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A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Bottom Line: We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames.Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles.As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.

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Split-and-pool assembly of DNA synthons.(A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.
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pone-0037617-g001: Split-and-pool assembly of DNA synthons.(A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.

Mentions: The application of IIS endonucleases in DNA assembly is not new, and a number of resourceful strategies have recently emerged [5], [7], [15], [16]. We here attempt to extend these methods by introducing a general protecting group strategy that exploits the unique properties of IIS endonucleases. In our general design, the ends of synthons are flanked by oppositely oriented recognition sites for two different IIS endonucleases (Fig. 1A). The choice of the endonuclease used for excision of the synthon from an entry vector determines which end of the synthon is protected and which end is reactive/cohesive. The orthogonal motif, together with appropriately selected overhang sequences, supports unidirectional and site-specific assembly of (composite) modules on either end of the entry synthon (Fig. 1B). In our design we have chosen IIS endonucleases with 6 base pair recognition sequences, whose theoretical frequency of occurrence is approximately once in 2048 base pairs (46/2), depending on the GC content of the donor genome. This is sufficiently rare to allow application of this approach to typical gene fragments; however, prior mutagenesis may occasionally be required to remove internal sites. From the wealth of commercially available IIS endonucleases, we chose BsmBI and BsaI for reasons of robustness and similarity in buffer and temperature requirements (Table 1). The recognition sequences of BsmBI and BsaI differ only in a single nucleotide. Conveniently, another IIS endonuclease, BsmAI, recognizes the common subset of these recognition sequences, so that one can optionally combine both cleavage reactions to yield synthons that are deprotected on both ends.


A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Split-and-pool assembly of DNA synthons.(A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369885&req=5

pone-0037617-g001: Split-and-pool assembly of DNA synthons.(A) Entry synthons are flanked on both sides by recognition sequences for the type IIS endonucleases BsaI and BsmBI. Restriction by either BsaI or BsmBI selectively exposes user-definable 4-base cohesive overhang sequences (5′-XXXX vs. 5′-xxxx) at one end of the synthon, while maintaining orthogonal protection groups (with 5′-YYYY vs. 5′-zzzz overhangs) at the opposite end. (B) Schematic representation of the ‘split-and-pool’ assembly principle. Cohesive ends of entry synthons are selectively deprotected by digestion with either BsaI or BsmBI. Pooling of the deprotected synthons in the presence of ligase results in unidirectional assembly, affording an idempotent tandem repeat synthon by restoration of orthogonal protecting groups on opposite ends. Each product module can recursively enter the assembly cycle (left panel) N times to yield concatameric synthons with 2N elements. The same strategy can be applied to the assembly of heterosynthons (dashed box), which allows for the engineering of chimeric and multimodular proteins or polycistronic genes.
Mentions: The application of IIS endonucleases in DNA assembly is not new, and a number of resourceful strategies have recently emerged [5], [7], [15], [16]. We here attempt to extend these methods by introducing a general protecting group strategy that exploits the unique properties of IIS endonucleases. In our general design, the ends of synthons are flanked by oppositely oriented recognition sites for two different IIS endonucleases (Fig. 1A). The choice of the endonuclease used for excision of the synthon from an entry vector determines which end of the synthon is protected and which end is reactive/cohesive. The orthogonal motif, together with appropriately selected overhang sequences, supports unidirectional and site-specific assembly of (composite) modules on either end of the entry synthon (Fig. 1B). In our design we have chosen IIS endonucleases with 6 base pair recognition sequences, whose theoretical frequency of occurrence is approximately once in 2048 base pairs (46/2), depending on the GC content of the donor genome. This is sufficiently rare to allow application of this approach to typical gene fragments; however, prior mutagenesis may occasionally be required to remove internal sites. From the wealth of commercially available IIS endonucleases, we chose BsmBI and BsaI for reasons of robustness and similarity in buffer and temperature requirements (Table 1). The recognition sequences of BsmBI and BsaI differ only in a single nucleotide. Conveniently, another IIS endonuclease, BsmAI, recognizes the common subset of these recognition sequences, so that one can optionally combine both cleavage reactions to yield synthons that are deprotected on both ends.

Bottom Line: We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames.Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles.As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.

Show MeSH