Research Highlights

Nature Chemical Biology
Published online: 25 February 2005 | doi: 10.1038/nchembio005

Nucleic Acid Biochemistry

Directed Evolution

Modified nucleic acids on display

Gregory Watt

Ichida et al. describe the development of a system for the in vitro selection of functional threose nucleic acid (TNA) molecules.

Non-natural nucleic acids have offered insight into natural DNA and RNA and provided experimental systems to test theories of nucleic acid evolution. Numerous structurally modified nucleic acids have been prepared in the laboratory that show a diverse range of genetic and structural properties. One in particular, threose nucleic acid (TNA), has attracted attention because of its chemical simplicity and its ability to base-pair with complementary DNA, RNA and other TNA. Ichida et al. now show, in the Journal of the American Chemical Society, that long stretches of TNA can be synthesized by an engineered DNA polymerase, and they suggest a system for the in vitro selection of TNA molecules with novel functions.

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The authors demonstrated previously that limited stretches of TNA nucleotides could be enzymatically polymerized on a DNA template using chemically synthesized threose nucleoside triphosphates. In the current work, they show that the mutant Therminator DNA polymerase (NEB), which is designed to incorporate a wide range of nucleotides, can polymerize more than 50 TNA nucleotides on a DNA template in yields above 20%, within 24 h. They assessed the fidelity of threose nucleotide incorporation and determined that more than 10% of TNA strands would be error free under selection conditions.

Having demonstrated the synthesis of longer TNA segments using a DNA polymerase, the authors proposed an in vitro selection method for TNA, similar in concept to the selection of peptides by mRNA display (Fig. 1). A DNA hairpin was constructed for use as a primer. After Therminator-mediated transcription of TNA on the hairpin template, the TNA strand was displaced by enzymatic synthesis of a complementary DNA beginning in the hairpin loop. Liberation of the TNA strand by cDNA synthesis should permit selection for function in the displayed TNA and subsequent PCR amplification of the selected DNA coding template. Although the authors did not include PCR or selection experiments in the current work, by showing that the double-stranded DNA product could be digested with a restriction enzyme, they validated the efficiency of the TNA strand-displacement step.

Figure 1: Proposed in vitro selection method for TNA display
Figure 1 : Proposed in vitro selection method for TNA display Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact

DNA polymerase−mediated TNA synthesis yields a hybrid DNA-TNA hairpin. The TNA strand is subsequently displaced by a cDNA strand synthesized from a primer targeted to the hairpin loop. Functional TNA sequences could be selected from a library and enriched by amplification of the duplex DNA tags of selected molecules.

Reprinted with permission from Ichida, J. K. et al. J. Am. Chem. Soc., published online 10 February 2005 (doi: 10.1021/ja045364w). Copyright 2005 American Chemical Society.

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The manuscript suggests that in vitro selection of functional TNA molecules may be feasible, providing the basis for future studies to assess whether TNA is capable of ligand binding or catalysis. In principle, this display approach may offer a general platform for the in vitro evolution of diverse non-natural biopolymers.



  1. Ichida, J.K. et al. An in vitro selection system for TNA. J. Am. Chem. Soc. , published online 10 February (2005) (doi:10.1021/ja045364w). | Article | ChemPort |

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