Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides

Stemmer, Willem P.C.; Crameri, Andreas; Ha, Kim D.; Brennan, Thomas M.; Heyneker, Herbert L.

doi:10.1016/0378-1119(95)00511-4

Cited by 649 publications

(540 citation statements)

References 10 publications

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“…Unlike the previous work, in which the overall path of the scaffold strand could be routed such that it never forms intentional base pairs with itself, the scaffold strand for the binary counter design here must wrap from helix to helix along a diagonal, which requires hairpin sequences at periodic intervals. To reduce problematic interference of secondary structure during synthesis of the scaffold strand by assembly PCR, 37 we chose exclusively-AT sequences for the hairpin domains. Furthermore, to specifically amplify the single-stranded sense strand at the end of assembly PCR (which produces long periodic double-stranded DNA), the entire scaffold strand sequence consists of only A,T,C; the final stage of synthesis, therefore, is provided only those nucleotides.…”

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confidence: 99%

“…To compare to Table 1 Crossover points in the scaffold are stretched in the diagram to accommodate the exploded spacing, but the molecules contain no nucleotides at the crossover points; the secondary structure of the SCA scaffold tile is consistent with the DAE-E motif. The scaffold consists of a single long periodic scaffold strand (blue) and three scaffold tile strands (SCA; red, yellow, green, of lengths 37,26,42). The intrinsic curvature of the DAE-E tiles is such that the radius of curvature points up out of the page; red stars indicate diagram artifacts at the nicks due to flattening the structure.…”

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confidence: 99%

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Two Computational Primitives for Algorithmic Self-Assembly: Copying and Counting

2005

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Copying and counting are useful primitive operations for computation and construction. We have made DNA crystals that copy and crystals that count as they grow. For counting, 16 oligonucleotides assemble into four DNA Wang tiles that subsequently crystallize on a polymeric nucleating scaffold strand, arranging themselves in a binary counting pattern that could serve as a template for a molecular electronic demultiplexing circuit. Although the yield of counting crystals is low, and per-tile error rates in such crystals is roughly 10%, this work demonstrates the potential of algorithmic self-assembly to create complex nanoscale patterns of technological interest. A subset of the tiles for counting form information-bearing DNA tubes that copy bit strings from layer to layer along their length.

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confidence: 99%

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confidence: 99%

Two Computational Primitives for Algorithmic Self-Assembly: Copying and Counting

2005

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“…The EGF-like domain of HRG was generated by DNA polymerasemediated assembly of overlapping oligonucleotides (listed in Supplementary Table 1) as described by Stemmer and co-workers. 59 The resulting product was cloned between the XhoI and SpeI sites in the HI loop coding sequence in pFG23XS to yield the plasmid pFG23XS-HI-HRG (Figure 1). Insertion of HRG in the HI loop resulted in substitution of residues Q 540 -P 547 of the HI loop with a 15 aa 5 0 linker, 52 aa EGF-like domain and 4 aa 3 0 linker for a total of 68 aa.…”

Section: Plasmidsmentioning

confidence: 99%

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice

et al. 2012

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Despite the tremendous potential of adenovirus (Ad) as a delivery vector for cancer gene therapy, its use in clinical settings has been limited, mainly as a result of the limited infectivity in many tumors and the wide tissue tropism associated with Ad. To modify the tropism of the virus, we have inserted the epidermal growth factor-like domain of the human heregulin-a (HRG) into the HI loop of Ad5 fiber. This insertion had no adverse effect on fiber trimerization nor did it affect incorporation of the modified fiber into infectious viral particles. Virions bearing modified fiber displayed growth characteristics and viral yields indistinguishable from those of wild-type (wt) virus. Most importantly, HRG-tagged virions showed enhanced infection of cells expressing the cognate receptors HER3/ErbB3 and HER4/ErbB4. This was significantly reduced in the presence of soluble HRG. Furthermore, HER3-expressing Chinese hamster ovary (CHO) cells were transduced by the HRG-modified virus, but not by wt virus. In contrast, CHO cells expressing the coxsackie-Ad receptor were transduced with both viruses. However, infection of an in vivo breast cancer xenograft model after intratumoral injection was similar with both viruses, suggesting that the tumor microenvironment and/or the route of delivery have important roles in infection of target cells with fiber-modified Ads.Cancer Gene Therapy (2012) 2,3 In addition to being among the most extensively studied viral vectors, Ads can be easily and economically manipulated, maintained and propagated to produce high titer stocks. 4,5 Moreover, they have the capacity to carry relatively large fragments of exogenous DNA into a wide range of cell and tissue types. 4,5 Subgroup C Ads (exemplified by the most widely used Ad5 and Ad2) attach to and enter host cells in a two-step process. 6 The initial recognition/attachment step is mediated by interactions between the knob component of fiber protein on the Ad capsid and the extracellular domain of the coxsackieadenovirus receptor (CAR) on the host cell surface. 7-11 This attachment is followed by a second interaction between the RGD motif in the penton base of the virus capsid and a v b 3 or a v b 5 cell surface integrins (secondary Ad receptors), which allows viral entry via receptor-mediated endocytosis. [12][13][14][15] Both the primary and secondary Ad receptors are expressed on a wide range of tissues leading to the observed broad tissue tropism of Ads. 16,17 Unfortunately, a large number of tumor cells appear relatively refractory to Ad infection because of limited surface expression of the primary Ad receptor. 1,[18][19][20][21][22][23] This observation has been a driving force behind recent intensive efforts to generate Ad viral vectors with altered tropism.

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“…26 However, the formation of incorrect hybridizations during synthesis yields erroneous final sequences in a population of different gene lengths and sequences. 27 These mismatched hybridizations are typically observed even in genes without repeating sequences.…”

Section: Introductionmentioning

confidence: 99%

Recombinant Human Collagen and Biomimetic Variants Using a De Novo Gene Optimized for Modular Assembly

et al. 2010

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A collagen-mimetic polymer that can be easily engineered with specific cell-responsive and mechanical properties would be of significant interest for fundamental cell-matrix studies and applications in regenerative medicine. However, oligonucleotide-based synthesis of full-length collagen has been encumbered by the characteristic glycine-X-Y sequence repetition, which promotes mismatched oligonucleotide hybridizations during de novo gene assembly. In this work, we report a novel, modular synthesis strategy that yields full-length human collagen III and specifically defined variants. We used a computational algorithm that applies codon degeneracy to design oligonucleotides that favor correct hybridizations while disrupting incorrect ones for gene synthesis. The resulting recombinant polymers were expressed in Saccharomyces cereVisiae engineered with prolyl-4-hydroxylase. Our modular approach enabled mixing-and-matching domains to fabricate different combinations of collagen variants that contained different secretion signals at the N-terminus and cysteine residues imbedded within the triple-helical domain at precisely defined locations. This work shows the flexibility of our strategy for designing and assembling specifically tailored biomimetic collagen polymers with re-engineered properties.

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Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides

Cited by 649 publications

References 10 publications

Two Computational Primitives for Algorithmic Self-Assembly: Copying and Counting

Two Computational Primitives for Algorithmic Self-Assembly: Copying and Counting

HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice

Recombinant Human Collagen and Biomimetic Variants Using a De Novo Gene Optimized for Modular Assembly

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