Polymerase Chain Reaction Based Scaffold Preparation for the Production of Thin, Branched DNA Origami Nanostructures of Arbitrary Sizes

Pound, Elisabeth; Ashton, Jeffrey R.; Becerril, Héctor A.; Woolley, Adam T.

doi:10.1021/nl902535q

Cited by 121 publications

(120 citation statements)

References 21 publications

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“…Successful kanR insertion disrupted LacZ function, so proper targeting of the lacZTkanR cassette could be verified by selecting on kanamycin and assaying for the inability to cleave X-Gal in order to release a blue chromophore. This dsDNA construct was generated by PCR and converted into ssDNA using a biotin capture and DNA melting protocol (Pound et al 2009), as detailed in File S1. PAGE analysis confirmed the purity of the lacZTkanR ssDNA construct, as no dsDNA band was readily detected.…”

Section: Resultsmentioning

confidence: 99%

Lambda Red Recombineering inEscherichia coliOccurs Through a Fully Single-Stranded Intermediate

2010

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The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 59 ends of double-stranded DNA are recessed by l exonuclease, leaving behind 39 overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinasecatalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.

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Section: Resultsmentioning

confidence: 99%

Lambda Red Recombineering inEscherichia coliOccurs Through a Fully Single-Stranded Intermediate

2010

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“…TE scaffolding materials have been extensively explored for the regeneration of various parts of the human body, including skin [1], muscle [2], teeth [3], bone [4,5], cartilage [4], ligament [6], spinal cord [7], nerve [8], genome [9] and artificial organs, particularly cardiac tissues or heart [10], lung [11], kidney [12], liver [13], urinary bladder [14], blood vessels [15], stomach [16], intestine [17], breast [18], ear [19], eye [20] and nose [21]. TE includes the combined growth of living cells, tissues or organs, as well as methods and materials that can restrain the shape of a particular tissue for better functions.…”

Section: Introductionmentioning

confidence: 99%

Progress of key strategies in development of electrospun scaffolds: bone tissue

Pramanik

Pingguan‐Murphy

Osman

2012

Science and Technology of Advanced Materials

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There has been unprecedented development in tissue engineering (TE) over the last few years owing to its potential applications, particularly in bone reconstruction or regeneration. In this article, we illustrate several advantages and disadvantages of different approaches to the design of electrospun TE scaffolds. We also review the major benefits of electrospun fibers for three-dimensional scaffolds in hard connective TE applications and identify the key strategies that can improve the mechanical properties of scaffolds for bone TE applications. A few interesting results of recent investigations have been explained for future trends in TE scaffold research.

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“…The remaining formation of the structures was achieved by gradually removing the formamide by dialysis. In the second method Woolley and co-workers 4 used biotinylated primers in a PCR reaction to obtain single stranded DNA (ssDNA) products to be used as scaffolds for the assembly of origami structures. Using this approach they generated several different DNA origamis with sizes ranging from 756 to 4808 bps.…”

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confidence: 99%

Organizing DNA Origami Tiles into Larger Structures Using Preformed Scaffold Frames

2011

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Structural DNA nanotechnology utilizes DNA molecules as programmable information-coding polymers to create higher order structures at the nanometer scale. An important milestone in structural DNA nanotechnology was the development of scaffolded DNA origami in which a long single-stranded viral genome (scaffold strand) is folded into arbitrary shapes by hundreds of short synthetic oligonucleotides (staple strands). The achievable dimensions of the DNA origami tiles units are currently limited by the length of the scaffold strand. Here we demonstrate a strategy referred to as ‘super-origami’ or ‘origami of origami’ to scale up DNA origami technology. First, this method uses a collection of bridge strands to pre-fold a single stranded DNA scaffold into a loose framework. Subsequently, pre-formed individual DNA origami tiles are directed onto the loose framework so that each origami tile serves as a large staple. Using this strategy, we demonstrate the ability to organize DNA origami nanostructures into larger spatially addressable architectures.

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Polymerase Chain Reaction Based Scaffold Preparation for the Production of Thin, Branched DNA Origami Nanostructures of Arbitrary Sizes

Cited by 121 publications

References 21 publications

Lambda Red Recombineering inEscherichia coliOccurs Through a Fully Single-Stranded Intermediate

Lambda Red Recombineering inEscherichia coliOccurs Through a Fully Single-Stranded Intermediate

Progress of key strategies in development of electrospun scaffolds: bone tissue

Organizing DNA Origami Tiles into Larger Structures Using Preformed Scaffold Frames

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