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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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Assembly of chimeric constructs and one-pot concatamer formation.(A) Assembly of (FNIII)2-VWFA2-(FNIII)2 sandwich constructs from a modular assembly vector (top). PCR amplification with specific primers (indicated above the lanes; Table S1) show that the A2 synthon is sandwiched between two 13FNIII repeats. (B) One-pot concatamer formation with orthogonal chain stoppers. Fully deprotected synthons are mixed in different molar ratios with orthogonal chain stoppers (equivalent synthons with protecting groups on one end). Increasing the concentration of chain stoppers shifts the size distribution towards shorter concatamers. Molar ratios of unprotected synthons:chain stoppers are indicated at the top of the lanes. Bands marked with asterisks presumably correspond to circularized dimers.
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pone-0037617-g005: Assembly of chimeric constructs and one-pot concatamer formation.(A) Assembly of (FNIII)2-VWFA2-(FNIII)2 sandwich constructs from a modular assembly vector (top). PCR amplification with specific primers (indicated above the lanes; Table S1) show that the A2 synthon is sandwiched between two 13FNIII repeats. (B) One-pot concatamer formation with orthogonal chain stoppers. Fully deprotected synthons are mixed in different molar ratios with orthogonal chain stoppers (equivalent synthons with protecting groups on one end). Increasing the concentration of chain stoppers shifts the size distribution towards shorter concatamers. Molar ratios of unprotected synthons:chain stoppers are indicated at the top of the lanes. Bands marked with asterisks presumably correspond to circularized dimers.

Mentions: Repetitive constructs are frequently used in single-molecule experiments for reasons of accelerated data sampling and confidence in distinguishing specific tethers from non-specific background. However, such constructs can hamper the identification of intermediates owing to simultaneous unfolding of structural elements in other domains of the repetitive context. In such cases, the unambiguous assignment of unfolding traces is facilitated if individual domains of interest are sandwiched between a repetitive series of protein domains whose elastic properties are well characterized, such as immunoglobulin-like domains of titin [13], [14], Dyctostelium discoideum filamin (ddFLN) [22] or fibronectin FNIII domains [17]. Our donor and acceptor vectors support the rapid assembly of such constructs and we illustrate this process by sandwiching single VWFA2 domains between amino- and carboxyl-terminal 13(FNIII)2 repeats (Fig. 5A). For this purpose, a 13(FNIII)2-X-13(FNIII)2 vector construct was created that contains inversely oriented BsaI sites between the 13FNIII repeats to form the entry point for synthon insertion (see Fig. 2B). 13(FNIII)2 synthons were subcloned into both pDA-N and pDA-C vectors and subsequently recombined by ligating the BsmBI/BamHI fragment from the resulting pDA-N-13(FNIII)2 vector (acting as the donor) into the BsmBI/BamHI-linearized pDA-C-13(FNIII)2 vector (acting as the acceptor). A synthon encoding residues Val1478 to Gly1674 of the VWFA2 domain and flanking (GlySer)3 linkers was subsequently inserted between the 13(FNIII)2 repeats using the BsaI restriction sites. The successful assembly was verified by PCR (Fig. 5A).


A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Assembly of chimeric constructs and one-pot concatamer formation.(A) Assembly of (FNIII)2-VWFA2-(FNIII)2 sandwich constructs from a modular assembly vector (top). PCR amplification with specific primers (indicated above the lanes; Table S1) show that the A2 synthon is sandwiched between two 13FNIII repeats. (B) One-pot concatamer formation with orthogonal chain stoppers. Fully deprotected synthons are mixed in different molar ratios with orthogonal chain stoppers (equivalent synthons with protecting groups on one end). Increasing the concentration of chain stoppers shifts the size distribution towards shorter concatamers. Molar ratios of unprotected synthons:chain stoppers are indicated at the top of the lanes. Bands marked with asterisks presumably correspond to circularized dimers.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369885&req=5

pone-0037617-g005: Assembly of chimeric constructs and one-pot concatamer formation.(A) Assembly of (FNIII)2-VWFA2-(FNIII)2 sandwich constructs from a modular assembly vector (top). PCR amplification with specific primers (indicated above the lanes; Table S1) show that the A2 synthon is sandwiched between two 13FNIII repeats. (B) One-pot concatamer formation with orthogonal chain stoppers. Fully deprotected synthons are mixed in different molar ratios with orthogonal chain stoppers (equivalent synthons with protecting groups on one end). Increasing the concentration of chain stoppers shifts the size distribution towards shorter concatamers. Molar ratios of unprotected synthons:chain stoppers are indicated at the top of the lanes. Bands marked with asterisks presumably correspond to circularized dimers.
Mentions: Repetitive constructs are frequently used in single-molecule experiments for reasons of accelerated data sampling and confidence in distinguishing specific tethers from non-specific background. However, such constructs can hamper the identification of intermediates owing to simultaneous unfolding of structural elements in other domains of the repetitive context. In such cases, the unambiguous assignment of unfolding traces is facilitated if individual domains of interest are sandwiched between a repetitive series of protein domains whose elastic properties are well characterized, such as immunoglobulin-like domains of titin [13], [14], Dyctostelium discoideum filamin (ddFLN) [22] or fibronectin FNIII domains [17]. Our donor and acceptor vectors support the rapid assembly of such constructs and we illustrate this process by sandwiching single VWFA2 domains between amino- and carboxyl-terminal 13(FNIII)2 repeats (Fig. 5A). For this purpose, a 13(FNIII)2-X-13(FNIII)2 vector construct was created that contains inversely oriented BsaI sites between the 13FNIII repeats to form the entry point for synthon insertion (see Fig. 2B). 13(FNIII)2 synthons were subcloned into both pDA-N and pDA-C vectors and subsequently recombined by ligating the BsmBI/BamHI fragment from the resulting pDA-N-13(FNIII)2 vector (acting as the donor) into the BsmBI/BamHI-linearized pDA-C-13(FNIII)2 vector (acting as the acceptor). A synthon encoding residues Val1478 to Gly1674 of the VWFA2 domain and flanking (GlySer)3 linkers was subsequently inserted between the 13(FNIII)2 repeats using the BsaI restriction sites. The successful assembly was verified by PCR (Fig. 5A).

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