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.2023 Aug 16;145(32):17872-17880.
doi: 10.1021/jacs.3c04979. Epub 2023 Jul 19.

Rapid Chemical Ligation of DNA andAcyclic Threoninol Nucleic Acid (a TNA) for Effective Nonenzymatic Primer Extension

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Rapid Chemical Ligation of DNA andAcyclic Threoninol Nucleic Acid (a TNA) for Effective Nonenzymatic Primer Extension

Hikari Okita et al. J Am Chem Soc..

Abstract

Previously, nonenzymatic primer extension reaction ofacyclic l-threoninol nucleic acid (L-aTNA) was achieved in the presence ofN-cyanoimidazole (CNIm) and Mn2+; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-aTNA to L-aTNA and of DNA to DNA. We found that Cd2+, Ni2+, and Co2+ accelerated ligation of both L-aTNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-aTNA fragment and template, resulting in unexpectedly more effective L-aTNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-aTNA primer could be elongated by ligation to L-aTNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(a) Chemical structuresof DNA and L-aTNA. (b)Schematic illustration of the chemical ligation reaction by CNIm anda divalent metal cation. (c) Sequences of L-aTNAand DNA used for chemical ligation. (d) Denaturing PAGE analysis ofchemical ligation of 8-mer L-aTNA fragments (T8A/T8B-3′p)on the 16-mer template (T16t) in the presence of CNIm and chloridesalt of an indicated divalent metal cation (MCl2). Reactionconditions: 0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μMT16t, 100 mM NaCl, 5 mM MCl2, 20 mM CNIm, 25 °C. PAGEconditions: 20% acrylamide, 8 M urea, 1 × TBE, 4 W at room temperaturefor 1.5 or 2 h. Negative control (lane 1) included only T8A and NaCl.(e) Yield as a function of time for ligation of T8A to T8B-3′pon the T16t template (left) and of D16A-3′p to D16B on theD32t template (right) in the presence of indicated divalent metalcations. The graphs are based on data shown from panel d andFigure S1. (f) Calculatedkobs values for chemical ligation of indicated oligomers inthe presence of indicated metal ions. Reaction conditions: 0.9 μMfragment A, 1.1 μM fragment B, 1.0 μM template, 100 mMNaCl, 5 mM MCl2, 20 mM CNIm, 25 °C.kobs values were calculated from linearized plots of -ln([FragmentA]/[Fragment A]0) assuming a pseudo-first-order reaction.
Figure 2
Figure 2
(a) Scheme of reactionof PhP and CNIm in the presence of Cd2+. (b)1H NMR time course analysis.1H NMR peaks colored brown,red, and green were assigned to PhP, CNIm,and Im, respectively. Reaction conditions: 5 mM PhP, CdCl2, and CNIm, 25 °C. (c) Possible reaction scheme and intermediates(i) suggested by NMR and (ii) not observed. (d) Normalized peak intensityof CNIm (red) and Im (green) calculated from NMR spectra.
Figure 3
Figure 3
(a) Plot of -ln([FragmentA]/[Fragment A]0) as a functionof time over a range of CNIm concentrations. Reaction conditions:0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μM T16t,5 mM CdCl2, 1, 5, 10, or 20 mM CNIm, 25 °C. (b) Apparentk1 obtained assuming a pseudo-first-order reaction.k1,app of T8A/T8B-3′p/T16t and the otherk1,apps were calculated from plots shown in Figures3a andS11–S15, respectively. Reactionconditions: 0.9 μM fragment A, 1.1 μM fragment B, 1.0μM template, 5 mM CdCl2, 1, 5, 10, or 20 mM CNIm,25 °C. (c) Plots of apparentk1 valuesfor each component versus CNIm concentration based on the Ostwaldisolation method. (d) Energy-minimized structures of T8B-3′pand template (top) and T8A-1′p and template (bottom) illustratinginteractions of phosphate with the neighboring amide group. (e) Correlationbetween the concentration of Cd2+ ions and the rate constantk1. (f) Schematic illustration of the predictedmechanism involving divalent metal cations in activation of the phosphategroup and rates for steps 1 and 2 and the overall reaction.
Figure 4
Figure 4
(a andb) Schematic illustrations of elongation via chemical ligationof an L-aTNA primer in (a) 3′ → 1′direction and (b) 1′ → 3′ direction. (c) DenaturingPAGE of L-aTNA elongation reaction products in thepresence of Cd2+ with ligation in the 1′ →3′ direction. (d) Comparison of yields of full-length elongationproducts in different conditions. Reaction conditions: 0.9 μMprimer, 100 μM T3Bmix, 1.0 μM template, 100 mM NaCl, 5mM CdCl2 or 20 mM MnCl2, 20 mM CNIm, 4 °Cfor 6 h in the presence of Cd2+ or for 24 h in the presenceof Mn2+. PAGE conditions: 20% acrylamide, 8 M urea, 1 ×TBE, 3 h, 4 °C, 4 W. Negative control (lane 1) included onlyprimer, NaCl, and divalent metal cation salt (Lane 1). Markers wereprepared by the reaction using complementary fragments (Figure S23).
Figure 5
Figure 5
(a) Schematicillustration of chemical primer extension on 23-merand 29-mer L-aTNA templates with optimized conditions.(b) Denaturing PAGE of the reaction with T23t as the template (left)and T29t as the template (right). Reaction conditions: 0.45 μMRev-T8A-3′p, 200 μM T3Bmix for T23t or 400 μM T3Bmixfor T29t, 1.0 μM T23t or T29t, 100 mM NaCl, 5 mM CdCl2, 20 mM CNIm, 4 °C for 4 h. PAGE conditions: 20% acrylamide,8 M urea, 1 × TBE, 3 h with T23t as the template or 3.5 h withT29t as the template, 15 °C, 4 W. Negative controls (lanes 1and 6) included only Rev-T8A-3′p, NaCl, and CdCl2. (c) MALDI-TOF MS analyses of products of the elongation reactionfor 2 h with T23t as the template (left) and for 4 h with T29t asthe template (right).
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