Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance.
BACKGROUND
Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide "letters", the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson-Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so.


RESULTS
We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson-Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2'-deoxyisocytidine and 2'-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted.


CONCLUSIONS
AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.
Foundation for Applied Molecular Evolution, P.O. Box 13174, Gainesville, FL, 32604, USA ; The Westheimer Institute for Science and Technology, 720 S. W. 2nd Avenue, Suites 201-208, Gainesville, FL, 32601, USA.
Kristen K
KK
Merritt
Foundation for Applied Molecular Evolution, P.O. Box 13174, Gainesville, FL, 32604, USA ; The Westheimer Institute for Science and Technology, 720 S. W. 2nd Avenue, Suites 201-208, Gainesville, FL, 32601, USA.
Kevin M
KM
Bradley
Foundation for Applied Molecular Evolution, P.O. Box 13174, Gainesville, FL, 32604, USA ; The Westheimer Institute for Science and Technology, 720 S. W. 2nd Avenue, Suites 201-208, Gainesville, FL, 32601, USA ; Firebird Biomolecular Sciences LLC, 13709 Progress Blvd. Box 17, Alachua, FL 32615, USA.
Daniel
D
Hutter
Foundation for Applied Molecular Evolution, P.O. Box 13174, Gainesville, FL, 32604, USA ; The Westheimer Institute for Science and Technology, 720 S. W. 2nd Avenue, Suites 201-208, Gainesville, FL, 32601, USA ; Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA.
Mariko F
MF
Matsuura
Foundation for Applied Molecular Evolution, P.O. Box 13174, Gainesville, FL, 32604, USA ; The Westheimer Institute for Science and Technology, 720 S. W. 2nd Avenue, Suites 201-208, Gainesville, FL, 32601, USA.
Diane J
DJ
Rowold
Foundation for Applied Molecular Evolution, P.O. Box 13174, Gainesville, FL, 32604, USA ; The Westheimer Institute for Science and Technology, 720 S. W. 2nd Avenue, Suites 201-208, Gainesville, FL, 32601, USA ; Firebird Biomolecular Sciences LLC, 13709 Progress Blvd. Box 17, Alachua, FL 32615, USA.
Steven A
SA
Benner
artificially expanded genetic information systems; automated gene synthesis; solid-phase DNA synthesis; synthetic biology