# Patent

## Title
Modified renilla luciferase nucleic acids and methods of use

## Abstract

This invention provides modified nucleotide sequences encoding luciferase that have greater expression than wild type luciferase.

## Background
CROSS-REFERENCE
This application claims the benefit of the priority date of U.S. application 61/397,340, filed Jun. 10, 2010, the contents of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
Reporter genes are important tools that enable researchers to study DNA sequences that regulate gene expression. Light emitting proteins, such as luciferases, are useful reporter genes since the amount of protein produced can be measured as light output. Luciferase genes have been isolated from a variety of organisms such as insects and a variety of marine organisms. The luciferase gene from the sea pansy (Renilla reniformis) is a widely used luciferase reporter and is commonly referred to as “Renilla”. 
Marine luciferases have become popular alternatives to firefly luciferase as a genetic reporter based on assay simplicity, high sensitivity, and a broad linear range of signal that provides greater sensitivity over firefly luciferases. The Renilla luciferase protein catalyzes oxidation of its coelenterazine substrate in the reaction shown below to produce light at 480 nm, easily read by standard luminometers. U.S. Pat. No. 5,418,155 (CORMIER, M. J. et al., May 23, 1995) provides an amino acid sequence of Renilla luciferase and a nucleotide sequence encoding it. U.S. patent application 2006/0068395 (WOOD, K. V. et al., Mar. 30, 2006) provides another nucleotide sequence encoding Renilla luciferase.
SUMMARY OF THE INVENTION
In one aspect this invention provides an isolated nucleic acid molecule comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2. In one embodiment the luciferase nucleotide sequence has at least 95%, at least 98%, at least 99% or at least 99.9% sequence identity with SEQ ID NO: 2. In another embodiment the luciferase nucleotide sequence is SEQ ID NO: 2. In another embodiment the polypeptide is Renilla luciferase (SEQ ID NO: 1) (RenS). In another embodiment the luciferase nucleotide sequence is SEQ ID NO: 2. the polypeptide is a fusion protein comprising the Renilla luciferase. In another embodiment the luciferase nucleotide sequence is SEQ ID NO: 2. In another embodiment the fusion protein is RenSP (SEQ ID NO: 3). In another embodiment the reporter nucleotide sequence is SEQ ID NO: 4. In another embodiment the polypeptide comprises a protein destabilization domain that decreases the half-life of the luciferase protein. In another embodiment the luciferase nucleotide sequence contains no more than 10, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 or no regulatory motifs selected from the motifs of FIG. 3. In another embodiment the luciferase nucleotide sequence, when operably linked to a human RPL10 promoter in an expression plasmid, transfected into HT1080 cells and incubated for 30 minutes with a luciferase substrate, expresses at least 10% more signal than hRluc under the same conditions.
In another aspect this invention provides a recombinant nucleic acid comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2. In one embodiment the recombinant nucleic acid comprises an expression control sequence operably linked with the nucleotide sequence. In another embodiment the expression control sequence comprises a mammalian promoter. In another embodiment the expression control sequence comprises a human promoter. In another embodiment the recombinant nucleic acid is contained in a vector is selected from a plasmid vector, a viral vector, a cosmid, a transposon and an artificial chromosome. In another embodiment the recombinant nucleic acid of comprises a cloning site positioned relative to the nucleotide sequence such that an expression control sequence inserted into the cloning site becomes operably linked with the luciferase-encoding nucleotide sequence. In another embodiment the cloning site comprises a plurality of restriction sites. In another embodiment the cloning site is positioned to place a promoter in operable linkage with the luciferase-encoding nucleotide sequence. In another embodiment the cloning site is positioned to place a 5′ UTR or a 3′UTR in operable linkage with the luciferase-encoding nucleotide sequence. In another embodiment the recombinant nucleic acid further comprises a promoter operably linked with the luciferase-encoding nucleotide sequence. In another embodiment the promoter is a promoter of the RPL10 gene. In another embodiment the vector is a plasmid comprising an origin or replication, a selectable marker, an origin of replication and a polyA sequence operatively linked with the nucleotide sequence.
In another aspect this invention provides a vector comprising a recombinant nucleic acid comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2.
In another aspect this invention provides a recombinant cell comprising a recombinant nucleic acid comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2. In one embodiment the recombinant nucleic acid comprises an expression control sequence operably linked with the nucleotide sequence. In another embodiment the recombinant nucleic acid is contained in a vector is selected from a plasmid vector, a viral vector, a cosmid, a transposon and an artificial chromosome. In another embodiment the recombinant cell is a mammalian cell, e.g. a human cell. In another embodiment the expression construct is stably integrated in the genome of the host cell. In another embodiment the recombinant nucleic acid is maintained in the cell as a stable episomal vector. In another embodiment the recombinant nucleic acid is integrated into the genome of the host cell.
In another aspect this invention provides a kit comprising a vector comprising a recombinant nucleic acid comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2 and a luciferase substrate.
In another aspect this invention provides a device comprising a plurality of receptacles, wherein each receptacle comprises a vector comprising a recombinant nucleic acid comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2. In one embodiment a plurality of the expression constructs comprises a different promoter. In another embodiment the plurality of the expression constructs comprises the same promoter.
In another aspect this invention provides a method comprising measuring a signal produced by a recombinant cell comprising an expression construct comprising an expression control sequence operably linked with the nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2.
In another aspect this invention provides a method of screening a test condition for its effect on expression of a reporter polypeptide comprising: a) exposing a recombinant cell comprising an expression construct comprising an expression control sequence operably linked with the nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2 to a test condition; b) measuring expression of the polypeptide; and c) comparing the measurement with a measurement of expression of the cell not exposed to the test condition. In one embodiment the test condition is selected from the group consisting of contacting the cell with a test compound, exposing the cell to an environmental condition and inducing or repressing expression of one or more genes in the cell. In one embodiment the test condition comprises contacting the cell with a test compound selected from: (1) a small organic molecule; (2) a nucleic acid derivative (e.g., a small interfering RNA, micro RNA mimic or micro RNA inhibitor); and (3) an expression construct that contains an open reading frame of a gene.
In another aspect this invention provides a method comprising: a) exposing a plurality of recombinant cells comprising an expression construct comprising an expression control sequence operably linked with the nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence (SEQ ID NO: 1), wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2 to a test condition; and b) measuring expression of the polypeptide in the cells. In one embodiment the method further comprises: c) comparing the measurements. In another embodiment the cells are exposed to a test condition.
INCORPORATION BY REFERENCE
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows results from the functional screen of Renilla codon variants.
FIG. 2 shows differences in expression of wild type luciferase and the luciferase of SEQ ID NO: 2.
FIG. 3 provides nucleotide and amino acid sequences.



DETAILED DESCRIPTION OF THE INVENTION
It is desirable to maximize the expression of a reporter protein to maximize signal. One approach to maximize expression is to optimize the codon usage of a reporter gene to maximize the translation rate in an organism other than the native organism from which the gene was isolated. Furthermore, it is desirable to remove potential regulatory sequences from the gene itself that may confound measurements of other regulatory elements of interest. There are many different approaches for optimizing the expression of a reporter gene. In the present invention, we identified a novel and improved nucleotide sequence for Renilla by screening hundreds of codon variants of a cDNA coding for the Renilla protein in human cells and eliminated many putative transcription factor binding sites from the nucleotide sequence as well.
1. Nucleotide Sequences Encoding Luciferase
This invention provides nucleotide sequences encoding luciferase. The nucleotide sequence can encode a Renilla luciferase having the amino acid sequence of SEQ ID NO: 1 (RenS), or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 1. Such amino acid sequences are referred to herein as “luciferase polypeptides”. A nucleotide sequence encoding a luciferase polypeptide is referred to herein as a “luciferase nucleotide sequence.” The nucleotide sequence encoding a luciferase polypeptide can have the nucleotide sequence of SEQ ID NO: 2 or it can have at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity with SEQ ID NO: 2. Alternatively, a nucleic acid having a nucleotide sequence encoding a luciferase polypeptide can hybridize under stringent conditions to a nucleic acid of SEQ ID NO: 2.
Nucleotide sequences that are different from SEQ ID NO: 2 and that encode SEQ ID NO: 1 can be generated by choosing an alternative codon consistent with the genetic code for one that encodes a particular amino acid in the amino acid sequence on luciferase. Accordingly, generating nucleotide sequences encoding the luciferase polypeptide and having a selected level of sequence homology with SEQ ID NO: 2 is a straightforward matter.
Nucleic acids can be made by any methods known in the art. This includes the use of nucleic acid synthesizers, or commercially available services, such as DNA2.0 (Menlo Park, Calif.).
The luciferase sequences of this invention express enzymatically active protein at higher levels than wild type Renilla luciferase genes. More specifically, the nucleotide sequences of this invention exhibit at least 10%, at least 15%, at least 20%, at least 50%, at least 75% or at least 100% greater expression than hRluc. hRluc is a humanized luciferase gene disclosed in U.S. patent application 2006/0068395 (WOOD, K. V. et al., Mar. 30, 2006). It is presented here has SEQ ID NO: 153, and with a PEST sequence attached as SEQ ID NO: 152. Relative expression can be determined in the following assay. hRlucP (SEQ ID NO: 153) and a luciferase gene to be tested are cloned into a vector, such as pLightSwitch prom (SEQ ID NO:5) (available from SwitchGear Genomics, Menlo Park, Calif.) operably linked with a human RPL10 promoter. 50 ng of each plasmid is transfected with FuGENE HD into human HT1080 cells. After 24 hours of incubation, 100 uL of LightSwitch Reagent (SwitchGear Genomics) is added to each well and incubated for 30 minutes. Expression is measured as a function of light emitted (read for 2 seconds on an LmaxII-384 luminometer). Alternatively, the assay can use luciferase genes with PEST sequence attached.
The nucleotide sequence of SEQ ID NO: 2 does not include any of the regulatory motifs of SEQ ID NOs: 9-134. The nucleotide sequences of this invention that are not identical to SEQ ID NO: 2 can have, for example, at most one, at most two, at most three, at most four, at most five or at most 10% of the sequence motifs of SEQ ID NOs: 9-134.
The nucleotide sequence of SEQ ID NO: 2 also does not have any of the restriction sites of SEQ ID NOs: 135-149. The nucleotide sequences of this invention that are not identical to SEQ ID NO: 2 can have, for example, at most one, at most two, at most three, at most four or at most five of the restriction sites of SEQ ID NOs: 9-134.
This invention also provides nucleotide sequences encoding a luciferase fusion protein. In such fusion proteins a luciferase polypeptide is attached at its amino terminus or its carboxy terminus to another amino acid sequence. A polypeptide comprising a luciferase polypeptide amino acid sequence is referred to herein as a “reporter polypeptide”. A nucleotide sequence encoding a reporter polypeptide is referred to herein as a “reporter nucleotide sequence.” Luciferase polypeptides and luciferase nucleotide sequences are, themselves, examples of reporter polypeptides and reporter nucleotide sequences, respectively.
In one embodiment the luciferase fusion protein comprises luciferase having a protein destabilization domain. For example, the protein destabilization domain can be a PEST sequence attached at the carboxy terminus of luciferase. Such an amino acid sequence is RenSP, provided as SEQ ID NO: 3. A nucleotide sequence encoding this amino acid sequence is provided as SEQ ID NO: 4.
In another embodiment the luciferase fusion protein comprises a gene-encoded polypeptide tagged with luciferase. Typically, luciferase is attached at the carboxy terminus of the polypeptide. The nucleotide sequences encoding such proteins are useful, for example, in understanding the regulation of a gene in its native genomic context.
Terms used to describe sequence relationships between two or more nucleotide sequences or amino acid sequences include “reference sequence,” “selected from,” “comparison window,” “identical,” “percentage of sequence identity,” “substantially identical,” “complementary,” and “substantially complementary.”
A “reference sequence” is a defined sequence used as a basis for a sequence comparison and may be a subset of a larger sequence, e.g., a complete cDNA, protein, or gene sequence.
Because two polynucleotides or polypeptides each may comprise (1) a sequence (i.e., only a portion of the complete polynucleotide or polypeptide sequence) that is similar between the two polynucleotides, or (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
A “comparison window” refers to a conceptual segment of typically at least 12 consecutive nucleotides or 4 consecutive amino acid residues that is compared to a reference sequence. The comparison window frequently has a length of at least 15 or at least 25 nucleotides or at least 5 or at least 8 amino acids. The comparison window may comprise additions or deletions (i.e., gaps) of about 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by inspection, and the best alignment (i.e., resulting in the highest percentage of similarity over the comparison window) generated by any of the various methods is selected.
A subject nucleotide sequence or amino acid sequence is “identical” to a reference sequence if the two sequences are the same when aligned for maximum correspondence over the length of the nucleotide or amino acid sequence.
The “percentage of sequence identity” between two sequences is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleotide or amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
Unless otherwise specified, the comparison window used to compare two sequences is the length of the shorter sequence.
Methods are described further in Natl. Acad. Sci. USA 85:2444; Higgins & Sharp (1988) Gene 73:237-244; Higgins & Sharp, CABIOS 5:151-153 (1989); Corpet et al. (1988) Nucleic Acids Research 16:10881-90; Huang et al. (1992) Computer Applications in the Biosciences 8:155-65; and Pearson et al. (1994) Methods in Molecular Biology 24:307-31. Alignment is also often performed by inspection and manual alignment.
A subject nucleotide sequence or amino acid sequence is “substantially identical” to a reference sequence if the subject amino acid sequence or nucleotide sequence has at least 70% sequence identity over a comparison window. Thus, sequences that have at least 70%, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity or at least 99% sequence identity with the reference sequence are also “substantially identical. Two sequences that are identical to each other are, of course, also “substantially identical”.
“Hybridizing specifically to” or “specific hybridization” or “selectively hybridize to”, refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
The term “stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. “Stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York.
Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al. for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
In certain embodiments, the nucleic acids of this invention are isolated nucleic acids. “Substantially pure” or “isolated” means an object species is the predominant species present (i.e., on a molar basis, more abundant than any other individual macromolecular species in the composition), and a substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition is the purified species of interest. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition.
The luciferase-encoding nucleic acids of this invention are not naturally occurring. “Naturally occurring” as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
2. Recombinant Nucleic Acid Molecules
2.1 Expression Constructs
An expression construct is a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression constructs typically include sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in vitro expression system.
This invention also provides recombinant nucleic acid molecules in which a nucleotide sequence encoding a luciferase or a luciferase fusion protein is linked with other functional nucleotide sequences. Recombinant nucleic acid molecules, also referred to as recombinant polynucleotides, are polynucleotides having sequences that are not naturally joined together.
In particular, this invention provides recombinant nucleic acid molecules in which a nucleotide sequence of this invention is operably linked with an expression control sequence. A recombinant nucleic acid molecule comprising an expression control sequence operably linked with a nucleotide sequence encoding a polypeptide is referred to herein as an expression construct. The expression control sequence can be positioned upstream or downstream of the luciferase-encoding nucleotide sequence.
2.2 Expression Control Sequences
An expression control sequence is a nucleotide sequence in a polynucleotide that regulates the expression (transcription and/or translation) of a nucleotide sequence operably linked to it. “Operably linked” refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence). Expression control sequences include, for example, promoters (e.g., inducible or constitutive), enhancers, transcription terminators and splicing signals for introns.
2.2.1 Promoters
Promoters are the best-characterized transcriptional regulatory sequences because of their predictable location immediately upstream of transcription start sites. Promoters include sequences that modulate the recognition, binding and transcription initiation activity of the RNA polymerase. These sequences can be cis acting or can be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, can be constitutive or regulated. They are often described as having two separate segments: core and extended promoter regions.
The core promoter includes sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. The core promoter includes the transcriptional start site, an RNA polymerase binding site and other general transcription binding sites and is where the pre-initiation complex forms and the general transcription machinery assembles. It is generally within 50 nucleotides (nt) of the transcription start site (TSS).
The extended promoter region includes the so-called proximal promoter, which extends to about 250 nucleotides upstream of the transcriptional start site (i.e., −250 nt). It includes primary regulatory elements such as specific transcription factor binding sites. It has been found that many genes have transcription regulatory elements located further up-stream. In particular, a fragment that includes most of the transcription regulatory elements of a gene can extend up to 700 nt or more up-stream of the transcription start site. (See, e.g., U.S. 2007-0161031.) In certain genes, transcription regulatory sequences have been found thousands of nucleotides upstream of the transcriptional start site.
Any promoter of interest can be operably linked with a luciferase-encoding nucleotide sequence. For example, the promoter can be a constitutive promoter. Alternatively, the promoter can be inducible promoter. This invention contemplates the choice of any promoter of interest to the artisan. A variety of promoters are commercially available from Switchgear Genomics (www.switchgeargenomics.com) and also are described in U.S. patent publications U.S. 2007-0161031 (Jul. 12, 2007, TRINKLEIN, N. D. et al.), U.S. 2009-0018031 (Jan. 15, 2009, TRINKLEIN, N. D. et al.) and U.S. 2011-0065100 (Mar. 17, 2011, ALDRED, S. F. et al.).
2.2.2 Other Transcription Regulatory Sequences
Transcription regulatory sequences include nucleotide sequences that confer inducible expression of a gene (i.e., that require a substance or stimulus for increased transcription). When an inducer is present, or present at increased concentration, gene expression increases. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression decreases. Regulatory regions typically bind one or more trans-acting or cis-acting proteins. Enhancers are known to influence gene expression when positioned 5′ or 3′ of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.
Regulatory regions also include, in addition to transcription regulatory sequences, sequences in DNA or RNA molecules that regulate transcript stability, transcript localization, facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding sites (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons and can be optionally included in an expression vector.
The expression control sequence can be a 5′- or a 3′-untranslated region (UTR) of a messenger RNA. Such UTRs also are commercially available from Switchgear Genomics and are described in U.S. patent publication U.S. 2008-0220983 (Sep. 11, 2008, TRINKLEIN, N. D. et al.).
2.3 Vectors
This invention also provides vectors including a luciferase-encoding nucleotide sequence. As used herein, a vector is a nucleic acid molecule capable carrying a nucleic acid insert and of replicating in a cell. Vectors include, for example, plasmids, viruses, cosmids, transposons, and artificial chromosomes. Vectors typically comprise an origin of replication (e.g., bacterial or eukaryotic (mammalian)), a selectable marker and a cloning site (e.g., a multiple cloning site).
Examples of specific vectors that may be useful in the practice of the present invention include, but are not limited to, E. coli bacteriophages, for example, lambda derivatives, or plasmids, for example, pBR322 derivatives or pUC plasmid derivatives; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast vectors such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, for example, vectors useful in insect cells, such as baculovirus vectors, vectors useful in mammalian cells such as retroviral vectors, adenoviral vectors, adenovirus viral vectors, adeno-associated viral vectors, lentivirus vectors, SV40 viral vectors, herpes simplex viral vectors and vaccinia viral vectors; vectors derived from combinations of plasmids and phage DNAs, plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
This invention contemplates vectors comprising an expression construct that includes a luciferase-encoding nucleotide sequence. This invention also contemplates vectors (referred to herein as “empty vectors”) comprising a luciferase-encoding nucleotide sequence that is not operably linked with a promoter or either or both of a 5′ or 3′ untranslated expression control sequence and that is positioned in the vector relative to a cloning site such that an expression control sequence inserted into the cloning site becomes operably linked with the luciferase-encoding nucleotide sequence. Accordingly, this invention provides empty vectors in a cloning site is positioned either upstream or downstream of the luciferase in the coming nucleotide sequence. Such vectors are configured to allow insertion of expression control sequences either or both of upstream or downstream of the luciferase encoding nucleotide sequence and operably linked to it. For example, a promoter and or a 5′ UTR sequence can be cloned into the vector upstream of the luciferase encoding nucleotide sequence and any 3′ UTR sequence can be introduced downstream of the gene. In certain embodiments, the expression construct comprises a promoter for the ACTB gene that includes a 5′ UTR operably linked with a luciferase sequence of this invention. In certain embodiments, the promoter for the RPL10 gene can be used to control the expression of a luciferase gene operably linked to a 3′UTR sequence that is cloned downstream of luciferase. In particular, the promoter for the RPL10 gene may have the sequence of SEQ ID NO: 151 or the promoter can comprise at least nucleotides 150 to 200 of SEQ ID NO:151.
2.3.1 Plasmids
In certain embodiments, the vector is a plasmid. A variety of plasmids are known and are commercially available for use with luciferase, particularly for use in mammalian cell systems.
SwitchGear Genomics commercializes plasmid vectors that include a multiple cloning site comprising BglI, SacI, MluI, NheI, XhoI, BglII and Hind III cloning sites that can be operably linked with a luciferase-encoding gene of this invention, a polyA region, an origin of replication and an ampicillin resistance gene.
Promega commercializes the pGL-2, pGL-3 and pGL-4 luciferase reporter vectors. (See, e.g., www.promega.com/products/imaging-and-immunological-detection/in-vivo-imaging/pg14-in-vivo-imaging-vectors/.) Certain of these vectors include a Hind III cloning site, a luc2 gene, which can be replaced with a luciferase encoding nucleotide sequence of this invention, an SV40 late polyA region, an SV40 early enhancer/promoter, a neomycin resistance gene, synthetic Poly(A), an origin of replication, an ampicillin resistance gene, a synthetic poly(A) signal/transcription pause site and a CMV early enhancer promoter.
5. Recombinant Cells
A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant cell.” The gene is then expressed in the recombinant host cell to produce, e.g., a “recombinant polypeptide.” A recombinant polynucleotide may serve a non-coding function. (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
This invention also provides recombinant cells into which are transiently or stably introduced a vector of this invention. Nucleic acids can be introduced into cells by any methods known in the art, e.g., transfection or transduction by chemical or non-chemical means. The transfected cells can be adherent cells or cells in suspension.
This invention also provides a recombinant cell whose genome has been engineered to include the recombinant polynucleotide sequence. Genome engineering can be accomplished with zinc finger nucleases, site directed transposons, homologous recombination and other techniques available to those skilled in the art.
The cells used can be any of those desired by the practitioner. This includes prokaryotic or eukaryotic cells, including bacterial cells, mammalian cells, human cells and cell lines of any of these. The cells can be cultured cells.
Typically a recombinant cell having an expression construct of this invention will include expression control sequences, e.g. promoters, operable in that cell type.
6. Methods of Use
The luciferase nucleic acids of this invention are useful in reporter assays for activity of expression control sequences, in particular, of promoters.
In a typical assay and expression vector on this invention is introduced into a cell. The expression vector can comprise a luciferase-encoding nucleotide sequence operably linked to an expression control sequence of interest to the practitioner. For example, a recombinant cell can be provided having an expression construct in which the promoter is a biomarker for a biological pathway. The cells can be provided as a set in which each cell contains a different promoter.
The cells can be cultured alone or in parallel and/or with control cells. These cells can be cultured under different incubation conditions. For example, a different culture conditions can't involve exposure to a different test condition. A test condition can be any condition to which a cell is exposed. Test conditions include, for example, contacting the cell with a test composition, exposing the cell to a test environmental condition, over-expressing or knocking down expression of a particular gene of interest in the cell, or combinations of these.
A test compound can be any element or molecule, for example, small organic molecules and biopolymers. Drug candidates useful as test compositions in this invention include small organic molecules and biological molecules, e.g., biologics. Organic molecules used as pharmaceuticals generally are small organic molecules typically having a size up to about 5000 Da, up to about 2000 Da, or up to about 1000 Da. Organic biopolymers also are used as test compositions.
This invention contemplates a number of different types of assays. In one assay, a cell comprising an expression construct of this invention is exposed to a test condition and the activity of the expression control sequence is determined based on expression of the reporter sequence. In another assay, a expression control sequence for a single biological response is exposed to a plurality of different test conditions, e.g., a plurality of different test compositions, and the effect of each test condition on the activity of the expression control sequence is determined. This can be, for example, a high throughput assay in which tens, hundreds or thousands of conditions are tested.
In certain methods, the assay is multiplexed. A number of compounds, e.g., a library of compounds can be tested. This can involve testing each compound against a recombinant cell of this invention.
A container (e.g., a multiwell plate array) containing cells harboring an expression construct of this invention is useful for high-throughput screening of promoter activity. A cell comprising an expression construct that comprises a biological response biomarker promoter operably linked with a reporter gene is exposed to a test condition under conditions chosen by the operator. Cells in which the promoters are “turned on” will express the reporter sequences under their transcriptional control. The investigator then checks each well of the device to measure the amount of reporter transcribed. Generally, this involves measuring the signal produced by a reporter protein encoded by the reporter sequence.
Measuring a response includes quantitative and qualitative determinations. Qualitative measurement includes measuring a response or lack of response, regardless of intensity. A quantitative response generally involves measuring the intensity of a response.
For example, after incubation cells can be lysed and luciferase assay substrate is added. Then the amount of light generated is detected with a luminometer.
The choice of a proper detection system for a particular application is well within the abilities of one of skill in the art. Exemplary detection means include, but are not limited to, detection by unaided eye, light microscopy using the eye or an optical sensor as the detector, confocal microscopy, laser scanning confocal microscopy, imaging using quantum dot color, fluorescence spectrum or other quantum dot property and wide-field imaging with a 2D CCD camera. In an exemplary embodiment, the device is a luminescent plate reader. For example, the assay can be performed in a multiwell plate, e.g., a plate with multiples of 96 wells (e.g., 96-well plate, 384-well plate, 1536-well plate). In assays in which the reporter is luminescent or fluorescent molecule, the reporter may have to be induced to produce light. Commercially available microplate luminometers can be used to detect the signal. These are available from, for example, Tecan, Molecular Devices and Berthold.
Assays can involve creation of a standard curve against which measurements are compared to quantify the amount of expression.
It also can be useful to identify differences in transcription regulatory sequence activity in two cell types. For example gene expression differs when cells transform from normal to cancerous. Promoters that are overactive in cancer cells may be targets of pharmacological intervention. The arrays of this invention are useful to identify such transcription regulatory sequences. Accordingly, the investigator provides two sets of arrays comprising expression constructs in the wells. Once cell type is used for transformation in a first device and a second cell type, for transformation in a second device. The expression of reporter sequences between the two devices is compared to identify those expressed differently in the two cell types.
Using expression constructs in which the transcription regulatory sequences are operably linked to unique reporter sequences opens the possibility of performing tests without the use of multiwell plates. In such situations a single culture of cells contains the entire expression library distributed among the cells. The culture can be incubated under conditions chosen by the investigator. Then the expression products are isolated. Reporters that emit different colors can be used. As described, if the reporter is a barcode, because each expression vector has a unique nucleotide sequence tag or barcode associated with its partner nucleic acid segment, the amount of each of the reporter sequences can be measured by measuring the amount of transcript comprising each unique sequence. For example, the molecules can be detected on a DNA array that contains probes complementary to the unique sequences. The amount of hybridization to each probe indicates the amount of the reporter sequence expressed, which, in turn, reflects the activity of the transcription regulatory sequences.
EXAMPLES
Example 1
From a parental Renilla cDNA sequence, a semi-random library of different cDNAs were created representing different codon variants of the same protein coding sequence. A specific list of regulatory motifs and restriction sites were also excluded from these codon variant sequences (see Table 1).
Each of these codon variant cDNAs was cloned into an expression vector using the human beta actin promoter to drive expression of the cDNA. 94 random clones were picked from this variant library, the plasmids were purified, and 50 ng of each plasmid was then transiently transfected into human HT1080 cells to measure the level of expression of the codon variants. Each clone was then sequence verified in both directions to identify the sequence of the Renilla cDNA that was assayed in the transient reporter assay. The results of the functional screen are shown in FIG. 1.
The sequence of the highest expressing codon variant, labeled “RenS”, is shown below. To measure various biological responses with reporter assays, it is often useful to include a protein degradation sequences such as the PEST sequence to decrease the half-life of the reporter protein. We also show below the sequence of RenS fused with the PEST domain, labeled “RenSP”.
Example 2
To determine the relative brightness of RenSP compared to hRlucP, another humanized form of the Renilla luciferase, the RenSP and hRlucP genes were cloned into separate vectors each containing the human RPL10 promoter. Three independent plasmid purifications were conducted for each vector, and 50 ng of each plasmid was transfected with FuGENE HID in triplicate in human HT1080 cells in 96-well format. After 24 hours of incubation, 100 uL of LightSwitch Reagent (SwitchGear Genomics, Menlo Park, Calif.) was added to each well and incubated for 30 minutes before being read for 2 seconds on an Lmaxll-384 luminometer. These results show that RenSP is significantly brighter than hRlucP.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

## Claims

1. A nucleic acid molecule comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence having at least 99% sequence identity with SEQ ID NO:1, wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 95% sequence identity with SEQ ID NO:2.

2. The nucleic acid molecule of claim 1 wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 98% sequence identity with SEQ ID NO:2.

3. The nucleic acid molecule of claim 1 wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 99% sequence identity with SEQ ID NO:2.

4. The nucleic acid molecule of claim 1 wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 99.9% sequence identity with SEQ ID NO:2.

5. The nucleic acid molecule of claim 1 wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence identical with SEQ ID NO:2.

6. The nucleic acid molecule of claim 1 wherein the polypeptide is Renilla luciferase (SEQ ID NO:1) (RenS).

7. The nucleic acid molecule of claim 6 wherein RenS is encoded by the nucleotide sequence of SEQ ID NO:2.

8. The nucleic acid molecule of claim 1 wherein the polypeptide is a fusion protein comprising the Renilla luciferase amino acid sequence of SEQ ID NO:1.

9. The nucleic acid molecule of claim 8 wherein the Renilla luciferase amino acid sequence is encoded by the nucleotide sequence of SEQ ID NO:2.

10. The nucleic acid molecule of claim 8 wherein the fusion protein is RenSP (SEQ ID NO: 3).

11. The nucleic acid of claim 10 wherein the RenSP is encoded by the nucleotide sequence of SEQ ID NO: 4.

12. The nucleic acid molecule of claim 8 wherein the fusion polypeptide comprises a protein destabilization domain that decreases the half-life of Renilla luciferase.

13. The nucleic acid molecule of claim 1 wherein the luciferase nucleotide sequence contains no more than 10 regulatory motifs selected from the motifs of FIG. 3.

14. A recombinant nucleic acid comprising a promoter operably linked with a reporter nucleotide sequence, wherein the reporter nucleotide sequence encodes a polypeptide comprising a Renilla luciferase amino acid sequence having at least 99% sequence identity with SEQ ID NO:1, wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 95% sequence identity with SEQ ID NO:2.

15. The recombinant nucleic acid of claim 14 wherein the polypeptide comprises Renilla luciferase (SEQ ID NO:1) (RenS) encoded by a luciferase nucleotide sequence having at least 99% sequence identity with SEQ ID NO:2.

16. The recombinant nucleic acid of claim 14 wherein the Renilla Luciferase amino acid sequence is encoded by the luciferase nucleotide sequence of SEQ ID NO:2.

17. The recombinant nucleic acid of claim 14 wherein the polypeptide is RenSP (SEQ ID NO: 3).

18. The recombinant nucleic acid of claim 14 comprised in a plasmid.

19. A recombinant cell comprising a recombinant nucleic acid comprising a promoter operably linked with a reporter nucleotide sequence, wherein the reporter nucleotide sequence encodes a polypeptide comprising a Renilla luciferase amino acid sequence having at least 99% sequence identity with SEQ ID NO:1, wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 95% sequence identity with SEQ ID NO:2.

20. The recombinant cell of claim 19 wherein the polypeptide comprises Renilla luciferase (SEQ ID NO:1) (RenS) encoded by a luciferase nucleotide sequence having at least 99% sequence identity with SEQ ID NO:2.

21. The recombinant cell of claim 19 wherein the luciferase nucleotide sequence has at least 98% sequence identity with SEQ ID NO:2.

22. The recombinant cell of claim 19 wherein the luciferase nucleotide sequence is SEQ ID NO:2.

23. The recombinant cell of claim 19 which is a mammalian cell.

24. A kit comprising: (1) a vector containing a nucleic acid molecule comprising a reporter nucleotide sequence encoding a polypeptide comprising a Renilla luciferase amino acid sequence having at least 99% sequence identity with SEQ ID NO:1, wherein the Renilla luciferase amino acid sequence is encoded by a luciferase nucleotide sequence having at least 95% sequence identity with SEQ ID NO:2; and (2) a luciferase substrate.

25. The kit of claim 24 wherein the polypeptide comprises Renilla luciferase (SEQ ID NO:1) (RenS) encoded by a luciferase nucleotide sequence having at least 99% sequence identity with SEQ ID NO:2.

26. The kit of claim 24 wherein the luciferase nucleotide sequence has at least 98% sequence identity with SEQ ID NO:2.

27. The kit of claim 24 wherein the luciferase nucleotide sequence is SEQ ID NO:2.

28. The kit of claim 24 wherein the Luciferase substrate is coelenterazine.

29. The nucleic acid molecule of claim 1 further comprising a cloning site positioned relative to the reporter nucleotide sequence such that an expression control sequence inserted into the cloning site becomes operably linked with the luciferase-encoding nucleotide sequence.

30. The nucleic acid molecule of claim 1, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

31. The nucleic acid molecule of claim 2, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

32. The nucleic acid molecule of claim 3, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

33. The nucleic acid molecule of claim 4, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

34. The nucleic acid molecule of claim 13, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

35. The recombinant nucleic acid of claim 14, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

36. The recombinant cell of claim 19, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

37. The recombinant cell of claim 21, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

38. The recombinant cell of claim 23, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

39. The kit of claim 24, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

40. The kit of claim 26, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.

41. The kit of claim 28, wherein the Renilla luciferase amino acid sequence is SEQ ID NO:1.