or targeting signal or both from the nucleic acid reporter.
- Linking Chemistry Of the various linking chemistries that can be used to link molecules with other molecules or reagents, the most common are amine, carbonyl, carboxyl, and thiol. It will be appreciated by those of skill in the art, that any linking chemistry may be utilized. Indirect crosslinking of the amines in one molecule to the thiols in a second molecule is the predominant method for forming a heteroconjugate. If the nucleic acid reporter, the linker, or the TKHR DOCKET NO. 820701.1135
- thiol groups can be introduce using a thiolation procedure.
- Thiol groups also called mercaptans or sulfhydryls
- Thiols are present in cysteine residues of proteins.
- Thiols can also be generated by selectively reducing cystine disulfides with reagents such as dithiothreitol (DTT) or - mercaptoethanol. Removal of DTT or -mercaptoethanol is sometimes accompanied by air oxidation of the thiols back to the disulfides.
- DTT dithiothreitol
- TCEP tris-(2- carboxyethyl)phosphine
- TCEP is generally impermeable to cell membranes and to the hydrophobic protein core, permitting its use for the selective reduction of disulfides that have aqueous exposure.
- the pH-insensitive and less polar phosphine derivative tris-(2-cyanoethyl)phosphine may yield greater reactivity with buried disulfides.
- Disulfide crosslinks for example of cystines in proteins, can be reduced to cysteine residues by dithiothreitol, tris-(2-carboxyethyl)phosphine or tris-(2- cyanoethyl)phosphine.
- Amines can be indirectly thiolated by reaction with succinimidyl 3-(2- pyridyldithio)propionate, followed by reduction of the 3-(2- pyridyldithio)propionyl conjugate with DTT or TCEP.
- amines can be indirectly thiolated by reaction with succinimidyl acetylthioacetate, followed by removal of the acetyl group with 50 mM hydroxylamine or hydrazine at near-neutral pH.
- Thiols can also be incorporated at carboxylic acid groups by an EDAC- mediated reaction with cystamine, followed by reduction of the disulfide with DTT or TCEP.
- Tryptophan residues in thiol-free proteins can be oxidized to mercaptotryptophan residues, which can then be modified by iodoacetamides or maleimides.
- TKHR DOCKET NO. 820701.1135
- Thiol-reactive functional groups are primarily alkylating reagents, including iodoacetamides, maleimides, benzylic halides and bromomethylketones.
- Arylating reagents such as NBD halides react with thiols or amines by a similar substitution of the aromatic halide. Reaction of any of these functional groups with thiols usually proceeds rapidly at or below room temperature in the physiological pH range (pH 6.5-8.0) to yield chemically stable thioethers.
- Thiols also react with many of the amine-reactive reagents described in including isothiocyanates and succinimidyl esters.
- thiol— isothiocyanate product (a dithiocarbamate) can react with an adjacent amine to yield a thiourea, the dithiocarbamate is more likely to react with water, consuming the reactive reagent without forming a covalent adduct.
- Iodoacetamides readily react with all thiols, including those found in peptides, proteins and thiolated polynucleotides, to form thioethers. Iodoacetamides can sometimes react with methionine residues. They may also react with histidine or tyrosine, but generally only if free thiols are absent.
- iodoacetamides can react with the free base form of amines, most aliphatic amines, except the -amino group at a protein's N-terminus, are protonated and thus relatively unreactive below pH 8.
- iodoacetamides react with thiolated oligonucleotide primers, as well as with thiophosphates and thiouridine residues present in certain nucleic acids, but usually not with the common nucleotides. Iodoacetamides are intrinsically unstable in light, especially in solution; reactions should therefore be carried out under subdued light.
- Adding cysteine, glutathione or mercaptosuccinic acid to the reaction mixture will quench the reaction of thiol-reactive probes, forming highly water-soluble adducts that are easily removed by dialysis or gel filtration.
- the thioether bond formed when an iodoacetamide reacts with a protein thiol is very stable, during amino acid hydrolysis the bioconjugate loses its fluorophore to yield S-carboxymethylcysteine.
- Maleimides are excellent reagents for thiol-selective modification, quantitation and analysis.
- the reaction involves addition of the thiol across the TKHR DOCKET NO. 820701.1135
- maleimides apparently do not react with methionine, histidine or tyrosine. Reaction of maleimides with amines usually requires a higher pH than reaction of maleimides with thiols. Hydrolysis of maleimides to a mixture of isomeric nonreactive maleamic acids can compete significantly with thiol modification, particularly above pH 8. Furthermore, maleimide adducts can hydrolyze or they can ring-open by nucleophilic reaction with an adjacent amine to yield crosslinked products. This latter reaction can potentially be enhanced by raising the pH above 9 after conjugation.
- a disulfide-containing linker or spacer including but not limited to an alkyl linker or spacer of about 1 to about 12 carbon atoms, is photo- or thermally coupled to the target nucleobase or polynucleotide using conventional chemistry, for example azide chemistry.
- the disulfide bond is reduced, yielding a free thiol.
- a covalent bond is formed between the reagent thiol and a thiol-reactive linker, hapten, fluorochrome, sugar, affinity ligand, or other molecule.
- the linking of two molecules can be achieved using heterobifunctional crosslinkers.
- heterobifunctional crosslinkers include, but are not limited to, p-maleimidophenyl isocyanate; succinimidyl acetylthioacetate; succinimidyI-trans-4(maleimidylmeyt yl)-cyclohexane-1 carboxylate (SMCC); succinimidyl acetylthioacetate (SATA); succinimidyl 3-(2- pyridyldithio)propionate (SPDP); ⁇ /-((2-pyridyldithio)ethyl)-4-azidosaIicylamide (PEAS; AET); 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester (ATFB, SE); 4-azido-2,3,5,6-tetrafluorobenzoic acid, STP ester, sodium salt (ATFB, STP ester); 4-azido-2,3,5,6-tetrafluorobenzy
- the heterobifunctional crosslinkers can be photoreactive, amine and/or thiol reactive, or aldehyde/ketone reactive, or a combination thereof.
- the disclosed nucleic acid reporters can also include targeting signals or domains that target the nucleic acid reporter to a specific cell, tissue or TKHR DOCKET NO. 820701.1135
- nuclear targeting signals can be found in the Nuclear Localization Signal Database at http://cubic.bioc.columbia.edu/db/NLSdb/ which is incorporated by reference herein in its entirety.
- Representative nuclear localization signals include, but are not limited to, SV 40 T antigen or a fragment thereof, such as PKKKRKV (SEQ ID NO. 9).
- the NLS can be simple cationic sequences of about 4 to about 8 amino acids, or can be bipartite having two interdependent positively charged clusters separated by a mutation resistant linker region of about 10-12 amino acids. Additional representative NLS include but are not limited to GKKRSKV (SEQ ID NO.
- the targeting signal can also target the nucleic acid reporter to the mitochondria which are also known in the art.
- Representative mitochondrial targeting signals include, but are not limited to include the mitochondrial localization signal of subunit VIII of human cytochrome oxidase, the yeast cytochrome c oxidase subunit IV presequence and the amino-terminal leader peptide of the rat ornithine-transcarbamylase.
- the identification of the specific sequences necessary for translocation of a linked nucleic acid reporter into a chloroplast or mitochondria can be determined using predictive software known to those skilled in the art, including the tools located at http://www.mips.biochem.mpg.de/cqi- bin/proj/medgen/mitofilter.
- Targeting signals can also include vitamins such as folate to target the nucleic acid reporters to cells having a high number of folate receptors, including but not limited to, cancer cells.
- Asialoglycoprotein receptor ligands can also be linked to the disclosed nucleic acid reporters to target them to the liver. For example, N-acetylgalactosamine containing peptides, asialoorosomucoid, and galactoside-containing cluster ligands can be used.
- Dual FRET/LRET Nucleic Acid Reporters Another embodiment of the present disclosure provides dual FRET nucleic acid reporters or luminesence resonance energy transfer (LRET) operably linked to a protein transduction domain, and optionally to a targeting signal. Dual FRET nucleic acid reporters include molecular beacons that operate in pairs. A first molecular beacon is labeled with a donor dye and a quencher and a second molecular beacon is labeled with an acceptor dye and a quencher. In the absence of their complements, the dyes are quenched by their respective quenchers.
- LRET luminesence resonance energy transfer
- Each molecular beacon in the dual FRET pair includes sequences complementary to adjacent regions of a target nucleic acid such that FRET between the donor dye of the first molecular beacon and the acceptor dye of the second molecular beacon only occurs when both beacons are hybridized to the target nucleic acid.
- the beacons must hybridize to the target nucleic acid so that the donor dye and the acceptor dye are apart by about 6 nm or less. See Tsourkas et al (2003) Anal Chem 75 (3697-3703) and Santagelo, P. et al. (2004) Nucleic Acid Res. 32(6) e57 which are incorporated by reference herein in their entirety. 5.
- Embodiments of the present disclosure can be used to detect, localize, or quantify a target nucleic acid in a cell, in particular a living cell, tissue, or organ.
- Representative cells include plant and animal cells.
- Target nucleic acids can be any polymer of nucleotides, DNA, RNA or combinations thereof, genomic, mRNA, nuclear RNA, enzymatic RNA, enzymatic DNA, or ribosomal RNA.
- Embodiments of the disclosed methods provide real-time visualization of specific endogenous mRNA expression in vivo.
- One embodiment provides a method for detecting cells expressing a target nucleic acid including contacting a cell suspected of expressing the target nucleic acid with a molecular beacon operably linked to a protein TKHR DOCKET NO. 820701.1135
- the molecular beacon includes a region complementary to the target nucleic acid.
- the molecular beacons can be added to a cell culture of immortalized cells, primary culture cells, transfected cells, or administered to a organism or tissue.
- Preferred tissues are those tissues with optical characteristics that enable the detection of the molecular beacons or it may involve the use of endoscopic instruments in combination with appropriate optical instrumentation for excitation and detection of electromagnetic emission in vivo.
- Preferred organisms include humans and zebra fish. When used with tissues, near-infrared labels are preferred.
- the PTD on the nucleic acid reporter facilitates translocation of cell membranes including, but not limited to, lipid bilayers, micelles, vesicles, and organelles such as the nucleus, mitochondria or chloroplast.
- the PTD enables the nucleic acid reporter to travel from extracellular space to intracellular space including the interior of organelles.
- the nucleic acid reporters can be irradiated with an exciting amount of electromagnetic radiation.
- An exciting amount of radiation is that amount of radiation sufficient to enable the nucleic acid reporter to emit electromagnet radiation.
- the electromagnetic energy emitted by the nucleic acid reporter indicates that nucleic acid reporter has bound its complement and that the cell expresses the target nucleic acid.
- Another embodiment provides a method for sorting cells expressing a target nucleic acid.
- a plurality of cells suspected of expressing the target nucleic acid is contacted with at least one nucleic acid reporter, for example a molecular beacon, operably linked to a protein transduction domain, wherein the protein transduction domain facilitates translocation of the at least one molecular beacon to at least one of the plurality of cells' interior.
- the molecular beacon includes a region complementary to the target nucleic acid.
- the plurality of cells are irradiated with an exciting amount of electromagnetic energy and electromagnetic energy emitted in response to the exciting amount of electromagnetic radiation by the molecular beacon in the interior of at least of the plurality of cells can be detected. Cells emitting a detectable amount of electromagnetic radiation can then be separated from cells, which TKHR DOCKET NO. 820701.1135
- Another embodiment provides a method for detecting a target nucleic acid in a host.
- This embodiment includes administering to the host a molecular beacon operably linked to a protein transduction domain, and optionally linked to a targeting signal.
- the molecular beacon is in the form of a pharmaceutical composition.
- the molecular beacons are irradiated with an exciting amount of electromagnetic radiation, and the electromagnetic radiation emitted by the molecular beacons in response to the exciting amount of electromagnetic radiation is detected.
- the detected electromagnetic radiation can be correlated with the presence, location, and quantity ofthe target nucleic acid in the host, cell, or tissue.
- Still another embodiment provides a method for detecting expression of a target nucleic acid in a living cell by contacting the cell with a molecular beacon operably linked to a protein transduction domain optionally linked to a targeting signal, wherein the molecular beacon is specific for the target nucleic acid; irradiating the molecular beacon with an exciting amount of electromagnetic radiation, and detecting the emission of electromagnetic radiation from the molecular beacon, wherein detectable emission from the molecular beacon is indicative of expression or location of the target nucleic acid in the cell.
- Still another embodiment provides an approach to study the transport of RNA in living cells and model organisms such as oocytes using molecular beacon operably linked to a protein transduction domain as well as a targeting signal such as a nuclear localization signal (NLS) whenever necessary.
- molecular beacons may be delivered specifically to the nuclear compartment, where they hybridize with the target RNA molecules which may be subsequently transported to the cytoplasm. This real time imaging of RNA transport may be used to gain a better understanding of RNA biology and developmental biology.
- Further molecular beacon may be linked to a targeting sequence such as NLS and modified using caged fluorophore.
- TKHR DOCKET NO. 820701.1135
- the caged molecule may be released, emitting a signal that can be imaged for studying transport of RNA molecules from nucleus to cytoplasm.
- a molecular beacon operably linked to a protein transduction domain optionally linked to a targeting signal is used in conjugation with fusion protein of interest (GFP- protein) or any flourescently labeled protein (fluorescent labeled antibody targeted protein) or subcellular organelle.
- the molecular beacons target the nucleic acid molecules of interest (RNA or DNA) and, upon hybridization with the target, provide electromagnetic emission in particular wavelength range, while the fusion protein or any flourescently labeled protein or organelle provides the non-overlapping wavelength upon excitation. This allows one to study the RNA-protein interactions and its dynamics in living cells.
- Yet another embodiment provides a method for identifying compounds that interfere with the expression of a target nucleic acid, for example a nucleic acid suspected to be involved with a pathological condition, for example cancer or other disease states.
- the method includes contacting a living cell or a plurality of living cells with compound suspected of modulating the expression a gene, for example a small organic molecule or antisense drug, contacting the cell with a nucleic acid reporter having a region complementary to a transcript of the gene, irradiating the nucleic acid reporter or the cell with an exciting amount of electromagnetic radiation, and detecting the emission of electromagnetic radiation from the nucleic acid reporter, wherein detectable emission from the nucleic acid reporter is indicative of expression or the location of the gene.
- compound suspected of modulating the expression a gene for example a small organic molecule or antisense drug
- the cell can be a primary culture, immortalized cell, or transfected cell.
- a cell is transfected with a gene of interest and the nucleic acid reporter is specific for transcripts of the gene of interest.
- a test compound can be selected based on its ability to decrease the expression of a gene or increase the expression of a gene when TKHR DOCKET NO. 820701.1135
- a control sample includes a cell contacted with the nucleic acid reporter in the absence of the test compound. Decreased expression of a gene is indicated by little to no detectable emissions from the nucleic acid reporter; whereas, increased gene expression is indicated by detectable emissions from the nucleic acid reporter which are greater from cells treated with the test compound compared to cells not treated with the test compound.
- This screening method is adaptable to high-throughput screening and imaging, for example using a fluorescence activated cell sorter, cell arrays, tissue arrays, or other microfluidic devices, which allow for high throughput detection and imaging. Combinatorial libraries can be used with this method to identify compounds that modulate gene expression in vivo.
- a first and a second nucleic acid reporter are independently operably linked to a PTD and optionally to a targeting signal.
- the first and second nucleic acid reporters each include different target recognition sequences.
- the target recognition sequences can be directed to transcripts of different forms or alleles of the same gene, or can be for transcripts of different genes.
- the first and second nucleic acid reporters typically provide different detectable signals, for example emit at different wavelengths.
- Another embodiment provides a method for determining the effectiveness of a therapeutic, for example a therapeutic that modulates gene expression of a host or suspected of modulating gene expression.
- the disclosed nucleic acid reporters can be used to obtain in vivo gene expression data from a individual or cell specific gene expression data of an individual or host. Such data can be used to determine whether the therapeutic is effective in the individual based on the level of specific gene expression in a host.
- One embodiment provides a method including the steps of administering a therapeutic to a host, obtaining cells from the host, contacting the cells with a nucleic acid reporter operably linked to a PTD and optionally to a targeting signal, wherein the nucleic acid reporter includes a target recognition sequence complementary to a predetermined target nucleic acid, irradiating TKHR DOCKET NO. 820701.1135
- a therapeutic includes compounds, molecules, drugs or combinations thereof, administered to a host to treat, alleviated, mitigate, a pathology of the host or a symptom of a pathology.
- one or more cells from a host can be contacted in vitro with an agent, for example a therapeutic agent, suspected of modulating expression of a target nucleic acid.
- the cells can be incubated for a period of time to allow the therapeutic agent to have a biological effect.
- the cells can then be contacted with a molecular beacon operably linked to a protein transduction domain, wherein the molecular beacon includes a target recognition sequence complementary to the target nucleic acid.
- the cells containing the nucleic acid reporter are irradiated with an exciting amount of radiation and electromagnetic emission from the cells emitted in response to the exciting mount of radiation can be detected. Emission from the cells exposed to the therapeutic agent and containing the molecular beacon can be compared with emissions from a control sample of cells containing the molecular beacon but which were not exposed to the therapeutic agent.
- a difference in emission between the cells exposed to the therapeutic agent and containing the molecular beacon compared with emissions from a control sample of cells containing the molecular beacon but which were not exposed to the therapeutic agent indicates that the therapeutic agent modulates expression of the target nucleic acid in the host.
- more than one nucleic acid reporter specific for more than one target nucleic acid can be used to establish a profile of gene expression modulation by a particular agent or compound for an individual host. Such data can provide information to a medical practitioner to assist in determining course of treatment and use of specific medicines personalized to one individual or host.
- the agent or compound will be an agent TKHR DOCKET NO. 820701.1135
- antisense oligonucleiotide probes When delivered through the endocytic pathway (e.g., liposome-based transfection), antisense oligonucleiotide probes tend to be trapped inside endocytic vesicles and degraded in the endosomes and lysosomes by nucleases (Dokka, S., Rojanasakul, Y. (2000) Adv. Drug Deliv. Rev. 44, 35-49). Peptide-based internalization has the potential to avoid the endocytic pathway, therefore reducing false-positive signals due to nuclease degradation. Consequently, ODN backbone modifications of the probe such as the use of 2'-O-methyl modified molecular beacons may not be necessary.
- peptide-Iinked molecular beacons approach is faster (-30 min) and simpler, which may be more suitable for certain applications, including basic biological studies of mRNA expression level in living cells in response to drug molecules, toxin and external stimuli, the knock-down effect of RNAi, and disease detection and diagnosis.
- NIR fluorophores as the reporter, peptide-Iinked molecular beacons have the potential to become a powerful TKHR DOCKET NO. 820701.1135
- compositions and dosage forms of the disclosure comprise a pharmaceutically acceptable salt of disclosed or a pharmaceutically acceptable polymorph, solvate, hydrate, dehydrate, co- crystal, anhydrous, or amorphous form thereof.
- Specific salts of disclosed compounds include, but are not limited to, sodium, lithium, potassium salts, and hydrates thereof.
- Pharmaceutical compositions and unit dosage forms of the disclosure typically also comprise one or more pharmaceutically acceptable excipients or diluents.
- Advantages provided by specific compounds of the disclosure such as, but not limited to, increased solubility and/or enhanced flow, purity, or stability (e.g., hygroscopicity) characteristics can make them better suited for pharmaceutical formulation and/or administration to patients than the prior art.
- Pharmaceutical unit dosage forms of the compounds of this disclosure are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., intramuscular, subcutaneous, intravenous, intraarterial, or bolus injection), topical, or transdermal administration to a patient.
- mucosal e.g., nasal, sublingual, vaginal, buccal, or rectal
- parenteral e.g., intramuscular, subcutaneous, intravenous, intraarterial, or bolus injection
- topical e.g., topical, or transdermal administration to a patient.
- dosage forms include, but are not limited to: tablets; caplets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
- suspensions e.g.,
- compositions, shape, and type of dosage forms of the compositions of the disclosure will typically vary depending on their use.
- a dosage form used in the detection of nucleic acids involved in a disease or disorder but expressed at a low level may contain larger amounts of the TKHR DOCKET NO. 820701.1135
- nucleic acid reporter for example the disclosed compounds or combinations thereof, than a dosage form used to detect nucleic acids that are overexpressed in a disease or disorder.
- a parenteral dosage form may contain smaller amounts of the nucleic acid reporter than an oral dosage form used to detect the same disease or disorder.
- Suitable excipients are well known to those skilled in the art of pharmacy or pharmaceutics, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient. For example, oral dosage forms such as tablets or capsules may contain excipients not suited for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the specific active ingredients in the dosage form. For example, the decomposition of some active ingredients can be accelerated by some excipients such as lactose, or when exposed to water.
- compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose.
- Such compounds which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.
- pharmaceutical compositions or dosage forms of the disclosure may contain one or more solubility modulators, such as sodium chloride, sodium sulfate, sodium or potassium phosphate or organic acids. A specific solubility modulator is tartaric acid.
- solubility modulators such as sodium chloride, sodium sulfate, sodium or potassium phosphate or organic acids.
- a specific solubility modulator is tartaric acid.
- the amounts and specific type of active ingredient in a dosage form may differ depending on factors such as, but not limited to, the route by which it is to be administered to patients.
- typical dosage forms of the compounds of the disclosure comprise a pharmaceutically acceptable salt, or a pharmaceutically acceptable polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous, or amorphous form thereof, in an amount sufficient to detect the target nucleic acid and include a range of from about 10 mg to about 1000 mg, preferably in an amount of from about 25 mg to about 750 mg, and more preferably in an amount of from 50 mg to 500 mg.
- the compounds and/or compositions can be delivered using lipid- or polymer-based nanoparticles.
- the nanoparticles can be designed to improve the pharmacological and therapeutic properties of drugs administered parenterally (Allen, T.M., Cullis, P.R. Drug delivery systems: entering the mainstream. Science. 303(5665): 1818-22 (2004)).
- Parenteral Dosage Forms can be administered to patients by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.
- parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
- controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, administration DUROS®-type dosage forms, and dose-dumping.
- Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; Water for Injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, Sodium TKHR DOCKET NO. 820701.1135
- Chloride Injection Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
- Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a disclosed composition herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms. 6.2.
- Topical dosage forms of the disclosure include, but are not limited to, creams, lotions, ointments, gels, shampoos, sprays, aerosols, solutions, emulsions, and other forms know to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, Pa. (1985).
- viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed.
- suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure.
- Suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon), or in a squeeze bottle.
- a pressurized volatile e.g., a gaseous propellant, such as freon
- Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990). TKHR DOCKET NO. 820701.1135
- Transdermal and mucosal dosage forms of the compositions of the disclosure include, but are not limited to, ophthalmic solutions, patches, sprays, aerosols, creams, lotions, suppositories, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th Ed., Lea & Febiger, Philadelphia, Pa. (1985).
- Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes, as oral gels, or as buccal patches.
- transdermal dosage forms include "reservoir type” or “matrix type” patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredient.
- transdermal dosage forms and methods of administration that can be used to administer the active ingredient(s) of the disclosure include, but are not limited to, those disclosed in U.S. Pat. Nos.: 4,624,665;
- Suitable excipients e.g., carriers and diluents
- other materials that can be used to provide transdermal and mucosal dosage forms encompassed by this disclosure are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue or organ to which a given pharmaceutical composition or dosage form will be applied.
- typical excipients include, but are not limited to water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1 ,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof, to form dosage forms that are non-toxic and pharmaceutically acceptable.
- penetration enhancers can be used to assist in delivering the active ingredients to or across the tissue.
- Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, an tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as TWEEN 80 (polysorbate 80) and SPAN 60 (sorbitan monostearate).
- the pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied may also be adjusted to improve delivery of the active ingredient(s).
- the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery.
- Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of the active ingredient(s) so as to improve delivery.
- stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery- enhancing or penetration-enhancing agent.
- Different hydrates, dehydrates, co-crystals, solvates, polymorphs, anhydrous, or amorphous forms of the pharmaceutically acceptable salt of a disclosed composition can be used to further adjust the properties of the resulting composition.
- kits which include the discloses nucleic acid reporters and directions, for example written instructions, for their use.
- a typical kit comprises a unit of nucleic acid reporter having a region complementary to a predetermined target nucleic acid.
- the kit can further include appropriate buffers and reagents known in the art for administering the nucleic acids to cell cultures or hosts.
- the kits can also include a plurality ofthe disclosed nucleic acid reporters specific for more than one target nucleic acid.
- Example 1 Design of peptide-Iinked molecular beacons Peptide-Iinked molecular beacons targeting the human GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and survivin mRNAs, as well as molecular beacons with a 'random' probe sequence were designed and synthesized. The specific design of these molecular beacons, and the sequence of the 11 amino-acid TAT-1 peptide used in the study are shown in Table 1.
- GAPDH is a glycolytic protein that has a diverse range of activities in mammalian cells including membrane fusion, microtubule bundling, nuclear RNA export, DNA replication and repair (Sirover, M.A. (1999) Biophys.
- GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis.
- Survivin is a member of the inhibitor of apoptosis protein (IAP) family and also regulates cell division Chiou, S.K. et al. (2003) Med. Sci. Monit. 9PI25-29).
- the GAPDH beacon is comprised of a 19-base probe domain targeting the Exon 6 region of GAPDH gene flanked by complementary 5-base sequences that hybridize to form the stem.
- the survivin beacon has a 16-base target sequence with a similar design of the stem.
- the 'random' beacon was designed as a negative control, with a 17- base probe sequence that does not have any match in the entire human genome.
- molecular beacons complementary to different segments of the same mRNA molecule are TKHR DOCKET NO. 820701.1135 typically designed and tested to avoid targeting sequences that are occupied by RNA-binding proteins or where double stranded RNA is formed.
- Table 1 Design of peptide-Iinked molecular beacons Peptide TAT (N terminus) TyrGlyArgLysLysArgArgGlnArgArgArg (C terminus) (SEQ ID NO. 1) Unmodified Molecular Beacon
- GAPDH 5'-Cv3-CGACGGAGTCCTTCCACGATACCACG/thiol-dT/CG-BHQ2-3' SEQ ID NO. 18
- peptides were linked to a molecular beacon through a streptavidin- biotin bridge by introducing a modified oligonucleotide, biotin-dT, to the quencher arm of the stem through a carbon-12 linker.
- exemplary peptide- linked molecular beacons included the biotin-modified molecular beacon, a streptavidin molecule, and biotin-modified TAT-1 peptides.
- the TAT-1 peptide was functionalized by adding a cysteine residue at the C terminus which forms a disulfide bridge with the thiol-modified molecular beacon as shown in Figure 1C. This cleavable design was based on the rationale that the reducing environment of the cytoplasm will cleave the disulfide bond once the construct enters the cell, thereby separating peptide from probe.
- Example 2 Peptide conjugation
- streptavidin was chosen as a linker molecule because of its high affinity for biotin and availability of multiple biotin- binding sites.
- the modified nucleotide (dT) at the third base from the 3' end on the quencher-arm of the stem was linked to biotin through a 12-carbon linker, and the biotin-modified peptides were conjugated to the biotin-modified molecular beacon through binding between biotin and streptavidin.
- the peptide-Iinked molecular beacon complex was dialyzed with PBS (1X) overnight using a Slide-A-Lyzer Dialysis Unit, 10K MWCO (Pierce Biotech Inc., Rockford, IL) to exchange the buffer and to remove the unconjugated peptide.
- the modified molecular beacons were synthesized at Integrated DNA Technologies, Inc (Coralville, IA) and MWG Biotech, Inc (High Point, NC), and the modified peptides were synthesized by Invitrogen Inc (Carlsbad, CA) and Synpep Corporation (Dublin, CA).
- Example 3 Solution assays of hybridization kinetics and signal-to-background ratio Measurement of hybridization kinetics and signal-to-background ratio of peptide linked molecular beacons was carried out using a SAFIRE microplate monochromator reader (TECAN, Austria).
- 200 nM peptide-Iinked molecular beacons were mixed with 1 ⁇ M complementary oligonucleotide target at 37°C and the fluorescence intensity was recorded as a function of time for conventional molecular beacons, direct linked (stable and cleavable) peptide-molecular beacon complexes, and streptavidin-Iinked peptide-molecular beacon complex.
- 200 nM conventional and peptide-Iinked molecular beacons were mixed with 200 nM of complementary target respectively in the microplate reader, and the fluorescence intensity at equilibrium was recorded.
- Example 4 Cellular delivery of peptide-Iinked molecular beacons Primary human dermal fibroblast (HDF) cells (Cambrex, NJ) and a pancreatic cancer cell line MiaPaca-2 (ATCC, VA) were used. These cells TKHR DOCKET NO. 820701.1135
- peptide-Iinked molecular beacons were cultured in an 8-well Nalge Nunc culture plate with a glass coverslip bottom in their respective cell culture media for 24 h prior to experiments. Delivery assays were performed by incubating cells at 37°C with the media containing peptide-Iinked molecular beacons.
- peptide-Iinked molecular beacons targeting GAPDH peptide-Iinked molecular beacons with three different concentrations (0.25 ⁇ M, 0.5 ⁇ M and 1.0 ⁇ M) were incubated with HDF cells for 30, 60 and 90 min.
- molecular beacons targeting survivin 0.5 ⁇ M of peptide-Iinked molecular beacons were incubated with HDF and MiaPaca-2 cells for 30 min.
- Example 5 Fluorescence in-situ hybridization. Normal human dermal fibroblast cells were cultured in 8-well chambered coverslides for 24 hours in normal growth medium (FGM-2).
- normal HDF cells were fixed in 100% methanol at -20°C for 10 min, allowed to dry and kept at -80°C for 12 h.
- In-situ hybridization assays were performed as described above with 400 nM of fluorescently labeled linear probes targeting wild-type K-ras and GAPDH. The cells were imaged after removing the hybridization solution with washing and adding 1x PBS.
- Example 6 Delivery of molecular beacons using commercial transfection reagents To compare the efficiency and functionality of different delivery methods, three commercially available transfection reagents were used:EFFECTINE® (Qiagen), SUPERFECT® (Qiagen), and
- OLIGOFECTIMINETM (Invitrogen). Transfection assays were carried out according to the procedure recommended by respective suppliers; both primary HDF cells and MiaPaca-2 pancreatic cancer cells were incubated with conventional (unmodified) molecular beacons for 0.5, 2 and 3.5 h.
- Example 7 Hybridization kinetics of peptide-Iinked molecular beacons To determine the effect of peptide conjugation on molecular beacon function, in-solution hybridization assays were carried out for the binding kinetics of peptide-Iinked molecular beacons with different conjugation methods.
- TAT-1 peptide when the positively charged TAT-1 peptide is conjugated to a molecular beacon, it might interact with the negatively charged hairpin oligonucleotide, thus interfering with proper probe-target binding.
- Shown in Figure 2A are normalized fluorescence intensity verses time curves as a result of probe-target hybridization for unmodified GAPDH- targeting molecular beacons and peptide-Iinked molecular beacons with the streptavidin-biotin linkage, the stable thiol-maleimide linkage, and the cleavable disulfide bridge.
- Peptide-Iinked molecular beacons with the thiol- maleimide linkage had almost exactly the same probe-target hybridization kinetics as unmodified molecular beacons (black and green curves respectively in Fig. 2A), indicating that the conjugation of peptide using the thiol-maleimide linkage has essentially no effect on the functionality of molecular beacons.
- Molecular beacons with the cleavable disulfide bridge also behaved similarly to the unmodified ones. With streptavidin-biotin linkage, the hybridization kinetics of peptide-Iinked molecular beacons was slightly slower, but it did not affect the signal level, as can be seen from Figure 2A.
- streptavidin molecule whose size is comparable to that of the molecule beacon, may have sterically hindered binding between the target and the hairpin probe, leading to a slightly reduced hybridization TKHR DOCKET NO. 820701.1135
- Example 8 Detection of GAPDH mRNA using peptide-Iinked molecular beacons
- detected mRNA of a housekeeping gene human GAPDH in normal human dermal fibroblast (HDF) cells was detected.
- TAT-peptide conjugated GAPDH-targeting molecular beacons clear and localized fluorescence signal in HDF cells as a result of molecular beacon-target mRNA hybridization for all three conjugation schemes, i.e., thiol-maleimide (Fig. 3A), disulfide bridge (Fig. 3B) and streptavidin-biotin (Fig.
- molecular beacons with the cleavable (thiol-cysteine disulfide bridge) design seemed to give better localization patterns than those with the thiol-maleimide linkage, and the latter seemed to perform better than molecular beacons with the streptavidin-biotin linkage. Cleavage of the delivery peptide from the construct may have provided molecular beacons a better access to target mRNA molecules, although more studies of this phenomenon are required to validate this assumption. It is likely that a molecular beacon with a relatively bulky streptavidin molecule is TKHR DOCKET NO. 820701.1135
- Example 9 Comparison With in situ hybridization To correlate the results of present method with a traditional method, fluorescence in situ hybridization (FISH) assays targeting GAPDH mRNA in fixed HDF cells were performed.
- the probes used in the FISH assays were fluorescently labeled linear probes (5'-Cy5-GAGTCCTTCCACGATACCA-3') (SEQ ID NO. 21) that have the same probe sequence as the GAPDH- targeting molecular beacon.
- Example 11 Comparison with conventional transfection methods As mentioned above, cellular delivery of molecular beacons using conventional transfection methods, either liposome based or dendrimer based, typically requires 3-4 hours of incubation during which a high level of background signal is generated.
- Example 12 Molecular beacons targeting K-ras
- the K-ras-targeting and 'random' sequence molecular beacon pairs adopted the shared-stem design (Tsourkas et al., 2003).
- the K-ras-targeting, GAPDH-targeting molecular beacons and Cy5-labeled random beacons were synthesized by Biosource International (Camarillo, CA) and MWG Biotech (High Point, NC).
- the Cy3-labeled random beacon and all of the synthetic targets were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).
- the GAPDH-targeting molecular beacon (see Table 2) was designed such that the stem sequence is independent of the target sequence.
- the underlined bases in all the beacon designs shown in Table 2 indicate the bases added to form the stem of a molecular beacon.
- TKHR DOCKET NO. 820701.1135
- K-ras dual FRET molecular beacons Donor MB: 5'-/Cv3/CC 4CGCC CC ⁇ GCrCCGTAGG/BHQ-2/-3' (SEQ ID NO. 23) Acceptor MB: 5'-/BHQ-3/AGTGCGCrGT ⁇ rCGTCA GGC/ACT/Cv5/-3' (SEQ ID NO. 24)
- GAPDH molecular beacon '-Cv3-CGACGGAGTCCTTCCACGATACCACG ⁇ hiol-dT/CG-BHQ2-3' (SEQ ID NO. 25) 'Random' sequence molecular beacons
- Donor MB 5'-/Cv3/CACGTCG ⁇ CA4GCGC/4CCG ⁇ TACGTG/BHQ-2/-3' (SEQ ID NO. 26)
- Acceptor MB 5WBHQ-3/ACGTGCG/AC/4 ⁇ GCGC/4CCG 7OACGT/Cv5/-3' (SEQ ID NO. 27).
- Example 13 SLO delivery of molecular beacons and organelle labeling
- Molecular beacons targeting K-ras mRNA were delivered into cytoplasm of living cells using a reversible permeabilization method with SLO. The detailed protocol was described elsewhere (Santangelo el al. (2004) Nucleic Acids Res. 32(6): e57). Briefly, SLO was activated first by adding 5 mM of TCEP to 2 U/ml of SLO for 30 min at 37° C. Cells were grown in 24- well plates were incubated for 10 min in 200 ⁇ l of serum free medium contaiing 0.2 U/ml of activated SLO (0.5 U SLO per 10 6 cells) and 5 ⁇ l of each molecular beacon.
- Example 14 Dual FRET molecular beacons
- Cy3 and Cy5 are selected as the donor and acceptor fluorophores, respectively, with excitation at 545 nm and emission detection at 670 nm for the FRET signal detection.
- MitoFlour Green was selected to label mitochondria to avoid possible spectral overlap with the TKHR DOCKET NO. 820701.1135
- Cy3-Cy5 FRET pair Specifically, the fluorescence signal of MitoFlour Green was detected using a bandpass filter of 510-530 nm under the excitation wavelength of 450 ⁇ 25 nm. Since MitoFlour Green has essentially no excitation at 545 nm (Cy3 excitation) and no emission at 670 nm (Cy5 emission detection), as demonstrated in Fig. 8, the optics used for detecting the FRET signal due to molecular beacons should have no effect on the image of mitochondria shown in Fig. 7A. Likewise, there is very limited excitation of Cy3 at 488 nm and no fluorescence emission of Cy3 at 510-530 nm (Fig. 8).
- ER-Tracker was chosen in ER labeling because it is specific to the ER (unlike other ER staining dyes) and has essentially no spectral overlap with MitoFluor Green. The fluorescence emission of ER-Tracker was detected at 460 nm under the excitation of 360 nm. This ensured that there was no cross-talk in the fluorescence imaging of mitochondrial and ER.
- Example 16 Fluorescence microscopy imaging Fluorescence imaging of live and fixed HDF cells was performed using a Zeiss Axiovert 100 TV epifluorescence microscope coupled to a Cooke Sensicam SVGA cooled CCD camera or a Zeiss Axiovert LSM-100 confocal microscope.
- Nuclear RNAs are non-coding RNA molecules that perform their functions in the cell nucleus including the splicing, processing, modification and biogenesis of mRNA, rRNA, tRNA and ribosomal complexes, which are transported to cell cytoplasm to produce proteins.
- a combination of reversible permeablization of cell membrane and NLS (nuclear localization signal) peptide was used.
- NLS peptides which can deliver different cargos into nucleus via the nuclear import pathway, were linked to molecular beacons; the peptide- Iinked molecular beacons were delivered into the cytoplasm of living cells using Streptolysin O (SLO) (Cheung, CY. et al. (2001) Bioconjug. Chem. 12: 906-910). NLS-peptide linked molecular beacons were delivered into the nucleas of living cells of the whole cell population with near 100% efficiency, making this approach far more effective compared with microinjection.
- SLO Streptolysin O
- U3 small nucleolar RNA (U3 snoRNA), one of the best characterized nuclear RNAs, was selected as a representative target.
- U3 snoRNA is a class of nuclear RNAs that are associated with coiled bodies and transported to nucleolus for processing of rRNA, a critical step in biogenesis of ribosomes.
- U3-snoRNA-targeting molecular beacons were designed based on the secondary structure of U3 snoRNA and checked the target sequence using BLAST search to ensure high specificity
- 'random'-sequence molecular beacon ('random beacon') was designed whose specific target sequence does not match with any mammalian gene.
- the design of molecular beacons are provided in Table 3, with underlined bases represent based added to form the stem of a molecular beacon.
- Both the U3- snoRNA-targeting and 'random' sequence molecular beacons have Cy3 TKHR DOCKET NO. 820701.1135
- the stem domain of the molecular beacon was modified to introduce a functional amine group using dT-(C6)-NH 2 (Table 3).
- the specific NLS peptide sequence selected in this study was a segment of the SV40 large T antigen NLS which was shown to be able to deliver different cargos such as plasmids and nanoparticles to cell nucleus (Sebestyen, M.G. et al. (1998) Nat Biotechnol. 16: 80-5; Zanta, M.A. et al. (1999) Proc. Natl. Acad. Sci. U. S. A. 96: 91-6; Luo, D. and Saltzman, W.M. (2000) Nat. Biotechnol., 18, 33-7).
- Example 18 Tat-linked molecular beacons for detecting RNA in cytoplasm and nucleus Tat-linked molecular beacons were used to detect both the cytoplasmic and nuclear population of RNA in live cells within 45 minutes (about half the time required for combination of SLO and NLS peptide as described above).
- This approach allows detection of both cytoplasmic and nuclear RNA simultaneously using a single probe and, since Tat peptide is used, it has the ability to translocate the probe across plasma as well as nuclear membranes, i.e., without the addition of a targeting signal.
- U1 snRNA was detected. U1 snRNA is transported from nucleus to cytoplasm where its 5' cap is processed, and then transported back to the nucleus.
- RNA molecule is an essential component of the mRNA splicing machinery in cell nucleus.
- SnRNAs are transcribed by RNA polymerase II and, consequently, the resulting RNAs are capped. However, they are methylated differently from other mRNAs.
- the guanine base is methylated at position N7 as normal but, in addition, it is dimethylated at position 2. Thus most snRNAs have a characteristic 2,2,7-trimethyl-guanosine (m3G) cap.
- m3G 2,2,7-trimethyl-guanosine
- Tat peptide to molecular beacons were used to detect both the cytoplasmic and nuclear population of U1 snRNA.
- the results of the assay are shown in Figure 8.
- U1 snRNA was observed to be mainly in the perinuclear region, while in nucleus it is in a discrete, spotlike localization pattern in HDF cells.
- images were collected at different positions using z-stacks in confocal microscopy. Scans of different z -positions are shown in Figure 8.
- a single probe can be used to determine different expression levels and localization patterns of the target nucleic acid in the nucleus and cytoplasm respectively. Similar approaches may also be used to study the transport of RNA from nucleus to cytoplasm.
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JP2010520990A (en) * | 2007-02-09 | 2010-06-17 | ノースウェスタン ユニバーシティ | Particles for detecting intracellular targets |
EP2920305A1 (en) * | 2012-11-15 | 2015-09-23 | Friedrich-Schiller-Universität Jena | New cell-specifically active nucleotide molecules and application kit for the application thereof |
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WO1997023607A2 (en) * | 1995-12-21 | 1997-07-03 | The University Court Of The University Of Dundee | Presentation of antigen, introduced into cells by stimulation of macropinocytosis |
WO1999005302A1 (en) * | 1997-07-24 | 1999-02-04 | The Perkin-Elmer Corporation | Conjugates of transporter peptides and nucleic acid analogs, and their use |
US6103476A (en) * | 1993-11-12 | 2000-08-15 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled, dual conformation oligonucleotide probes, assays and kits |
US20020110826A1 (en) * | 2000-07-14 | 2002-08-15 | Nanibhushan Dattagupta | Nucleic acid hairpin probes and uses thereof |
US20030219375A1 (en) * | 1998-06-20 | 2003-11-27 | David Piwnica-Worms | Membrane-permeant peptide complexes for medical imaging, diagnostics, and pharmaceutical therapy |
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US6103476A (en) * | 1993-11-12 | 2000-08-15 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled, dual conformation oligonucleotide probes, assays and kits |
WO1997023607A2 (en) * | 1995-12-21 | 1997-07-03 | The University Court Of The University Of Dundee | Presentation of antigen, introduced into cells by stimulation of macropinocytosis |
WO1999005302A1 (en) * | 1997-07-24 | 1999-02-04 | The Perkin-Elmer Corporation | Conjugates of transporter peptides and nucleic acid analogs, and their use |
US6025140A (en) * | 1997-07-24 | 2000-02-15 | Perseptive Biosystems, Inc. | Membrane-permeable constructs for transport across a lipid membrane |
US20030219375A1 (en) * | 1998-06-20 | 2003-11-27 | David Piwnica-Worms | Membrane-permeant peptide complexes for medical imaging, diagnostics, and pharmaceutical therapy |
US20020110826A1 (en) * | 2000-07-14 | 2002-08-15 | Nanibhushan Dattagupta | Nucleic acid hairpin probes and uses thereof |
Cited By (2)
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JP2010520990A (en) * | 2007-02-09 | 2010-06-17 | ノースウェスタン ユニバーシティ | Particles for detecting intracellular targets |
EP2920305A1 (en) * | 2012-11-15 | 2015-09-23 | Friedrich-Schiller-Universität Jena | New cell-specifically active nucleotide molecules and application kit for the application thereof |
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