Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers

Nucleic Acids Res. 2012 Aug;40(15):e117. doi: 10.1093/nar/gks624. Epub 2012 Jun 26.

Abstract

DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin 'capping' oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line
  • DNA / biosynthesis*
  • DNA / chemistry
  • DNA-Binding Proteins / genetics
  • Deoxyribonucleases, Type II Site-Specific / genetics*
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Genes, Reporter
  • Genes, Synthetic*
  • Homologous Recombination
  • Humans
  • Molecular Sequence Data
  • Protein Structure, Tertiary / genetics
  • Repetitive Sequences, Nucleic Acid

Substances

  • DNA-Binding Proteins
  • DNA
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific