Nucleic acid evolution and minimization by nonhomologous random recombination.
We have developed a simple method for exploring nucleic acid sequence space by nonhomologous random recombination (NRR) that enables DNA fragments to randomly recombine in a length-controlled manner without the need for sequence homology. We compared the results of using NRR and error-prone PCR to evolve DNA aptamers that bind streptavidin. Starting with two parental sequences of modest avidin affinity, evolution using NRR resulted in aptamers with 15- to 20-fold higher affinity than the highest-affinity aptamers evolved using error-prone PCR, and 27- or 46-fold higher affinities than parental sequences derived using systematic evolution of ligands by exponential enrichment (SELEX). NRR also facilitates the identification of functional regions within evolved sequences. Inspection of a small number of NRR-evolved clones identified a 40-base DNA sequence, present in multiple copies in each clone, that binds streptavidin. Our findings suggest that NRR may enhance the effectiveness of nucleic acid evolution and the ease of identifying structure-activity relationships among evolved sequences.
Bittker
Joshua A
JA
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Le
Brian V
BV
Liu
David R
DR
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D019020:Directed Molecular Evolution / Q000379:methods; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D019809:Streptavidin / Q000235:genetics
D017931:DNA Primers; D004247:DNA; D019809:Streptavidin