by Harald Zähringer Labtimes 06/2014
Paper clips are used to fasten documents and paper sheets. A new DNA assembling method applies molecular paper clips to staple multiple DNA parts.
Assembling DNA sequences (parts) such as promoters, open reading frames, terminators, etc. into functional biological constructs or devices is one of the essential techniques in synthetic biology. The DNA parts may be put together by different cloning methods based on homologous recombination (Gibson assembly, SLIC, SLiCE, CPEC), TypIIS restriction enzymes (Golden Gate cloning and its variants Golden Braid and MoClo), special enzymes like USER or various BioBrick assembly systems.
These methods depend on either restriction enzymes, preparation of homologous ends, restriction sites, mutagenesis of internal restriction sites or special enzymes and often require a set of new oligos for each assembly reaction.
These handicaps may be avoided with the PaperClip assembly technique described by a Scottish group in Nucleic Acids Research (Trubitsyna et al., NAR, doi: 10.1093/nar/gku829).
The crucial elements of this new assembly system are four especially-designed oligonucleotides required for each DNA part to be assembled: an upstream forward (UF) oligo and an upstream reverse (UR) oligo, corresponding to the upstream region of the DNA part, as well as a downstream forward (DF) oligo and a downstream reverse (DR) oligo, referring to the downstream region.
Stitching together two and three DNA parts with clips. The succession of the parts in three or multiple part assemblies is guided by the order of the half-clips (1-2-3 or 1-3-2 in the example shown above). Image: Trubitsyna et al.
To work according to paper clips, the oligos have to be designed in the following way: select the first 40 bases of the forward strand of the DNA part and add the trinucleotide GCC at the 5’-end to get the UF-oligo. Choose the first 37 nucleotides of this strand to create the reverse complement UR-oligo. Pick the last 40 bases of the DNA part to design the DF-oligo (the first three bases should not be GCC or GGC). Finally, take the last 37 bases of the sequence to create a reverse complement DR-oligo and add GGC at its 5’-end.
Once you’ve got these four oligos together, you’re almost ready to assemble the parts. Phosphorylating the oligos with T4 polynucleotide kinase and subsequent annealing leads to two half-clip pairs, corresponding to the upstream and downstream regions of the DNA part respectively, with GCC-extensions at the outer ends. The half-clip pairs are stored together with the respective DNA part and may be ligated prior to assembly with T4 DNA ligase. GCC-tails allow easy ligation of adjacent half-clips to form double-stranded oligo-clips.
The clips “stitch” together the DNA parts, which may be assembled by various methods. The Scottish group received the best assembly results with a PCR approach based on circular polymerase extension cloning, however, any other assembly method may work as well.
Short DNA sequences, such as tags or linkers, may be inserted between the two DNA parts in a similar fashion. Again four oligos are applied with two of them creating GCC- and GGC-overhangs. In this case, however, the ligation of the clip oligo takes two steps. In the first step, the downstream half-clip is ligated with the upstream half of the insert in one tube and the downstream half of the intervening sequence is ligated with the upstream half-clip in another. The contents of both tubes are, subsequently, mixed in the second step and further incubated to complete ligation of the whole construct.
Cloning experts may have noticed that PaperClip assembly introduces a GCC-scare encoding alanine between each DNA part, which may not be tolerable in some cases. However, this minor concern is more than compensated by the cost and time reduction offered by this new assembly method.
Last Changed: 20.11.2014