Darwinian evolution of an alternative genetic system provides support for TNA as an RNA progenitor

Journal name:
Nature Chemistry
Year published:
Published online


The pre-RNA world hypothesis postulates that RNA was preceded in the evolution of life by a simpler genetic material, but it is not known if such systems can fold into structures capable of eliciting a desired function. Presumably, whatever chemistry gave rise to RNA would have produced other RNA analogues, some of which may have preceded or competed directly with RNA. Threose nucleic acid (TNA), a potentially natural derivative of RNA, has received considerable interest as a possible RNA progenitor due to its chemical simplicity and ability to exchange genetic information with itself and RNA. Here, we have applied Darwinian evolution methods to evolve, in vitro, a TNA receptor that binds to an arbitrary target with high affinity and specificity. This demonstration shows that TNA has the ability to fold into tertiary structures with sophisticated chemical functions, which provides evidence that TNA could have served as an ancestral genetic system during an early stage of life.

At a glance


  1. Structure of TNA.
    Figure 1: Structure of TNA.

    a, Constitutional (left) and configurational (right) structures for the linearized backbone of an α-L-threofuranosyl-(3′ → 2′)-oligonucleotide. TNA contains one less atom per backbone repeat unit than natural RNA and DNA. B represents a nucleotase. b, Solution NMR structure for the duplex formed from the self-pairing complementary sequence 3′-CGAATTCG-2′ (ref. 5). Duplex structures were rendered in PyMol with models showing ball and stick (left) and surface (right) images. TNA adopts a right-handed structure with helical parameters similar to A-form RNA.

  2. Synthesis of TNA libraries by enzyme-mediated primer extension.
    Figure 2: Synthesis of TNA libraries by enzyme-mediated primer extension.

    a, Chemical structure of TNA triphosphates (tNTPs): tDTP, 1; tTTP, 2; tGTP, 3; tCTP, 4. b, Schematic diagram of the primer-extension assay used to evaluate DNA libraries L1–L3. Each library contains a central random region of 50 nts flanked on either side by a 20 nt constant region. Library compositions: L1, equal distribution of A, C, G and T; L2, equal distribution of A, C and T; L3, one-half equivalent of G relative to A, C and T. c, Therminator-mediated TNA transcription assays analysed by denaturing polyacrylamide gel electrophoresis. Primer extension of L1 with tNTPs 14 yields only trace amounts of full-length product (left panel). Primer extension of L1 using defined combinations of dNTPs (black) and tNTPs (red) leads to full-length product when tCTP is replaced with dCTP (centre panel). Primer extension across libraries L2 and L3 leads to full-length product in ~60% and ~30% yield, respectively (right panel). M: DNA marker.

  3. Evolution of TNA receptors in vitro.
    Figure 3: Evolution of TNA receptors in vitro.

    a, In vitro selection strategy designed to isolate TNA aptamers with affinity to human thrombin. The DNA library encodes a random region of 50 nt positions flanked on the 3′ end with a stem-loop structure that serves as a DNA primer and a fixed-sequence primer-binding site located at the 5′ end. The DNA primer is extended with tNTPs to produce a chimeric TNA–DNA hairpin. A separate DNA primer modified with 6-carboxy-fluorescein (star) is annealed to the stem-loop region and extended with DNA to displace the TNA strand. The resulting pool of TNA–DNA fusion molecules is incubated with the protein target. Bound molecules are separated from the unbound pool by capillary electrophoresis and amplified by PCR. The dsDNA is made single-stranded and the coding strand is annealed to generate a new pool of DNA templates for the next selection cycle. b, Equilibrium binding affinity measurement of the core binding domain of TNA aptamer 3.12. The aptamer (3′-TGTTDTDGDDDDDDTDDT GGTGGGGGGTTTDGDTDDDGGGG-2′) binds to human thrombin with a Kd of 200 nM and shows no detectable affinity for BSA or streptavidin. Error bars, standard deviation of each data point (n = 3).


4 compounds View all compounds
  1. (4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl hydrogen triphosphate
    Compound 1 (4R,5R)-5-(2,6-Diamino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-3-yl hydrogen triphosphate
  2. (4R,5R)-4-Hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl hydrogen triphosphate
    Compound 2 (4R,5R)-4-Hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl hydrogen triphosphate
  3. (4R,5R)-5-(2-Amino-6-oxo-1H-purin-9(6H)-yl)-4-hydroxytetrahydrofuran-3-yl hydrogen triphosphate
    Compound 3 (4R,5R)-5-(2-Amino-6-oxo-1H-purin-9(6H)-yl)-4-hydroxytetrahydrofuran-3-yl hydrogen triphosphate
  4. (4R,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl hydrogen triphosphate
    Compound 4 (4R,5R)-5-(4-Amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-3-yl hydrogen triphosphate


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Author information

  1. These authors contributed equally to the project

    • Hanyang Yu &
    • Su Zhang


  1. Center for Evolutionary Medicine and Informatics in the Biodesign Institute and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-5301, USA

    • Hanyang Yu,
    • Su Zhang &
    • John C. Chaput


J.C. conceived the project and wrote the manuscript. H.Y., S.Z. and J.C. designed the experiments. H.Y. and S.Z. performed the experiments and wrote initial drafts of the manuscript. All authors discussed the results and commented on the manuscript.

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