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Nucleic acid evolution and minimization by nonhomologous random recombination

Abstract

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.

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Figure 1: Diversification methods for nucleic acid evolution.
Figure 2: Overview of the nonhomologous random recombination (NRR) method.
Figure 3: Nucleic acid evolution by NRR or error-prone PCR.
Figure 4: Streptavidin binding affinities of DNA aptamers evolved by error-prone PCR (EPPCR) or by NRR.
Figure 5: Diversification by error-prone PCR and by NRR.
Figure 6: Isolation of a minimal streptavidin binding motif by inspection of NRR-generated clones.

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Acknowledgements

Funding was provided by US National Science Foundation CAREER award MCB-0094128 and by Harvard University. J.A.B. is a Howard Hughes Medical Institute predoctoral fellow. B.V.L. was supported by the Harvard College Research Program. We are grateful to P. Patten (Maxygen, Redwood City, CA) for helpful discussions.

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Correspondence to David R. Liu.

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Bittker, J., Le, B. & Liu, D. Nucleic acid evolution and minimization by nonhomologous random recombination. Nat Biotechnol 20, 1024–1029 (2002). https://doi.org/10.1038/nbt736

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