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NumXL allows you to easily make forecasts, back-track and analyze them. Pixillion Premium Edition v. Allavsoft for Mac v. In some embodiments, the protein of interest is a naturally occurring prokaryotic protein or a variant thereof. In some embodiments, the protein of interest is a naturally occurring archaeal protein or a variant thereof.

In some embodiments, the protein of interest is a naturally occurring mammalian protein or a variant thereof. In some embodiments, the protein of interest comprises a human protein or a variant thereof. A nucleic acid encoding a protein of interest comprises a polynucleotide sequence that codes for the protein of interest, which may or may not include introns.

A nucleic acid encoding a protein of interest may also further comprise one or more regulatory element that is operatively-linked to the polynucleotide sequence encoding the protein of interest. The one or more regulatory elements may be selected on the basis of the host cell to be used for expression. Suitable regulatory elements are described in further detail in Section IV.

Any method known in the art can be used to modify a nucleic acid sequence at codons 3, 4 and 5, provided the change does not result in a stop codon encoded by codon 3, 4 or 5 of the modified nucleic acid sequence, or in a shift in the reading frame of the modified nucleic acid sequence at any position corresponding to codons 6 and greater in the nucleic acid sequence encoding the protein of interest.

Non-limiting examples include in vitro and in vivo methods to insert into a nucleic acid sequence encoding a protein of interest a polynucleotide sequence consisting of 3, 6, or 9 nucleotides, and in vitro and in vivo methods to make on or more nucleotide changes in a nucleic acid sequence.

In another example, a polynucleotide sequence selected from Table A, Table B, or Table C may be inserted between the second and third codons of a nucleic acid sequence encoding a protein of interest. The above modifications may be made to the nucleic acid sequence in vitro using standard molecular cloning techniques that rely on restriction endonucleases, PCR-based methods, etc.

For example, 1, 2, or 3 nucleotides changes may be made at codon three; 1, 2, or 3 nucleotides changes may be made at codon four; or 1, 2, or 3 nucleotides changes may be made at codon five. In another example, 2, 3, 4, 5, or 6 nucleotides changes may be made at codon three and codon four; 2, 3, 4, 5, or 6 nucleotides changes may be made at codon three and codon five; or 2, 3, 4, 5, or 6 nucleotides changes may be made at codon four and codon five.

In another example, 3, 4, 5, 6, 7, 8, or 9 nucleotides changes may be made at codon three, codon four, and codon five. In still further examples, one or more changes may be made at the second codon, the third codon, the fourth codon, or any combination thereof, to produce a modified nucleic acid sequence that has a polynucleotide sequence of Table D, at codons 3, 4 and 5.

In still further examples, one or more changes may be made at the second codon, the third codon, the fourth codon, or any combination thereof, to produce a modified nucleic acid sequence encoding a peptide selected from Table I. Modifying a nucleic acid sequence encoding a protein of interest by interesting a polynucleotide sequence consisting of 3, 6, or 9 nucleotides will result in a modified nucleic acid sequence encoding a second protein that has a different amino acid sequence than the protein of interest.

Modifying a nucleic acid sequence encoding a protein of interest by changing one or more nucleotides at the codon three, codon four, codon five, or any combination thereof, may or may not result in a modified nucleic acid sequence encoding a second protein that has a different amino acid sequence than the protein of interest.

Importantly, the polynucleotide sequence at codons 3, 4, and 5 of the modified nucleic acid sequence does not encode a stop codon recognizable by the cell or the cell-free system.

Preferably, the polynucleotide sequence at codons 3, 4, and 5 of the modified nucleic acid sequence also does not encode a methionine. The polynucleotide sequences in Tables A-F do not encode a stop codon when inserted into a nucleic acid sequence in-frame. For example, the modified nucleic acid may need to be cloned into a suitable expression vector, packaged into a viral particle for delivery into a cell, etc.

Advantageously, the method of the present disclosure can be used with any cell type e. Compared to the protein of interest, the level of expression of the protein encoded by the modified nucleic acid may be higher or lower by up to about orders of magnitude difference.

The direction and magnitude of the change depends upon the original polynucleotide sequence at codons 3, 4, and 5 and the modified polynucleotide sequence at codons 3, 4, and 5. Advantageously, the present disclosure provides methods to modulate the level of expression of a protein of interest in a deliberate manner i. For example, to achieve the greatest change in protein expression, the difference between the GFP score or RFU value for codons 3, 4 and 5 of the nucleic acid sequence encoding a protein of interest and the GFP score or RFU value for the selected polynucleotide sequence should be maximized.

Although the correlation between RFU and protein abundance measured by quantitative western blot is not strictly linear, the RFU values can be used as an estimate of magnitude of increase or decrease if the desired goal is to not maximize the change but to achieve an intermediate level of change.

In certain embodiments, the present disclosure provides a method to increase the level of expression of a protein. In certain embodiments, the present disclosure provides a method to decrease the level of expression of a protein. When selecting a polynucleotide sequence for codons 3, 4 and 5 of the modified nucleic acid sequence, it may be preferable to select a sequence that encodes an amino acid sequence that has a narrow range for the GFP score.

The range takes into account all possible codons encoding that amino acid sequence and their spread, and serves as a proxy for standard deviation.

Amino acid sequences with a narrow range will have a high expression value regardless of the codon used. Alternatively, or in addition, it may be preferable to not select a sequence that codes for a methionine to avoid the creation of an alternative translation start site. Codon optimization programs are available as freeware or from commercial sources. In still further embodiments, the present disclosure provides a method to increase or decrease the level of expression of a protein.

In other embodiments, the amino acid sequence selected from Table E, F, or G will have a range for its score that is less than about In other embodiments, the amino acid sequence selected from Table E, F, or G will have a range for its score that is about 7. In another aspect, the present disclosure provides isolated, non-natural nucleic acid molecules comprising a polynucleotide sequence selected from Table A, Table B, or Table C, at codons 3, 4, and 5.

The isolated nucleic acid molecule may further comprise one or more regulatory element s that are operatively-linked to the polynucleotide sequence. The one or more regulatory elements may be selected, at least in part, on the basis of the host cells to be used for expression. In another aspect, the present disclosure provides an isolated, non-natural nucleic acid molecule comprising a polynucleotide sequence selected from Table D at codons 3, 4, and 5.

The isolated nucleic acid molecule may further comprise one or more regulatory element that is operatively-linked to the polynucleotide sequence. In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising R 5 -R 6 -R 7 , wherein R 5 , R 6 , and R 7 are joined by phosphodiester bonds. R 5 is i a first fragment of a polynucleotide sequence of interest, wherein the first fragment consists of the first and second codons of the sequence of interest, or ii a polynucleotide sequence consisting of a start codon and second codon encoding any natural or non-natural amino acid; optionally the polynucleotide sequence does not encode a methionine.

R 7 is a second fragment of a polynucleotide sequence of interest, wherein the second fragment lacks the first and second codons of the sequence of interest. The isolated nucleic acid molecule may further comprise one or more regulatory element that is operatively-linked to R 5 -R 6 -R 7. The one or more regulatory element s may be selected, at least in part, on the basis of the host cells to be used for expression.

R 5 is i a first fragment of a polynucleotide sequence of interest, wherein the first fragment consists of the first and second codons of the sequence of interest, or ii a polynucleotide sequence consisting of a start codon and second codon encoding any natural or non-natural amino acid. R 6 is a polynucleotide sequence selected from Table D. The one or more regulatory element may be selected, at least in part, on the basis of the host cells to be used for expression.

R 6 is a polynucleotide sequence selected from Table A, Table B, or Table C with the proviso that R 6 is not a polynucleotide sequence consisting of codons 3, 4, and 5 of the polynucleotide sequence of interest, and optionally codons 3, 4, and 5 do not encode a methionine.

R 7 is a second fragment of the polynucleotide sequence of interest, the second fragment lacking the first and second codons of the sequence of interest. R 6 is a polynucleotide sequence selected from Table D, with the proviso that R 6 is not a polynucleotide sequence consisting of codons 3, 4, and 5 of the polynucleotide sequence of interest. Isolated polynucleotides of the present disclosure may be produced by any method known in the art.

In another aspect, the present disclosure provides an array comprising a substrate, wherein the substrate comprises an oligonucleotide probe as described above or an epitope binding agent. Several substrates suitable for the construction of arrays are known in the art, and one skilled in the art will appreciate that other substrates may become available as the art progresses.

The substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of an epitope binding agent and is amenable to at least one detection method. Non-limiting examples of substrate materials include glass, modified or functionalized glass, plastics including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.

In an exemplary embodiment, the substrate may allow optical detection without appreciably fluorescing. A substrate may be planar, a substrate may be a well, e. Additionally, the substrate may be the inner surface of a tube for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.

An oligonucleotide probe or an epitope binding agent may be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art. An oligonucleotide probe or epitope binding agent may either be synthesized first, with subsequent attachment to the substrate, or may be directly synthesized on the substrate.

For example, the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, an oligonucleotide probe or epitope binding agent may be attached using functional groups on the nucleic acid or epitope binding agent either directly or indirectly using linkers. An oligonucleotide probe or epitope binding agent may also be attached to the substrate non-covalently.

For example, a biotinylated oligonucleotide probe or epitope binding agent may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, an oligonucleotide probe or epitope binding agent may be synthesized on the surface using techniques such as photopolymerization and photolithography.

Additional methods of attaching epitope binding agents to arrays and methods of synthesizing biomolecules on substrates are well known in the art, i. An oligonucleotide probe or epitope binding agent may be attached to the substrate at a spatially defined address of the array. Arrays may comprise from about 1 to about several hundred thousand addresses.

In one embodiment, the array may be comprised of less than 10, addresses. In another alternative embodiment, the array may be comprised of at least 10, addresses.

In yet another alternative embodiment, the array may be comprised of less than 5, addresses. In still another alternative embodiment, the array may be comprised of at least 5, addresses.

In a further embodiment, the array may be comprised of less than addresses. In yet a further embodiment, the array may be comprised of at least addresses. An oligonucleotide probe or epitope binding agent may be represented more than once on a given array. In other words, more than one address of an array may be comprised of the same oligonucleotide probe or epitope binding agent. In some embodiments, two, three, or more than three addresses of the array may be comprised of the same oligonucleotide probe or epitope binding agent.

The controls may be internal controls, positive controls, negative controls, or background controls. Arrays of the present disclosure may be utilized in several suitable applications. For example, an array may be used in a method for detecting association between an epitope binding agent and a target. This method typically comprises incubating a sample comprising a target with the array under conditions such that the target may associate with the epitope binding agent attached to the array.

The association may then be detected, using means commonly known in the art, such as fluorescence. A skilled artisan will appreciate that conditions under which association may occur will vary depending on the epitope binding agent, the substrate, the sample, and the detection method utilized. As such, suitable conditions may have to be optimized for each individual array created. In yet another embodiment, the array may be used as a tool in a method for determining whether a subject has a genomic mutation in a gene that results in increased or decreased expression of the protein encoded by the gene.

Typically, such a method comprises incubating the array with a biological sample from the subject. If the biological sample comprises a polynucleotide sequence selected from Table A, Table B or Table C, then an association between the array and the sample may be detected, and the subject may have a genomic mutation in a gene that results in increased or decreased expression of the protein encoded by the gene.

In another aspect, the present disclosure provides vectors for the tunable expression of polypeptides of interest in cells. The presently disclosed vectors comprise a nucleic acid molecule of Section II. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends e.

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