BACKGROUND OF THE INVENTION

Generation of DNA libraries for mutational scanning, directed evolution, and related techniques requires trading-off control over library content against diversity. Traditional site-directed mutagenesis offers a high level of control, but requires one independent reaction per mutation or site, which limits how much diversity can be generated in a cost-efficient manner. Error-prone PCR, spiked oligonucleotide synthesis, and chemical mutagenesis can generate highly diverse libraries in a single reaction, but provide limited control over their contents. In particular, introducing unbiased nucleotide substitutions into a protein-coding sequence produces a highly biased mutational spectrum at the amino acid level, and introducing multiple substitutions into one sequence forces incomplete sampling of the sequence space and may obscure the effect of individual substitutions through non-additive effects.

There is a need for methods of generating mixtures of variants of a template DNA molecule in a short time and in a relatively inexpensive manner, such that the mixtures include, e.g., every possible variant of every nucleotide of the template DNA molecule.

SUMMARY OF THE INVENTION

The invention provides methods of generating mixtures of variants of template DNA molecules. In general, the methods include: a) providing a plurality of oligonucleotides, where each of the oligonucleotides: i) corresponds to a first region of a template DNA molecule, ii) has at least one sequence variation as compared to the sequence of the first region of the template DNA molecule, and, optionally, iii) includes a central region that has the at least one sequence variation and two end regions, wherein the end regions are invariant; b) providing a plurality of linearized plasmid vectors comprising the remainder of the template DNA molecule, but lacking the first region of the template DNA molecule at the ends of the linearized plasmid vectors; and c) joining the plurality of oligonucleotides to the plurality of linearized plasmid vectors, thereby generating plasmids including a mixture of variants of the first region of the template DNA molecule.

In one embodiment of the methods of the invention, the template DNA molecule encodes a polypeptide, a non-coding RNA, or an untranslated regulatory sequence. Additionally or alternatively, the template DNA sequence can include a promoter sequence and/or an enhancer sequence. The variation can be, for example, a substitution of at least one nucleotide (e.g., 1, 2, 3, or more nucleotides). The methods of the invention can, in various examples, include the use of a plurality of oligonucleotides including oligonucleotides encoding at least one variation of every amino acid encoded by the first region of the template DNA molecule.

The methods of the invention also can include: a) providing a second plurality of oligonucleotides, wherein each of the oligonucleotides: i) corresponds to a second region of a template DNA molecule, ii) has at least one sequence variation as compared to the sequence of the second region of the template DNA molecule, and, optionally, iii) includes a central region that has the at least one sequence variation and two end regions, wherein the end regions are invariant; b) providing a plurality of linearized plasmid vectors including the remainder of the template DNA molecule, but lacking the second region of the template DNA molecule at the ends of the linearized plasmid vectors; c) joining the second plurality of oligonucleotides to the plurality of linearized plasmid vectors, thereby generating plasmids including a mixture of variants of the second region of the template DNA molecule; and d) mixing the mixture of plasmids including variants of the first region of the template DNA molecule with the mixture of plasmids including variants of the second region of the template DNA molecule to generate a mixture of variants of the first and second regions of the template DNA molecule. The first and second regions of the template DNA molecule may or may not overlap.

The plurality of oligonucleotides used in the methods of the invention can be oligonucleotides that include at least one or more variations (e.g., one, two, three, or more) of every nucleotide of the first region and/or the second region of the template DNA molecule.

The methods of the invention can be used to generate variants of template DNA molecules which have “n” regions, where “n” is designated arbitrarily and some or all of “n” regions may overlap. In such a case, the method includes performing steps a) to d) mentioned above to generate a mixture of variants of each of the “n” regions. In any of the methods of the invention, “n” can be greater than, e.g., 1, 2, 3, 4, 5, 10, 20, 25, 30, 50, or 100. For example, “n” can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 50. In various embodiments of the invention, the mixture of plasmids of each of the “n” regions is mixed in equimolar amounts.

The methods of the invention can be used to generate mixtures of variants that include at least one variation (e.g., 1, 2, 3, 4, or more) in every nucleotide along the entire length of a template DNA molecule. The variations in template DNA sequence can be substitutions, insertions, or deletions of one or more nucleotides. If a template DNA molecule encodes a polypeptide, then the variation in amino acid sequence encoded by the template DNA molecule, resulting from the methods of the invention, can encode a naturally occurring amino acid or an unnatural amino acid.

The methods of the invention also include synthesizing a plurality of oligonucleotides, as described herein, on a micro-scale solid support (e.g., a microarray). Oligonucleotides corresponding to overlapping “n” regions can optionally be synthesized on different microarrays. Each oligonucleotide in the above-mentioned plurality of oligonucleotides can be, for example, 150 to 250 nucleotides in length, and the plurality of oligonucleotides can be amplified using the polymerase chain reaction prior to the above-mentioned joining step.

The invention also includes methods of generating linearized plasm id vectors by carrying out the polymerase chain reaction or restriction enzyme digestion reaction prior to the joining step. Prior to the above-mentioned mixing step, the methods can further include: a) transforming into a host cell (e.g., a bacteria or yeast), the mixture of plasmids generated by the joining; b) selecting (e.g., by antibiotic selection or nutrient selection) for recombinant clones containing the plasmids; and c) isolating plasmids containing the variants. The linearized plasmid vectors may, for example, encode an antibiotic selection protein or a protein for nutrient selection, which can be used for selecting recombinant clones.

The invention also includes methods involving generating one or more plasmids in a mixture of plasmids where each plasmid includes one or more variation in two or more different regions of a template DNA molecule. The mixture of plasmids can be generated by creating a first mixture of variant-containing plasmids corresponding to a first region of the template DNA molecule, followed by linearization of the first mixture of variant-containing plasmids and joining of a second plurality of variation-containing oligonucleotides corresponding to a second region of the template DNA molecule to create a mixture of plasmids that includes variations in two different regions of the template DNA molecule. The methods can be used successively to generate mixtures of plasmids wherein each plasmid comprises variations in the ‘in’ different regions of a template DNA molecule.

The invention also includes methods of generating mixtures of variants of a template DNA molecule by: a) providing a plurality of oligonucleotides, wherein each of the oligonucleotides: i) corresponds to one of multiple regions of the template DNA molecule; ii) has at least one sequence variation as compared to the sequence of the one of multiple regions of the template DNA molecule; and iii) the plurality of oligonucleotides comprises oligonucleotides that correspond to each of the multiple regions of the template DNA molecule; b) providing a plurality of linearized plasmid vectors including the remainder of the template DNA molecule, but lacking the multiple regions of the template DNA molecule as described herein; and c) joining the plurality of oligonucleotides to each other and the plurality of linearized plasmid vectors, thereby generating plasmids including a mixture of variants of the multiple regions of the template DNA molecule. The plurality of oligonucleotides can further include oligonucleotides identical to at least one region of the template DNA molecule and the plurality of oligonucleotides can, optionally, not include oligonucleotides corresponding to the at least one region that have a variation as compared to the template DNA molecule.

In another aspect, the invention includes methods of identifying variant nucleic acid molecules (e.g., molecules encoding proteins, optionally an antibiotic or chemotherapeutic resistance protein) which, when introduced into a cell, selectively increase or decrease the sensitivity of the cell to an environmental factor. These methods include:a) introducing a mixture of variants of a template DNA molecule (e.g., a mixture generated by any of the foregoing methods) into both a first population of cells and a second population of cells,b) incubating the first population of cells in the presence of a first environmental factor;c) incubating the second population of cells in the absence of the first environmental factor;d) isolating cells exhibiting a phenotype associated with increased or decreased sensitivity to the first environmental factor; ande) determining which variants of the template DNA molecule are enriched or depleted in cells isolated from the first population of cells as compared to the second population of cells;
thereby identifying variant nucleic acid molecules that selectively increase or decrease the sensitivity of the cells to the first environmental factor.

In certain embodiments, the second population of cells are grown in the presence of a second environmental factor, thereby permitting the identification of variants that selectively increase or decrease the sensitivity of the cells to the first environmental factor relative to the second environmental factor.

In some embodiments, the population of cells are bacterial or fungal cells, and the first environmental factor is, e.g., an antibiotic. When present, a second environmental factor can be, e.g., an antibiotic, optionally an antibiotic from the same family as the first environmental factor.

In other embodiments, the cell populations can be mammalian cells and, e.g., the first environmental factor can be a chemotherapeutic agent. If present, the second environmental factor can be, e.g., a chemotherapeutic agent, optionally a chemotherapeutic agent from the same family as the first environmental factor.

In other embodiments, the environmental factor or factors can be selected from, e.g., cell differentiation agents, anti-viral compounds, growth factors, drugs (e.g., anti-cancer agents), nutrients (e.g., carbon sources and vitamins), cellular toxins, anti-aging compounds, anti-fungal compounds, anti-protozoan compounds, hormones, steroids, anti-inflammatory agents, mutagens (e.g., ultraviolet light, X-rays, gamma rays, and chemical carcinogens), temperature, pressure, pH, salinity, viscosity, pathogenic organisms or particles that attach to or enter cells, and components of such organisms or particles that mediate cell entry or other interactions with the host cell.

In any of the foregoing methods, the isolating of cells can include, e.g., collection of surviving cells or separation of cells having a particular morphological characteristic, optionally wherein the morphological characteristic is cell size, expression of a differentiation marker, cell adhesion, or cell membrane integrity. Such separation can include, e.g., fluorescent activation cell sorting (FACS).

Any of the foregoing methods can further include introducing a mixture of variants of a template DNA molecule into a third population of cells, incubating the third population of cells in the presence of a third environmental factor, and determining which variants of the template DNA molecule are enriched or depleted in cells incubated with the third environmental factor as compared to the first and/or second environmental factors, thereby identifying variants that selectively increase or decrease sensitivity of the cells to the third environmental factor.

Also, in any of the foregoing methods, the mixture of variants of a template DNA molecule can be, e.g., encapsulated in a viral particle when introduced into a population of cells. In some embodiments, the methods include isolating cells exhibiting a phenotype associated with increased or decreased sensitivity to the first environmental factor, and further can include isolating viral DNA or RNA from the population of cells.

Definitions

By “template DNA molecule” is meant a DNA polynucleotide that is, in general, 100 or more nucleotides in length (e.g., greater than 100, 150, 200, 300, 500, 800, 1000, 2000, 5000, 10000, or 20000 nucleotides in length, with the option of the upper limit being typically 1 megabase, 100 kb, 50 kb, or 15 kb). Thus, in various examples, the template DNA polynucleotide is 100-20000, 150-5000, 200-2000, 300-1000, or 500-800 nucleotides in length. A template DNA molecule can include, e.g., a sequence that encodes a polypeptide, DNA regulatory element, or RNA, and can be in the form of double or single stranded DNA.

By a “variant” template DNA molecule is meant a template DNA molecule with one or more (e.g., 2, 3, 4, 5, 10, or more) changes in nucleotide sequence relative to an original template DNA molecule. Thus, in various examples, the variant can have 1-20, 2-15, 3-12, 4-10, or 5-8 changes. The variant can be created by substitution, insertion, or deletion of one or more nucleotides within the original template DNA molecule sequence.

A variant is “enriched” in a first population of cells (e.g., an isolated population of cells) relative to a second population of cells if the variant is present at a greater frequency in the first population of cells as compared to the second population of cells.

A variant is “depleted” in a first population of cells (e.g., an isolated population of cells) relative to a second population of cells if the variant is present at a lower frequency in the first population of cells as compared to a second population of cells. A lower frequency includes (but does not require) the complete absence of a particular variant in the first population of cells.

By “oligonucleotide” or “polynucleotide” is meant a polymer of nucleotides, with oligonucleotides in general being relatively short (e.g., 25-500, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides in length) and polynucleotides being longer. Oligonucleotides and polynucleotides can contain natural or synthetic nucleotides. Unless indicated otherwise, these terms are interchangeable as used herein.

By “linearized plasmid vector” is meant a plasmid vector that is made linear (e.g., by a polymerase chain reaction or by a restriction digestion reaction). The linearized plasmid vector has a 5′ end and a 3′ end. The 5′ and 3′ ends can be, e.g., single stranded or blunt.

By “overlap” is meant a region of nucleotide sequence that is identical between two oligonucleotides such that the 5′ end of one oligonucleotide has 3 or more nucleotide (e.g., 3, 4, 5, 10 or more) identity to the 3′ end of another oligonucleotide.

By “nutrient selection” is meant a technique known in the art for selecting plasmids containing a gene required for the growth and propagation of a bacterium, in a medium which lacks or is supplemented with a specific nutrient.

By “phenotype associated with increased or decreased sensitivity” to an environmental factor is meant a cell characteristic that is associated with exposure to the environmental factor. For example, the phenotype can be expression of a differentiation marker, increase cell growth, increased cell death, or decreased cell growth. Increases in sensitivity result can result in an increase or decrease in the presence of the associate phenotype. Similarly, a decrease in sensitivity can result in an increase or decrease in the presence of the associate phenotype. For example, in cases where the environmental factor is a particular toxic agent, a decrease in sensitivity to the toxic agent produces a phenotype of increased cell survival/growth, while an increase in sensitivity to the toxic agent produced a decrease in cell survival/growth.

By “isolating” is meant separating cells having a certain phenotype from those cells that lack that phenotype. Methods of isolation include physical separation of cells having a phenotype, depletion of cells lacking the phenotype, or enrichment of cells having a phenotype.

By “environmental factor” is meant any aspect of the cell culture conditions that can be controlled by an individual performing the methods of the invention. For example, environmental factors can be the presence or absence of particular nutrients, toxins, antibiotics, anti-viral agents, chemotherapeutic agents, anti-inflammatory agents, vitamins, hormones, differentiation factors, pathogens (including viruses or bacteria), or other cell types (e.g., feeder cells). Environmental factors can also include other aspects of cell culture, including temperature, atmospheric components, atmospheric pressure, electromagnetic radiation (e.g., visible light, UV light, X-rays, or gamma rays), or mechanical vibration.

Other features and advantages of the invention will be apparent from the detailed description, drawings, and claims.

