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Synthetic nucleic acid molecule compositions and methods of preparationRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Compound Containing Saccharide Radical, N-glycoside, , Nucleotide, Polynucleotide (e.g., Nucleic Acid, Oligonucleotide, Etc.)Synthetic nucleic acid molecule compositions and methods of preparation description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060127988, Synthetic nucleic acid molecule compositions and methods of preparation. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] Transcription, the synthesis of an RNA molecule from a sequence of DNA is the first step in gene expression. Sequences which regulate DNA transcription include promoter sequences, polyadenylation signals, transcription factor binding sites and enhancer elements. A promoter is a DNA sequence capable of specific initiation of transcription and consists of three general regions. The core promoter is the sequence where the RNA polymerase and its cofactors bind to the DNA. Immediately upstream of the core promoter is the proximal promoter which contains several transcription factor binding sites that are responsible for the assembly of an activation complex that in turn recruits the polymerase complex. The distal promoter, located further upstream of the proximal promoter also contains transcription factor binding sites. Transcription termination and polyadenylation, like transcription initiation, are site specific and encoded by defined sequences. Enhancers are regulatory regions, containing multiple transcription factor binding sites, that can significantly increase the level of transcription from a responsive promoter regardless of the enhancer's orientation and distance with respect to the promoter as long as the enhancer and promoter are located within the same DNA molecule. The amount of transcript produced from a gene may also be regulated by a post-transcriptional mechanism, the most important being RNA splicing that removes intervening sequences (introns) from a primary transcript between splice donor and splice acceptor sequences. [0003] Natural selection is the hypothesis that genotype-environment interactions occurring at the phenotypic level lead to differential reproductive success of individuals and therefore to modification of the gene pool of a population. Some properties of nucleic acid molecules that are acted upon by natural selection include codon usage frequency, RNA secondary structure, the efficiency of intron splicing, and interactions with transcription factors or other nucleic acid binding proteins. Because of the degenerate nature of the genetic code, these properties can be optimized by natural selection without altering the corresponding amino acid sequence. [0004] Under some conditions, it is useful to synthetically alter the natural nucleotide sequence encoding a polypeptide to better adapt the polypeptide for alternative applications. A common example is to alter the codon usage frequency of a gene when it is expressed in a foreign host cell. Although redundancy in the genetic code allows amino acids to be encoded by multiple codons, different organisms favor some codons over others. It has been found that the efficiency of protein translation in a non-native host cell can be substantially increased by adjusting the codon usage frequency but maintaining the same gene product (U.S. Pat. Nos. 5,096,825, 5,670,356, and 5,874,304). [0005] However, altering codon usage may, in turn, result in the unintentional introduction into a synthetic nucleic acid molecule of inappropriate transcription regulatory sequences. This may adversely effect transcription, resulting in anomalous expression of the synthetic DNA. Anomalous expression is defined as departure from normal or expected levels of expression. For example, transcription factor binding sites located downstream from a promoter have been demonstrated to effect promoter activity (Michael et al., 1990; Lamb et al., 1998; Johnson et al., 1998; Jones et al., 1997). Additionally, it is not uncommon for an enhancer element to exert activity and result in elevated levels of DNA transcription in the absence of a promoter sequence or for the presence of transcription regulatory sequences to increase the basal levels of gene expression in the absence of a promoter sequence. [0006] Thus, what is needed is a method for making synthetic nucleic acid molecules with altered codon usage without also introducing inappropriate or unintended transcription regulatory sequences for expression in a particular host cell. SUMMARY OF THE INVENTION [0007] The invention provides a synthetic nucleic acid molecule comprising at least 300 nucleotides of a coding region for a polypeptide, having a codon composition differing at more than 25% of the codons from a wild type nucleic acid sequence encoding a polypeptide, and having at least 3-fold fewer, preferably at least 5-fold fewer, transcription regulatory sequences than would result if the differing codons were randomly selected. Preferably, the synthetic nucleic acid molecule encodes a polypeptide that has an amino acid sequence that is at least 85%, preferably 90%, and most preferably 95% or 99% identical to the amino acid sequence of the naturally-occurring (native or wild type) polypeptide (protein) from which it is derived. Thus, it is recognized that some specific amino acid changes may also be desirable to alter a particular phenotypic characteristic of the polypeptide encoded by the synthetic nucleic acid molecule. Preferably, the amino acid sequence identity is over at least 100 contiguous amino acid residues. In one embodiment of the invention, the codons in the synthetic nucleic acid molecule that differ preferably encode the same amino acids as the corresponding codons in the wild type nucleic acid sequence. [0008] The transcription regulatory sequences which are reduced in the synthetic nucleic acid molecule include, but are not limited to, any combination of transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences and promoter sequences. Transcription regulatory sequences are well known in the art. [0009] It is preferred that the synthetic nucleic acid molecule of the invention has a codon composition that differs from that of the wild type nucleic acid sequence at more than 30%, 35%, 40% or more than 45%, e.g., 50%, 55%, 60% or more of the codons. Preferred codons for use in the invention are those which are employed more frequently than at least one other codon for the same amino acid in a particular organism and, more preferably, are also not low-usage codons in that organism and are not low-usage codons in the organism used to clone or screen for the expression of the synthetic nucleic acid molecule (for example, E. coli). Moreover, preferred codons for certain amino acids (i.e., those amino acids that have three or more codons,), may include two or more codons that are employed more frequently than the other (non-preferred) codon(s). The presence of codons in the synthetic nucleic acid molecule that are employed more frequently in one organism than in another organism results in a synthetic nucleic acid molecule which, when introduced into the cells of the organism that employs those codons more frequently, is expressed in those cells at a level that is greater than the expression of the wild type or parent nucleic acid sequence in those cells. For example, the synthetic nucleic acid molecule of the invention is expressed at a level that is at least about 110%, e.g., 150%, 200%, 500% or more (1000%, 5000%, or 10000%) of that of the wild type nucleic acid sequence in a cell or cell extract under identical conditions (such as cell culture conditions, vector backbone, and the like). [0010] In one embodiment of the invention, the codons that are different are those employed more frequently in a mammal, while in another embodiment the codons that are different are those employed more frequently in a plant. A particular type of mammal, e.g., human, may have a different set of preferred codons than another type of mammal. Likewise, a particular type of plant may have a different set of preferred codons than another type of plant. In one embodiment of the invention. the majority of the codons which differ are ones that are preferred codons in a desired host cell. Preferred codons for mammals (e.g., humans) and plants are known to the art (e.g., Wada et al., 1990). For example, preferred human codons include, but are not limited to, CGC (Arg), CTG (Leu), TCT (Ser), AGC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCC (Ala), GGC (Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAG (Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys) and TTC (Phe) (Wada et al., 1990). Thus, preferred "humanized" synthetic nucleic acid molecules of the invention have a codon composition which differs from a wild type nucleic acid sequence by having an increased number of the preferred human codons, e.g. CGC, CTG, TCT, AGC, ACC, CCA, CCT, GCC, GGC, GTG, ATC, ATT, AAG, AAC, CAG, CAC, GAG, GAC, TAC, TGC, TTC, or any combination thereof. For example, the synthetic nucleic acid molecule of the invention may have an increased number of CTG or TTG leucine-encoding codons, GTG or GTC valine-encoding codons, GGC or GGT glycine-encoding codons, ATC or ATT isoleucine-encoding codons, CCA or CCT proline-encoding codons, CGC or CGT arginine-encoding codons, AGC or TCT serine-encoding codons, ACC or ACT threonine-encoding codon, GCC or GCT alanine-encoding codons, or any combination thereof, relative to the wild type nucleic acid sequence. Similarly, synthetic nucleic acid molecules having an increased number of codons that are employed more frequently in plants, have a codon composition which differs from a wild type or parent nucleic acid sequence by having an increased number of the plant codons including, but not limited to, CGC (Arg), CTT (Leu), TCT (Ser), TCC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCT (Ser), GGA (Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAA (Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys), TTC (Phe), or any combination thereof (Murray et al., 1989). Preferred codons may differ for different types of plants (Wada et al., 1990). [0011] The choice of codon may be influenced by many factors such as, for example, the desire to have an increased number of nucleotide substitutions or decreased number of transcription regulatory sequences. Under some circumstances (e.g. to permit removal of a transcription factor binding site) it may be desirable to replace a non-preferred codon with a codon other than a preferred codon or a codon other than the most preferred codon. Under other circumstances, for example, to prepare codon distinct versions of a synthetic nucleic acid molecule, preferred codon pairs are selected based upon the largest number of mismatched bases, as well as the criteria described above. [0012] The presence of codons in the synthetic nucleic acid molecule that are employed more frequently in one organism than in another organism, results in a synthetic nucleic acid molecule which, when introduced into a cell of the organism that employs those codons, is expressed in that cell at a level which is greater than the level of expression of the wild type or parent nucleic acid sequence. [0013] A synthetic nucleic acid molecule of the invention may encode a selectable marker protein or a reporter molecule. However, the invention applies to any gene and is not limited to synthetic reporter genes or synthetic selectable marker genes. In one embodiment of a synthetic nucleic acid molecule of the invention that is a reporter molecule, the synthetic nucleic acid molecule encodes a luciferase having a codon composition different than that of a wild type or parent Renilla luciferase or a beetle luciferase nucleic acid sequence. A synthetic click beetle luciferase nucleic acid molecule of the invention may optionally encode the amino acid valine at position 224 (i.e., it emits green light), or may optionally encode the amino acid histidine at position 224, histidine at position 247, isoleucine at position 346, glutamine at position 348 or combination thereof (i.e., it emits red light). Preferred synthetic luciferase nucleic acid molecules that are related to a wild type Renilla luciferase nucleic acid sequence include, but are not limited to, SEQ ID NO:21 (Rlucver2) or SEQ ID NO:22 (Rluc-final). Preferred synthetic luciferase nucleic acid molecules that are related to click beetle luciferase nucleic acid sequences include, but are not limited to, SEQ ID NO:7 (GRver5), SEQ ID NO:8 (GR6), SEQ ID NO:9 (GRver5.1), SEQ ID NO:14 (RDver5), SEQ ID NO:15 (RD7), SEQ ID NO:16 (RDver5.1), SEQ ID NO:17 (RDver5.2) or SEQ ID NO:18 (RD156-1H9). [0014] The invention also provides an expression cassette. The expression cassette of the invention comprises a synthetic nucleic acid molecule of the invention operatively linked to a promoter that is functional in a cell. Preferred promoters are those functional in mammalian cells and those functional in plant cells. Optionally, the expression cassette may include other sequences, e.g., restriction enzyme recognition sequences and a Kozak sequence, and be a part of a larger polynucleotide molecule such as a plasmid, cosmid, artificial chromosome or vector, e.g., a viral vector. [0015] Also provided is a host cell comprising the synthetic nucleic acid molecule of the invention, an isolated polypeptide (e.g., a fusion polypeptide encoded by the synthetic nucleic acid molecule of the invention), and compositions and kits comprising the synthetic nucleic acid molecule of the invention or the polypeptide encoded thereby in suitable container means and, optionally, instruction means. Preferred isolated polypeptides include, but are not limited to, those comprising SEQ ID NO:31 (GRver5.1), SEQ ID NO:226 (Rluc-final), or SEQ ID NO:223 (RD156-1H9). [0016] The invention also provides a method to prepare a synthetic nucleic acid molecule of the invention by genetically altering a parent (either a wild type or another synthetic) nucleic acid sequence. The method may be used to prepare a synthetic nucleic acid molecule encoding a polypeptide comprising at least 100 amino acids. One embodiment of the invention is directed to the preparation of synthetic genes encoding reporter or selectable marker proteins. The method of the invention may be employed to alter the codon usage frequency and decrease the number of transcription regulatory sequences in any open reading frame or to decrease the number of transcription regulatory sites in a vector backbone. Preferably, the codon usage frequency in the synthetic nucleic acid molecule is altered to reflect that of the host organism desired for expression of that nucleic acid molecule while also decreasing the number of potential transcription regulatory sequences relative to the parent nucleic acid molecule. [0017] Thus, the invention provides a method to prepare a synthetic nucleic acid molecule comprising an open reading frame. The method comprises altering (e.g., decreasing or eliminating) a plurality of transcription regulatory sequences in a parent (wild type or a synthetic) nucleic acid sequence that encodes a polypeptide having at least 100 amino acids to yield a synthetic nucleic acid molecule which has a decreased number of transcription regulatory sequences and which preferably encodes the same amino acids as the parent nucleic acid molecule. The transcription regulatory sequences are selected from the group consisting of transcription factor binding sequences, intron splice sites, poly(A) addition sites, enhancer sequences and promoter sequences, and the resulting synthetic nucleic acid molecule has at least 3-fold fewer, preferably 5-fold fewer, transcription regulatory sequences relative to the parent nucleic acid sequence. The method also comprises altering greater than 25% of the codons in the synthetic nucleic acid sequence which has a decreased number of transcription regulatory sequences to yield a further synthetic nucleic acid molecule, wherein the codons that are altered encode the same amino acids as those in the corresponding position in the synthetic nucleic acid molecule which has a decreased number of transcription regulatory sequences and/or in the parent nucleic acid sequence. Preferably, the codons which are altered do not result in an increase in transcriptional regulatory sequences. Preferably, the further synthetic nucleic acid molecule encodes a polypeptide that has at least 85%, preferably 90%, and most preferably 95% or 99% contiguous amino acid sequence identity to the amino acid sequence of the polypeptide encoded by the parent nucleic acid sequence. [0018] Alternatively, the method comprises altering greater than 25% of the codons in a parent nucleic acid sequence which encodes a polypeptide having at least 100 amino acids to yield a codon-altered synthetic nucleic acid molecule, wherein the codons that are altered encode the same amino acids as those present in the corresponding positions in the parent nucleic acid sequence. Then, a plurality of transcription regulatory sequences in the codon-altered synthetic nucleic acid molecule are altered to yield a further synthetic nucleic acid molecule. Preferably, the codons which are altered do not result in an increase in transcriptional regulatory sequences. Also, preferably, the further synthetic nucleic acid molecule encodes a polypeptide that has at least 85%, preferably 90%, and most preferably 95% or 99% contiguous amino acid sequence identity to the amino acid sequence of the polypeptide encoded by the parent nucleic acid sequence. Also provided is a synthetic (including a further synthetic) nucleic acid molecule prepared by the methods of the invention. [0019] As described hereinbelow, the methods of the invention were employed with click beetle luciferase and Renilla luciferase nucleic acid sequences. While both of these nucleic acid molecules encode luciferase proteins, they are from entirely different families and are widely separated evolutionarily. These proteins have unrelated amino acid sequences, protein structures, and they utilize dissimilar chemical substrates. The fact that they share the name "luciferase" should not be interpreted to mean that they are from the same family, or even largely similar families. The methods produced synthetic luciferase nucleic acid molecules which exhibited significantly enhanced levels of mammalian expression without negatively effecting other desirable physical or biochemical properties (including protein half-life) and which were also largely devoid of known transcription regulatory elements. [0020] The invention also provides at least two synthetic nucleic acid molecules that encode highly related polypeptides, but which synthetic nucleic acid molecules have an increased number of nucleotide differences relative to each other. These differences decrease the recombination frequency between the two synthetic nucleic acid molecules when those molecules are both present in a cell (i.e., they are "codon distinct" versions of a synthetic nucleic acid molecule). Thus, the invention provides a method for preparing at least two synthetic nucleic acid molecules that are codon distinct versions of a parent nucleic acid sequence that encodes a polypeptide. The method comprises altering a parent nucleic acid sequence to yield a first synthetic nucleic acid molecule having an increased number of a first plurality of codons that are employed more frequently in a selected host cell relative to the number of those codons present in the parent nucleic acid sequence. Optionally, the first synthetic nucleic acid molecule also has a decreased number of transcription regulatory sequences relative to the parent nucleic acid sequence. The parent nucleic acid sequence is also altered to yield a second synthetic nucleic acid molecule having an increased number of a second plurality of codons that are employed more frequently in the host cell relative to the number of those codons in the parent nucleic acid sequence, wherein the first plurality of codons is different than the second plurality of codons, and wherein the first and the second synthetic nucleic acid molecules preferably encode the same polypeptide. Optionally, the second synthetic nucleic acid molecule has a decreased number of transcription regulatory sequences relative to the parent nucleic acid sequence. Either or both synthetic molecules can then be further modified. [0021] Clearly, the present invention has applications with many genes and across many fields of science including, but not limited to, life science research, agrigenetics, genetic therapy, developmental science and pharmaceutical development. BRIEF DESCRIPTION OF THE FIGURES [0022] FIG. 1. Codons and their corresponding amino acids. Continue reading about Synthetic nucleic acid molecule compositions and methods of preparation... 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