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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q2522/00—Reaction characterised by the use of non-enzymatic proteins
  • C12Q2522/10—Nucleic acid binding proteins
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  • C12Q2561/00—Nucleic acid detection characterised by assay method
  • C12Q2561/107—Enzyme complementation
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  • C12Q2561/00—Nucleic acid detection characterised by assay method
  • C12Q2561/113—Real time assay
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  • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
  • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence

Definitions

• the present invention is directed to compositions and methods for the in vivo detection of nucleic acids. More preferably, the compositions and methods allow for the sensitive and real time detection of RNA in vivo.
• RNA is an active participant in a multi-step process broadly determined as gene expression, which includes transcription and processing of RNA within nucleus, export from the nucleus, transport through cytoplasm and translation within ribosomes. Additionally, non-coding RNAs, an ever growing class of RNA molecules, participate in a variety of post-transcriptional and post-translational events concerning all cellular macromolecules, proteins, DNA and RNA: RNA editing, RNA modifications, DNA methylation and protein modifications (Kiss, 2002, Mattick, 2004; Huttenhofer et al, 2005). To perform these multiple functions, RNA must be in the correct cellular location at the correct time. In other words, spatial and temporal localization of RNA molecules within the cell has recently emerged as an important mechanism of cellular biology.
• RNA not only regulates protein synthesis, but also creates gradient of morphogens and determines formation of cell lineages and cell organelles (for review, see Kloc et al., 2002). IfRNA is not functioning properly, it may result in various forms of pathology.
• RNA localization in vivo One example of the difficulties currently faced in the field of RNA localization in vivo is the lack of adequate methods to estimate the diffusion coefficient of poly(A)+-mRNA within the nucleus. Values have been reported that differ by two orders of magnitude, namely, 0.03 -0.6 ⁇ 2 /sec and 9 ⁇ 2 /sec (Politz et al., 1998, Politz et al., 1999, Molenaar et al., 2004). Thus, it is evident that to better understand functions and behavior of various RNAs in vivo, new methods of studying their localization and movements within living cells are urgently needed.
• molecular beacons and caged probes do not produce signal unless they hybridize to the target or are illuminated, respectively; this allows lower background and consequently better sensitivity (Sokol et al., 1998; Perlette &Tan, 2001; Matsuo, 1998; Tsuji et al, 2001; Sei-Iida et al., 2000).
• RNA detection strategy The most severe limitation of this hybridization strategy is the low sensitivity of hybridization due to the low concentration of RNA within the cell. In most cases only highly abundant RNA species can be detected ( ⁇ -actin mRNA, c-fos niRNA, basic fibroblast growth factor RNA, or total poly (A)-RNA).
• RNA detection in vivo Another obstacle of using oligonucleotide probes for RNA detection in vivo is their fast accumulation in the nucleus (Tsuji et al., 2000; Molenaar et al., 2001).
• RNA-binding proteins and fluorescent proteins and introduction of the protein-binding tags within RNA- target.
• Two groups used systems based on interaction of the MS2 coat protein and corresponding RNA motif (Bertrand et al, 1998; Beach et al., 1999; Beach & Bloom, 2001) and another group used a system based on UlA splicing protein (Takizawa & Vale, 2000).
• RNA-specific fluorescent probes are either delivered to (e.g., molecular beacons) or are synthesized inside the cell (e.g., the enhanced green fluorescent protein, EGFP, fused to an RNA-binding protein, e.g. MS2 coat protein) [see (14,15) for reviews].
• RNA-binding protein e.g. MS2 coat protein
• the inventors of the present invention discovered a method to detect nucleic acid molecules, such as RNA molecules in vivo using real time protein complementation methods.
• the invention provides methods for detecting nucleic acids, for example RNA, in vivo with a high signal :background ratio, thus enabling detection of RNA with high sensitivity.
• the methods of the invention provide methods and components for the detection of DNA and RNA within a live cell using non-invasive methods.
• the compositions of the present invention comprise a detector molecule which comprises a detector protein that is split into two or more polypeptide fragments which are attached to nucleic acid binding motifs, which may function independently or cooperate to bind a single site.
• the detector protein is a fluorescent molecule, such as, for example EGFP.
• the fluorescent reporter is EGFP that is split into an alpha fragment (approximately amino acids 1-158) and a beta fragment (approximately amino acids 159-239).
• the alpha fragment contains mature fully formed chromophore, which does not fluoresce alone, but is primed to fluoresce and when paired with the beta fragment, immediately fluoresces.
• the immediacy of the fluorescence allows for the real-time detection of RNA in vivo which is currently unavailable in the art.
• the alpha and the beta fragments do not reassociate or fluoresce in the absence of target nucleic acid, for example target RNA.
• the fluorescent protein is green fluorescent protein (GFP) or enhanced green fluorescent protein (EGFP).
• the fluorescent protein is yellow fluorescent protein (YFP), an enhanced yellow fluorescent protein (EYFP), a blue fluorescent protein (BFP), an enhanced blue fluorescent protein (EBFP), a cyan fluorescent protein (CFP) 5 an enhanced cyan fluorescent protein (ECFP) or a red fluorescent protein (dsRED) or any other natural or genetically engineered fluorescent protein of those listed above.
• the reconstituted fluorescent proteins may comprise of a mixture of fragments from the same or a combination any of the above listed fluorescent proteins.
• the compositions and methods provide a reporter assay which allows for the real time detection of RNA in vivo.
• cells of interest are created to stably express a detector construct.
• the detector construct expresses a detector molecule which does not produce a signal from the detector protein in the absence of target RNA and thus does not fluoresce or show activity.
• the cells can also express a reporter construct encoding a reporter molecule comprising a binding site for the detector molecule's nucleic acid binding motif(s) and a gene of interest. The expression of such a reporter construct can be induced.
• the detector molecule When such a reporter molecule is expressed within a cell, the detector molecule will bind the target RNA and facilitate detector protein reconstitution and production of an active protein and signal. This allows for the real time detection of RNA expression in vivo and is exemplified in Figures 10 -11, and 18-19 and Examples 9 to 11.
• the cells of interest can express a bicistronic nucleic acid sequence comprising the reporter molecule where the detector construct is constitutively expressed and the expression of the reporter construct is operatively linked to a promoter of interest.
• the reporter construct (operatively linked to a promoter of interest) and detector construct are on separate nucleic acid sequences.
• the binding site(s) for the target nucleic acid sequence becomes available and binding of the nucleic acid binding protein of the detector molecule facilitates the association of the detector polypeptide fragments and formation of the active detector protein which can be immediately detected.
• This embodiment allows for promoter function to be studied in real time in vivo.
• the regulation of a gene can be studied in real time by transfecting a plasmid expressing a promoter driving an RNA binding site and analyzing any baseline detector construct activity.
• the cells are contacted with i) a compound or compounds; or ii) subjected to a variety of conditions that may alter the activity of the promoter, thereby increasing, decreasing or causing no change in the transcription of the RNA. This alteration is immediately detected by the detector construct.
• a "tet-on" or “tet-off system may be utilized in the methods of the present invention.
• cells of interest may be created to stably express the detector construct of the present invention.
• the cells may also stably express a tet-on or tet-off regulatable reporter construct enabling controlled transcription of the target binding site(s) and gene of interest for the detector molecules nucleic acid binding motifs).
• a tet-off system the cells would immediately express the gene of interest and the reporter construct would be active and immediately detectable.
• the transcription of the reporter construct (comprising the gene of interest and target nucleic acid binding site) is shut off and only the transcripts that have been already transcribed can be analyzed in real time.
• the stability of RNA such as for example, its half-life, in vivo in real time by detecting the diminishing reporter fluorescence or activity.
• the reporter assay is in vivo in an organism.
• animals, non-human animals, mouse, zebrafish, C. elegans, or frog may be created to express both a detector construct and a gene of interest with an RNA binding site that will bind to the detector construct.
• the expression of both constructs within the organism allows for a transcript of interest to be analyzed throughout development by taking samples from Day 1 embryo throughout adulthood.
• the organism is a mammal.
• the mammal is a mouse
• the organism is a transgenic organism, for example but not limited to a transgenic mouse.
• RNA probes used in traditional in situ hybridization are difficult to prepare due to the ease of RNA degradation and the use of radioactivity. In the methods of the present invention there is no need for radioactivity or RNA probes.
• the detector constructs are easily prepared, for example, from bacteria, as is known to those of skill in the art. Such an assay is similar to immunohisto- or immunocyto-chemistry techniques whereby antibodies are used to detect protein in vitro. Here, RNA is detected.
• compositions and methods of the present invention provide for the real time detection of nucleic acid, in particular RNA and DNA in vivo, which relates to use of the invention when both the RNA and protein constructs are manipulated in cells out side the body, for example within a test tube, as a non-limiting example, detection of RNA in cells that are ex vivo.
• the compositions and methods of the present invention may be used in embodiments to detect DNA in vivo in real time.
• the detector construct of the present invention may comprise a detector protein (either fluorescent or enzymatic) that is split into at least two inactivated polypeptide fragments, each fragment associated with a nucleic acid-binding motif.
• the polypeptide fragments when brought together by the presence of target nucleic acid, for example the aptamer, form a fully active detector protein, and can be immediately detected.
• the methods of the present invention allow for the real time detection of replication of various loci within the genome.
• the nucleic acid- binding motifs associated with the detector proteins in the detector construct are specific for various regions of the RNA or DNA and the replication of those loci can be detected in real time in vivo.
• the detector construct may comprise a detector protein associated with telomerase binding proteins and expressed within cells.
• telomerase binding proteins associated with telomerase binding proteins
• teleomeres can be detected in real time over the lifespan of the cell.
• the advantage of the compositions and methods presented herein is that the specificity is increased by providing a telomerase binding protein that is dissected into two halves, each half associated with one half of the detector protein. The coordinated binding of the two halves of the telomerase binding proteins to a single binding site ensures that there is little to no detector activity prior to target recognition.
• kits suitable for the method of the invention in particular detecting the RNA in vivo.
• the kits comprise the detector construct and the inducible reporter construct.
• the reporter construct and detector construct are on separate nucleic acid molecules. In another embodiment, they are on both part of bicistronic nucleic acid molecule.
• the reporter molecule can be modified to add the practitioner's gene of interest, and in another embodiment, the expression of the reporter molecule is operatively linked to a tet-on or tet-off promoter.
• the reporter construct, comprising the target nucleic acid sequence can be modified by the practitioner to be regulated by the practitioner's promoter of interest.
• kits comprise a choice of split polypeptides for the detector protein in the detector molecule, and also comprise instructions and reagents for detecting a signal from the detector protein.
• the kits also comprise reagents suitable for capturing and/or detecting the present or amount of target nucleic acid in a sample.
• the reagents for detecting the present and/or amount of target nucleic acid can include enzymatic activity reagents or an antibody specific for the assembled protein. The antibody can be labeled.
• the combination of protein complementation and an enzymatic reporter allows for a substantially reduced background and signal amplification. This results in an in vivo RNA detection technique capable of analyzing nucleic acids of moderate abundance.
• kits that comprise means for detecting RNA in vivo.
• Figure 1 Transcriptional system to study the effect of the aptamer on gene expression in Saccharomyces cervisiae (Blake et al., 2003).
• Figure 2 Insertion of the aptamer at the 3' end of the marker gene does not affect gene expression in yeast.
• An aptamer tag was inserted at the 3' end of EGFP (see Fig.l for plasmids description). Cultures were grown to a logarithmic phase in the presence of 0.2% galactose and 40 ng/ml ATc. Numbers 1-4 correspond to four different chromosomal integrations of the inducible construct. 50,000 cells were assayed by FACS in each culture.
• Figure 4 Expression of two chimeric proteins in bacterial cells in the absence of aptamer-tag does not reconstitute fluorescence.
• EGFP(A)-eIF4A(Fl) and EGFP(B)- eIF4A(Fl) were co-expressed in BL21 (DE3) cells. Cultures were induced with ImM IPTG for 3 hours at 37°C and complex formation was monitored by fluorescence flow cytometry.
• BL21 (DE3) cells expressing full-length EGFP were used as a positive control for induction (bEGFP), and expression of A-Fl and B-Fl was considered as a negative control.
• Figures 5 Schematics of protein complementation in vivo with two adjacent aptamer-tags and two peptides independently interacting with cognate aptamer.
• the split marker protein is in grey, and the RNA-binding peptides (probe protein) are in orange.
• Figure 5B Three aptamers found by in vitro selection against CQ peptide of HIV Tat protein (SEQ ID NO.2); HIV rev peptide (SED ID NO.l) and HTLV Rex peptide (SEQ ID NO.3), respectively (see Table 1 for Kd and refs). These or similar structures can be used in our project as RNA tags in an alternative design (as opposed to split-protein approach).
• Figure 6 Outline of the protein complementation assay supported by nucleic acids interactions for RNA studies in vivo.
• a protein with enzymatic activity or fluorogenic properties is split into two inactive parts (termed alpha- and beta-subunits). These parts are expressed in vivo as chimeras with another protein, which has an RNA-binding domain.
• An ideal RNA-binding protein would consist of two parts, which are inactive by itself but are active together. In the presence of an RNA, which contains a motif recognizable by the RNA-binding protein, the activity of the marker protein is restored. In case the protein is an enzyme, the signal is amplified. In case the protein is fluorogenic, there is no signal amplification, but there is also no background, because fluorescence is completely dependent on the presence of target RNA.
• Figure 7 Schematics of RNA detection in vivo.
• Target RNA is synthesized in vivo or injected into the cell.
• Two protein chimeras are synthesized in vivo or transfected into a cell.
• Each chimera contains a part of a marker protein and a domain, which specifically binds special secondary structure (or motif) within the target RNA.
• a protein marker is assembled, which is detected by its activity (fluorescence or enzymatic activity).
• Figure 8 Schematic of one embodiment of the invention whereby RNA expression is analyzed in vivo in real time.
• a gene of interest and a RNA binding site is encoded on a plasmid and transfected into cells that have the reporter construct of the present invention stably integrated.
• the reporter construct is activated.
• Figure 11 The kinetics of fluorescence facilitated by RNA detection in living cells, (b) Kinetics of fluorescence changes in different single cells (c) Kinetics of total fluorescence changes of all cells in the field, (d) Changes in fluorescence distributions in real-time in a single bacterial cell measured along the long axis of the cell.
• Figure 13 Cells expressing aptamer-containing target RNA from a higher copy vector (-100 copies) display higher fluorescence, (a) Schematics of plasmids expressing protein fusions and RNA transcript containing aptamer sequence, (b) Fluorescence distributions of the cells expressing EGFP complementing complexes obtained by FACS. Black, before IPTG induction; red, after IPTG induction. For comparison, fluorescence distribution of the cells with aptamer expressed from the lower copy number ( ⁇ 40) plasmid is shown in blue.
• Figure 17 Protein complementation of split EGFP based on binary aptamer/peptide interaction detects RNA in live bacterial cells. Low concentration of IPTG (0.01 niM) allows discrimination of signal from background. A, Fluorescence distributions of cells induced by 1.00 mM IPTG. B, Fluorescence distributions of the cells induced by decreasing concentrations of IPTG.
• Figure 21 Mg2+ ions affect differently fluorescence of native and reconstructed EGFP.
• Figures 22 A and 22B Figure 22A: Fast kinetics of fluorescence increase upon mixing together ⁇ - and ⁇ -fragments of EGFP with appended complementary 21-nt long oligonucleotides.
• Figure 22B Kinetic pathway of chromophore formationin S65T with tl/2 of all the steps (from Zimmer, 2002).
• Figures 23A-4B Folded ⁇ -fragment of EGFP may contain pre-formed chromophore.
• Figure 23 A Backbone representation often aligned typical folded structures of ⁇ -fragment from discrete molecular dynamics (DMD) simulations (Dokholyan, unpublished). Chromophore-forming amino acids are shown in blue. Note the tight packing of the N-terminal major part of the fragment and very flexible C-terminal part of the remaining fragment due to the small number of contacts with the rest of the molecule.
• Figure 23B Alignment of the folded ⁇ -domain and full-length EGFP. The full-length EGFP is colored yellow and the ⁇ -domain is colored blue.
• Chromophore-forming residues (#62-70) are shown in red.
• Figure 23 C The average root-mean-square-deviation (RMSD) of each residue in the ⁇ -fragment of EGFP compared to amino acids in full-size EGFP.
• the RMSD values are calculated from DMD simulations at low temperatures when the protein is folded.
• the chromophore-forming amino acids are in a shaded area and their deviation is ⁇ 2 A.
• Figures 24A-24D ⁇ -fragment of EGFP contains pre-formed chromophore.
• Figure 24 A Absorbance spectrum of EGFP
• Figure 24B Absorbance of ⁇ - and ⁇ - fragments of EGFP
• Figure 24C Fluorescence spectra of EGFP (blue- excitation, pink - emission)
• Figure 24D Fluorescence spectra of ⁇ -fragment (blue - excitation, pink - emission). Equimolar concentrations of all proteins were subjected to spectral analyses.
• Figures 25-25B Absorbance of the chromophore-containing peptide isolated from GFP by partial proteolysis (Figure 25A) and of the chemically synthesized chromophore at acidic/neutral pH ( Figure 25B) (from Niwa et al, 1996).
• Figure 26 Two possible arrangements of nucleic acid interactions as support for protein complementation assay.
• Figure 27 Purification of beta-fragment of EGFP (beta-cys) expressed in E.coli as chimera with self-splitting intein.
• the protein in lane 6 is pure ⁇ -subunit of EGFP obtained after self-splitting of intein.
• Figure 28 Purification of the alpha-fragment of EGFP expressed in E.coli as chimera with self-splitting intein. Protein in lane 5 is pure ⁇ -subunit of EGFP.
• Figure 29 Conjugation of the proteins with SH-containing oligonucleotides is almost 100% complete. Analysis using non-denaturing PAGE. After conjugation, the proteins are linked to oligonucleotides bearing negative charge. Therefore, modified proteins move faster than unmodified (compare lane 1 and 2, and 3 and 4). If oligonucleotide is biotinylated, it can make a complex with streptavidin. Such complex also moves faster than streptavidin alone (compare lanes 6 and 7). From this data we conclude that the efficiency of oligonucleotide coupling with protein is close to 100%.
• Figure 30 Fluorescence of the combined alpha+A and beta+B shows reconstruction of the active EGFP, while in the absence of oligonucleotides there is no active EGFP.
• Figure 31 Schematics of protein complementation with restoration of protein enzymatic activity. The principle is similar to Figure 6. Re-assembly of the enzyme is dependent on interaction of RNA with RNA-binding protein. An active re-assembled enzyme is splitting its substrate, which results in a chromogenic or fluorogenic product. Signal is thus amplified due to the enzymatic activity of the protein.
• the inventors have discovered a novel method for rapid detection of RNA within a living cell which can be detected with a high signal to background ratio that enables high sensitivity of detection.
• the present invention comprises compositions and methods for the sensitive detection of nucleic acids, for example RNA and DNA in vitro and in vivo in real time.
• the present invention comprises a method using a detector molecule comprising a split-detector protein into at least two polypeptide fragments which are attached to nucleic acid binding motifs, for example RNA-binding motifs, which may function independently or cooperate to bind a single nucleic acid binding site.
• Re-association of the detector protein fragments into a functional detector protein will only occur in the presence of a target nucleic acid molecule as a result of interaction of the RNA-binding motifs with their cognate binding site(s) on the nucleic acid. This interaction will bring together the complementary polypeptide fragments of the detector protein, allowing for signal detection.
• This invention provides an innovative method for visualizing nucleic acids, such as RNA, in vivo, is based on protein complementation, meaning the re-association of two or more polypeptide protein fragments into an active protein.
• the split polypeptides are in an active configuration, yet inactive alone and therefore their association with the complementary polypeptide fragment immediately forms an active detector protein.
• complementation takes place only if it is supported by additional nucleic acid/protein interactions.
• high-affinity and high-specificity protein/RNA aptamer interactions are described.
• An aptamer is expressed as a recognition tag within the target RNA 5 while the nucleic acid binding motif is synthesized as two inactive fragments fused to the fragments of the split detector protein. Interaction of nucleic acid binding motif fragments with the aptamer brings the detector protein fragments together, which results in enzymatic activity or fluorescence of the re-assembled detector protein inside within the cell.
• the methods described herein can be used for the real time detection of nucleic acids,
• a detector construct comprising a nucleic acid sequence encoding a detector molecule, the detector molecule comprising one polypeptide fragment of a detector protein conjugated with a nucleic acid binding motif and at least one other polypeptide fragment of the detector protein conjugated to a nucleic acid binding motif is described.
• the assay utilizes a reporter construct, encoding a reporter molecule comprising a nucleotide of interest and a nucleic acid binding sequence.
• the nucleic acid binding sequence is recognized by the nucleic acid binding motifs of the detector molecule. This recognition allows for the detector protein to be reconstituted and detected in real time and immediately.
• the detector protein does not have activity in the absence of target nucleic acid and thus is highly sensitive and exhibits low background.
• the fragmentation of the detector protein is designed so that on recognition in the presence of target nucleic acid sequences the activity is immediately detectable.
• the nucleic acid is RNA. Any RNA can be detected, selected from a group comprising mRNA, and miRNA. Alternatively, the nucleic acid is DNA.
• the assay of the present invention describes as a nucleic acid binding motif.
• the motif may be associated with the detector protein in a way in which the nucleic acid binding motif is split into a polypeptide fragment, and one fragment is conjugated with one fragment of the detector protein and rest of the nucleic acid polypeptide fragments are conjugated with one or more other fragments of the detector protein.
• the binding of the motif is coordinated and each fragment must be present to bind to one nucleic acid binding sequence.
• the nucleic acid binding motif that is associated with one polypeptide fragment of the detector protein may be a full-length motif that is independent from the other nucleic acid binding motif which is conjugated with one or more other fragments of the detector protein.
• the nucleic acid binding protein may be a small multi-domain nucleic acid binding protein, therefore each domain associated with one or more fragments of the detector protein.
• the binding of the nucleic acid binding motifs to their cognate nucleic acid sequences in the reporter molecule (or native nucleic acid) is independent.
• the detector and reporter molecules are expressed within a cell in vivo.
• the nucleic acid sequences encoding the detector and reporter molecules may be inserted into the cell by any suitable method known by persons skilled in the art, e.g. transformation or transfection.
• the constructs may, for example, be on a single nucleic acid sequence. In an alternative embodiment, for example, they may be on two constructs (one comprising the reporter construct and one comprising the detector construct). Such constructs may be co- transformed or co-transfected into the cell. Without being bound by theory, it is proposed that the co-transformed/co-transfected constructs may recombine during transformation/transfection with the result that both are integrated at the same site in the cell's genomic DNA.
• the detector and reporter constructs may alone or in combination, be stably expressed within a cell.
• Stable clones producing high levels of recombinant protein are obtained after transfection of cells with an expression vector encoding the desired genes of interest and a dominant genetic marker. Methods for stable integration into a variety of cell types are known to those of skill in the art.
• the nucleic acid may encode the split-polypeptide fragments (comprising the detector protein and nucleic acid binding motif) that reconstitute to form the active detector protein.
• the split-polypeptide fragments may, for example, be linked by an internal ribosome entry site (IRES).
• IRES allows for the production of a single transcript from two or more separate genes which can be translated into corresponding separate products due to the presence of an additional ribosome entry site(s) on the transcript.
• the split-polypeptide fragments and/or detector proteins and/or nucleic acid binding motifs may be encoded by one nucleic acid, with splittable sites between each nucleic acid sequence to enable separation of the desired polypeptides.
• a nucleic acid could comprise a sequence encoding the active detector protein comprising at least two nucleic acid binding motifs, where the sequence encoding the detector protein comprises a splittable site to enable generation of split-polypeptide fragments of the detector protein each comprising a nucleic acid binding motif.
• Methods to introduce nucleic acid sequences into cells are known by persons skilled in the art, and include for example the detector and reporter constructs may be introduced into the cell by multiple means including vectors, viral vectors, and non-viral means.
• Non-viral means include without limitation, fusion, electroporation, biolistics, transfection, lipofection, protoplast fusion, calcium phosphate transfection, microinjection, pressure-forced entry, naked DNA etc. or any other means known any person skilled in the art.
• Vectors comprising useful elements such as bacterial or yeast origins of replication, selectable and/amplifiable markers, promoter/enhancer elements for the expression in prokaryotes or eukaryotes, and mammalian expression control elements etc, may be used to prepare stocks of nucleic acid constructs and for carrying out transfections are well known in the art, and many are commercially available.
• split-polypeptide fragments of the detector construct which have a polypeptides as the nucleic acid binding moiety
• the entire split-polypeptide fragment and nucleic acid binding moiety molecule can be encoded by a single construct, including the polypeptide portion, a linker and the nucleic acid binding moiety polypeptide.
• This construct can either be expressed in the cell or microinjected into the cell. These constructs can also be used for in vitro detection of a nucleic acid of interest.
• Patent Publication 20050222400 when the oligonucleotide is expressed, the aptamer is released upon cleavage of the self-cleaving ribozymes, and the aptamer that is released has short flanking sequences that are remnants of the self-cleaving ribozymes, which are highly unlikely to interfere with the proper folding of the aptamer sequence, as opposed to prior art methodologies. Without being limited to any particular mechanism, it is believed that these short flanking sequences do not substantially interfere with the aptamer function.
• Examples of zinc fingers include Zif 2g8, SpI, finger 5 of Gfi-1, finger 3 of YYl, finger 4 and 6 of CF2II, and finger 2 of TTK (PNAS (2000) 97: 1495-1500; J Biol Chem (20010 276 (21): 29466-78; Nucl Acids Res (2001) 29 (24) :4920-9; Nucl Acid Res (2001) 29(11): 2427-36).
• Other polypeptides include polypeptides, obtained by in vitro selection, that bind to specific nucleic acids sequences.
• aptamers examples include platelet-derived growth factor (PDGF) (Nat Biotech (2002) 20:473-77) and thrombin (Nature(1992) 355: 564-6.
• PDGF platelet-derived growth factor
• thrombin Nile(1992) 355: 564-6.
• polypeptides are polypeptides which bind to DNA triplexes in vitro; examples include members of the heteronuclear ribonucleic particles (hnRNP) proteins such as hnRNP K, L 5 El, A2/B1 and I (Nucl Acids Res (2001)29(11): 2427-36).
• the nucleic acid binding moiety of a given pair of activated split-polypeptide fragment can be of the same kind of molecule, for example oligonucleotides, or they can be different, for example one split-polypeptide of a pair comprise an active protein can have an oligonucleotide nucleic acid binding moiety, and the other member of the pair can have a polypeptide nucleic acid binding moiety.
• the cognate non-fluorescent polypeptide fragment which combines with the mature chromophore-containing split-fluorescent fragment can comprise of more than one active non-fluorescent fragment.
• Such activated non-fluorescent polypeptides are usually produced by splitting the coding nucleotide sequence of one fluorescent protein at an appropriate site and expressing each nucleotide sequence fragment independently.
• the activated split-fluorescent protein fragments may be expressed alone or in fusion with one or more protein fusion partners.
• association of activated split- polypeptide fragments can form an assembled protein which contains a discontinuous epitope, which may be detected by use of an antibody which specifically recognizes the discontinuous epitope on the assembled protein but not the partial epitope present on either individual polypeptide.
• a discontinuous epitope is found in gpl20 of HIV.
• the detector proteins may be conjugated to the nucleic acid binding motif by any means known to persons skilled in the art.
• the detector protein is conjugated to the nucleic acid binding motif by gene fusion.
• conjugate used herein refers to the attachment of two or more proteins joined together to form one entity.
• the proteins may be attached together by linkers, chemical modification, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusion or by any means known to one skilled in the art.
• the joining may be permanent or reversible.
• several linkers may be included in order to take advantage of desired properties of each linker and each protein in the conjugate.
• Peptide linkers may be linked by expressing DNA encoding the linker to one or more proteins in the conjugate.
• Linkers may be acid cleavable, photocleavable and heat sensitive linkers.
• fusion protein refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by a nucleic acid sequence encoding one protein is joined to the nucleic acid encoding another protein such that they constitute a single open-reading frame that can be translated in the cells into a single polypeptide harboring all the intended proteins. The order of arrangement of the proteins can vary. As a non-limiting example, the nucleic acid sequence encoding the split-detector polypeptide fragment is fused in frame to an end, either the 5' or the 3' end, of a nucleic acid encoding the nucleic acid binding motif.
• nucleic acid sequence encoding the detector molecule and reporter molecule employs standard ligation techniques.
• the ligation mixtures may be used to transfect/transduce a host cell and successful genetically altered cells may be selected by antibiotic resistance where appropriate.
• Vectors from the transfected/transduced cells are prepared, analyzed by restriction and/or sequenced by, for example, the method of Messing, et al., (Nucleic Acids Res., 9: 309-.1981), the method of Maxam, et al., (Methods in Enzymology, 65: 499, 1980), the Sanger dideoxy-method or other suitable methods which will be known to those skilled in the art.
• LTR long terminal repeat
• MMV Moloney mouse leukemia virus
• RSV Rous sarcoma virus
• Other potent promoters include those derived from cytomegalovirus (CMV) and other wild-type viral promoters.
• promoter and enhancer regions of a number of non-viral promoters have also been described (Schmidt et al., Nature 314: 285 (1985); Rossi and decrombrugghe, Proc. Natl. Acad. Sci. USA 84: 5590-5594 (1987)).
• Methods for maintaining and increasing expression of transgenes in quiescent cells include the use of promoters including collagen type I (1 and 2) (Prockop and Kivirikko, N. Eng. J. Med. 311 : 376 (1984); Smith and Niles, Biochem. 19: 1820 (1980); de Wet et al, J. Biol. Chem., 258: 14385 (1983)), SV40 and LTR promoters.
• the promoter is a constitutive promoter selected from the group consisting of: ubiquitin promoter, CMV promoter, JeT promoter, SV40 promoter, Elongation Factor 1 alpha promoter (EFl -alpha), chick beta-actin, PGK, MT-I (Metallothionin).
• inducible/repressible promoters are also encompassed in the present invention.
• these constructs are the “Tet-On”, “Tet-Off” , and Rapamycin-inducible promoter, which are encompassed in the present invention.
• an enhancer sequence may be used to increase the level of transgene expression. Enhancers can increase the transcriptional activity not only of their native gene but also of some foreign genes (Armelor, Proc. Natl. Acad. Sci. USA 70 : 2702 (1973)).
• collagen enhancer sequences are used with the collagen promoter 2 (I) to increase transgene expression.
• the enhancer element found in SV40 viruses may be used to increase transgene expression. This enhancer sequence consists of a 72 base pair repeat as described by Grass et al., Proc. Natl. Acad. Sci.
• Transgene expression may also be increased for long term stable expression using cytokines to modulate promoter activity.
• cytokines have been reported to modulate the expression of transgene from collagen 2 (I) and LTR promoters (Chua et al., connective Tissue Res., 25: 161-170 (1990); Elias et al., Annals N. Y. Acad. Sci., 580 : 233- 244 (1990)); Seliger et al., J. Immunol. 141: 2138-2144 (1988) and Seliger et al., J. Virology 62: 619-621 (1988)).
• transforming growth factor TGF
• IL interleukin
• INF interferon
• TGF Tumor necrosis factor
• TGF 1 up regulate, and may be used to control, expression of transgenes driven by a promoter.
• Other cytokines include basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).
• a collagen promoter with the collagen enhancer sequence can also be used to increase transgene expression by suppressing further any immune response to the vector which may be generated in a treated brain notwithstanding its immune-protected status.
• a transgenic organism that expresses a reporter and detector construct for the real time detection of nucleic acids in vivo.
• the transgenic organisms, or animals may carry the transgene in all their cells or in some, but not all cells, i.e., mosaic animals.
• the transgene can be integrated as a single transgene or in tandem, e.g., head to head tandems, or head to tail, or tail to tail, or as multiple copies. Double, triple, or multimeric transgenic animals may preferably comprise at least two or more transgenes.
• the animal comprises the a detector construct comprising two halves of the EGFP transgene operably linked to a nucleic acid binding motif and a transgene encoding a nucleic acid binding sequence and a gene of interest.
• a transgene construct described herein may include a 3' untranslated region downstream of the DNA sequence. Such regions can stabilize the RNA transcript of the expression system and thus increase the yield of desired protein from the expression system.
• 3' untranslated regions useful in the constructs of this invention are sequences that provide a polyA signal. Such sequences may be derived, e.g., from the SV40 small t antigen, or other 3' untranslated sequences well known in the art.
• the length of the 3' untranslated region is not critical but the stabilizing effect of its polyA transcript appears important in stabilizing the RNA of the expression sequence.
• the method of the invention can be used in multiple applications known by persons skilled in the art.
• One important embodiment of the present invention is a reporter assay for the real time detection of RNA in vivo.
• cells of interest stably express a detector molecule which does not produce a signal from the detector protein in the absence of target RNA and thus does not fluoresce or show activity.
• the cells can also express an inducible reporter molecule comprising a binding site for the detector molecule's nucleic acid binding motif(s) and a gene of interest, which when expressed within a cell, the detector molecule will bind the target RNA and facilitate detector protein reconstitution and production of an active protein and signal.
• This allows for the real time detection of RNA expression in vivo and is exemplified in Figures 10-11 and 18-19 and Examples 9-11.
• compositions and methods of the present invention provide for the real time detection of RNA in an in vivo method similar to in situ hybridization, for example Fluorescence in situ Hybridization (FISH).
• FISH Fluorescence in situ Hybridization
• a cell expressing a reporter construct comprising a RNA of interest with a RNA binding sequence that will bind to the nucleic acid binding motif of the detector molecule is permeablized, and the detector constructs of the invention are administered to the cell. If the RNA of interest is expressed, the detector construct will bind to the RNA binding sequences and the localization and abundance of RNA can be determined.
• a method to detect PCR products in real time is disclosed.
• the products of a PCR reaction are detected with higher specificity than is present available and in real time.
• a PCR primer is designed so that the amplification product of the reaction incorporates an aptamer that can be specifically recognized by a nucleic acid-binding motif present on a detector construct.
• the detector protein can be detected in real time.
• the present invention provides advantages over currently used techniques, such as SYBR green, because the detector construct is specific for PCR products and would not detect template DNA.
• the present invention allows for the real-time detection of gene mutations, polymorphisms, or aberrations in an individual.
• an aptamer could tag or transiently attach to a specific mutation or polymorphism site, which could be detected by the split-polypeptide molecule of the present invention that is designed so that the nucleic acid binding motifs of the detector molecule recognizes the attached aptamer associated to a gene of interest comprising specific mutations, polymorphisms or aberration one is trying to detect.
• a pool of molecules may be used whereby many mutations, polymorphisms, or aberrations may be detected.
• Rotavirus C Sindbis virus, Simian hnmunodeficiency cirus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Enmunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.
• the target nucleic acid can be of human origin.
• the target nucleic acid can be DNA or RNA.
• the target nucleic acid can be free in solution or immobilized to a solid support.
• the methods of the invention can be used for protein complementation for multiple nucleic acid targets simultaneously.
• protein complementation of complementary split-polypeptide fragments which have associated different nucleic acid binding motifs.
• the presence of one target nucleic acid will facilitate protein complementation of one active split-polypeptide fragment pair, while the presence of another target will facilitate protein complementation of anther pair of activated split- polypeptide fragments, resulting in a different active protein and detectable signal.
• multiple nucleic acid targets can be detected simultaneously.
• simultaneous detection of target nucleic acids, such as RNA and DNA can be monitored by real-time protein complementation.
• the multiple protein complementation using split- fluorescent protein fragments from different fluorescent proteins enable real-time detection and identification of specific target nucleic among a variety of other putative but different nucleic acid targets (see Hu et al, Nature Biotechnology, 2003;21;539-545; Kerppola, 2006, 7;449-456, Hu, et al, Protein- Protein Interactions (Ed. P. Adams and E. Golemis), Cold Spring Harbor Laboratory Press. 2005, herein incorporated by reference in its entirety).
• IRES refers to internal ribosome entry sites (see Kozak (1991) J. Biol. Chem. 266'19867-70) are sequences encoding consensus ribosome binding sites, and can be inserted immediately 5' of the start codon and/or regulatory sequences or elsewhere 5' of nucleic acid sequences encoding genes or marker genes to enhance expression of the downstream nucleic acid sequence. The desirably of, or need for, such modification may be empirically determined.
• the ⁇ -fragment of EGFP comprises a pre-formed chromophore. Though with some small spectral differences, up to 100% fluorescence can be restored by nucleic acid- based protein complementation.
• the kinetics of the DNA-templated EGFP re-assembly was surprisingly fast with the 1 1/2 ⁇ 100 sec (see Fig.22), being close to kinetics of renaturation of EGFP from denatured protein with mature chromophore (Reid & Flynn, 1997, Zimmer, 2000). Of note, typical chromophore maturation in fluorescent proteins requires hours (Zimmer, 2000, Fig.22b).
• B-F2 Cloning of two chimeric proteins into two MOSs of pACYCDuet-1.
• the B-F2 fragment was next integrated into the second MCS of pMB12 already carrying A-FIl, according to the described protocol (Geiser et al, 2001). Briefly, B-F2 was PCR-amplified from the plasmid pMBI3 with flanking 5' and 3' vector sequences. The flanking sequences corresponded to 20 bp of DNA immediately upstream and downstream from the point of insertion in pMB12. A PCR reaction was performed with the recipient vector and the B-F2 fragment which would anneal to the uncut vector via the 20 bp flanking homology.
• Fluorescent measurements and cell imaging Total cell fluorescence will be measured with Becton-Dickinson fluorescence activated cell sorter (FACSCalibur, with the 488 nm excitation argon laser) and will be analyzed using Excel software. Cell imaging will be performed using a confocal microscope system with automated switching between fluorescence and differential interference contrast (DIC). The magnified specimen image xl50 will be directly transmitted onto the cooled, slow-scan CCD imaging device (C4880; Hamamatsu Photonics, Bridgewater, NJ).
• FACSCalibur Becton-Dickinson fluorescence activated cell sorter
• DIC differential interference contrast
• MALDI-TOF MS and Quantitative Analysis Prior to MALDI-TOF MS analysis, salts from the reactions were removed using SpectroCLEAN resin and 16 ⁇ L of water. ASV analysis was performed using the MassARRAY system (Sequenom) by dispensing approximately 10 nL of final product onto a 384-plate format MALDI-TOF MS SpectroCHIP using a SpectroPOINT nanodispenser (Sequenom). Mass spectrometries data were analyzed using TITAN (Elvidge et al, 2005) software set at the default values.

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