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.2021 Feb 5;12(1):804.
doi: 10.1038/s41467-021-21128-0.

Nonenzymatic polymerase-like template-directed synthesis of acyclic L-threoninol nucleic acid

Affiliations

Nonenzymatic polymerase-like template-directed synthesis of acyclic L-threoninol nucleic acid

Keiji Murayama et al. Nat Commun..

Abstract

Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chemical ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also observed. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Chemical ligation ofl-aTNA and DNA.
a Chemical structures of DNA andl-aTNA.b Sequences of 8-mer fragments and 16-mer templates.c Schematic illustration of the chemical ligation reaction. Denaturing PAGE analysis ofdl-aTNA ligation reaction ande DNA ligation reaction. Lane 1, X8a as a marker; lane 2, reaction products after 1 h; lane 3, reaction products after 4 h; lane 4, reaction products after 24 h; lane 5, products after reaction in the absence of template; lane 6, products after reaction in the absence of CNIm; and lane 7, products after reaction in the absence of Mn2+. Conditions: [X8a]  =  0.9 μM, [X8b-p] = 1.1 μM, [X16t] = 0 or 1.0 μM, [NaCl] = 100 mM, [MnCl2] = 0 or 20 mM, [CNIm] = 0 or 20 mM, 4 °C. Source data are provided as a Source Data file.f Chemical ligation yield ofl-aTNA (red circles) and DNA (black circles) after indicated times of reaction.g Chemical ligation yields ofl-aTNA at indicated temperatures. Conditions: [oligomers] = 1.0 μM each, [NaCl] = 100 mM, [MnCl2] = 20 mM, [CNIm] = 20 mM. Source data are provided as a Source Data file.h Ligation yields ofl-aTNA at indicated concentrations of CNIm and Mn2+. Conditions: [T8a] = 0.9 μM, [T8b-p] = 1.1 μM, [T16t] = 1.0 μM, [NaCl] = 100 mM 25 °C, 30 min. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Evaluation of rate of chemical ligation.
a Sequences of 8-mer fragments and 16-mer template ofl-aTNA and 16-mer fragments and 32-mer template of DNA.b Melting temperatures of fragment/template duplexes.c Yield as a function of time for the chemical ligation reactions ofl-aTNA (red circles) and of DNA (black circles) with either primary (closed circles) or secondary OH termini (open circles). Conditions: [oligomers] = 1.0 μM each, [NaCl] = 100 mM, [MnCl2] = 20 mM, [CNIm] = 20 mM, 25 °C. Source data are provided as a Source Data file.d Schematic illustration of chemical ligation focusing on condensation between OH and phosphate.
Fig. 3
Fig. 3. Homo- and heteroligations and template-directed incorporation of short fragment.
a Sequences of 8-mer fragments and 16-mer template.b Ligation yields of indicated pairs of fragments on different templates. T8a/T8b-p (red bars), T8a/D8b-p (yellow bars), D8a/T8b-p (green bars), and D8a/D8b-p (blue bars) were ligated. Conditions: [X8a] = 0.9 μM, [X8b-p] = 1.1 μM, [X16t] = 1.0 μM, [NaCl] = 100 mM, [MnCl2] = 20 mM, [CNIm] = 20 mM, 4 °C, 24 h. Source data are provided as a Source Data file.c Sequences of 8-mer primer and fragments and 16-mer template.d Ligation yields for fragments of different lengths as a function of time. 5-mer fragment (red circles), 4-mer fragment (blue circles), 3-mer fragment (yellow circles), and 2-mer fragment (green circles) were used. Conditions: [T8primer] = 0.9 μM, [T16t] = 1.0 μM, [NaCl] = 100 mM, [MnCl2] = 20 mM, [CNIm] = 20 mM, concentration of Tnb-p strands is indicated in legend, 4 °C. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Sequential, template-directed ligation of trimers.
a Sequences used for sequential ligation of trimers.b Schematic illustration of the sequential, template-directed ligation of trimers.c Denaturing PAGE analysis of sequential ligation. Lane 1, T8primer as a marker; lane 2, reaction products after 1 h; lane 3, reaction products after 2 h; lane 4, reaction products after 4 h; lane 5, reaction products after 8 h; lane 6, reaction products after 24 h; lane 7, reaction products in the absence of T3c-p and T3d-p; lane 8, reaction products in the absence of T3d-p; lane 9, reaction products in the absence of T3b-p and T3d-p; and lane 10, reaction products in the absence of T3b-p and T3c-p. Conditions: [T8primer] = 0.9 μM, [T3b-p] = [T3c-p] = [T3d-p] = 50 μM, [T17t] = 1.0 μM, [NaCl] = 100 mM, [MnCl2] = 20 mM, [CNIm] = 20 mM, 4 °C. Source data are provided as a Source Data file.d Ligation yield determined from PAGE. Blue triangle, green square, and red circle represent 11-mer intermediate, 14-mer intermediate, and 17-mer full-length product, respectively. Source data are provided as a Source Data file.e MALDI-TOF MS spectra and observedm/z of reaction mixture containing all components analyzed after 24 h (red), of reaction products in the absence of T3c-p and T3d-p, which yielded only 11-mer (blue line), and of reaction products in the absence of T3d-p, which yielded 14-mer (green line). Peaks corresponding to the template (T17t) and T8primer were also observed. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Template-directed, polymerase-like primer extension from random trimers.
a Illustration of primer extension in the presence of T3mix-p pool.b Denaturing PAGE analysis of reaction with randomized trimers. Lane 1, T8primer as a marker; lane 2, reaction with only T3b-p as substrate to yield 11-mer intermediate; lane 3, reaction with T3b-p and T3c-p as substrates to yield 14-mer intermediate; lane 4, reaction with T3b-p, T3c-p, and T3d-p as substrates to yield full-length 17-mer; lane 5, reaction with T3mix-p as substrates after 1 h; lane 6, reaction with T3mix-p as substrates after 3 h; lane 7, reaction with T3mix-p as substrates after 6 h; lane 8, reaction with T3mix-p as substrates after 24 h; and lane 9, control without template. Conditions: [T8primer] = 0.9 μM, [T3mix-p] = 100 μM (total), [T17t] = 1.0 μM, [NaCl] = 100 mM, [MnCl2] = 20 mM, [CNIm] = 20 mM, 4 °C. Source data are provided as a Source Data file.c MALDI-TOF MS of reaction with T3mix-p as substrates after 24 h. Source data are provided as a Source Data file.
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