Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

Abstract

When oligonucleotide bearing a hairpin near either its 3'- or 5'-end was treated with T4 DNA ligase, the intramolecular cyclization dominantly proceeded and its monomeric cyclic ring was obtained in extremely high selectivity. The selectivity was hardly dependent on the concentration of the oligonucleotide, and thus it could be added in one portion to the mixture at the beginning of the reaction. Without the hairpin, however, the formation of polymeric byproducts was dominant under the same conditions. Hairpin-bearing oligonucleotides primarily take the folded form, and the enzymatically reactive species (its open form) is minimal. As the result, the intermolecular reactions are efficiently suppressed due to both thermodynamic and kinetic factors. The 'terminal hairpin strategy' was applicable to large-scale preparation of a variety of DNA rings. The combination of this methodology with 'diluted buffer strategy', developed previously, is still more effective for the purpose. When large amount of l-DNA bearing a terminal hairpin (e.g. 40 μM) was treated in a diluted ligase buffer (0.1× buffer) with T4 DNA ligase, the DNA ring was prepared in 100% selectivity. Even at [l-DNA]0 = 100 μM in 0.1× buffer, the DNA ring was also obtained in pure form, simply by removing tiny quantity of linear byproducts by Exonuclease I.

Figure 1.

Outline of ‘terminal hairpin strategy’ for exclusive production of DNA rings. The single-stranded DNA (ssDNA) as active species for the ligation (the open form) is in equilibrium with l-DNA bearing a terminal hairpin (the folded form). This equilibrium is largely shifted towards the latter because of the steric requirements of hairpins so that the concentration of reactive ssDNA is sufficiently low to allow dominant occurrence of intramolecular ligation to DNA ring (pathway ).

Figure 2.

Dominant cyclization of l-DNA using hairpins as internal promoters. (A) The solution structures of L64_3-4,24-4, L64_16-4,37-4 and L64_3-4, determined by Mfold calculation under the conditions of [Mg²⁺] = 10 mM and 25°C. (B) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64_3-4,24-4 without the T4 ligase treatment; lane 2, L64_3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64_3-4,24–4; lane 4, L64_16-4,37-4 without the treatment; lane 5, L64_16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64_3-4 without the treatment; lane 8, L64_3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA]₀ = 5 μM, [splint]₀ = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

Figure 3.

Terminal hairpin strategy for T4 DNA ligase-mediated preparation of DNA rings of smaller sizes. (A) Solution structures of 74-, 64-, 54-, 44- and 34-nt l-DNAs. (B) Gel electrophoresis patterns of the T4 ligase ligation products. The conditions of T4 ligase reactions are the same as described in Figure 2.

Figure 4.

Effects of the distance between a hairpin and the ligation site on the magnitude of ‘terminal hairpin effect’ for the selective formation of single-stranded DNA ring. (A) The solution structures of l-DNAs used here. (B) Lane 1, L64_{2-4,23-4,51-2} without the treatment; lane 2, L64_{2-4,23-4,51-2} treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of l-DNA; lane 3, L64_3-4,24-4 alone; lane 4, L64_3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 5, L64_4-4,25-4 alone; lane 6, L64_4-4,25-4 treated with T4 DNA ligase in the presence of 12-nt splint; lane 7, L64_5-4,26-4 alone; lane 8, L64_5–4,26-4 treated with T4 DNA ligase in the presence of 12-nt splint. lane 9, L64_6-4,27-4 alone; lane 10, L64_6-4,27-4 treated with T4 DNA ligase in the presence of 12-nt splint. Reaction conditions are the same as described in Figure 2.

Figure 5.

Effects of the stability of hairpin on the cyclization by T4 DNA ligase. (A) The solution conformations of L64_1-4,24-4, L64_1-6,24-4, L64_1-7,24-4 and L60_1-7,20-4, determined by Mfold calculation. (B) Lane 1, L64_1-4,24-4 without T4 ligase treatment; lane 2, L64_1-4,24-4 with T4 ligase treatment; lane 3, L64_1-6,24-4 alone; lane 4, L64_1-6,24-4 with T4 ligase treatment; lane 5, L64_1-7,24-4 alone; lane 6, L64_1-7,24-4 with T4 ligase treatment; lane 7, L60_1-7,20-4 alone; lane 8, L60_1-7,20-4 with T4 ligase treatment. The enzymatic conditions are the same as described in Figure 2.

Figure 6.

Comparison of the reaction conversion and yield of monomeric cyclic ring between substrates with and without the terminal hairpin. (A) Time-courses for the T4 ligase-mediated ligation of L64_3-4,24-4 (circles) and L64_16-4,37-4 (rectangles). The total amounts of DNA, consumed in the presence of T4 ligase (by both intramolecular and intermolecular ligation), are plotted as a function of reaction time. In (B), the yield of DNA ring is shown as a function of reaction time. Reaction conditions: [l-DNA]₀ = 5 μM, [splint]₀ = 10 μM, and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C.

Figure 7.

Highly selective cyclization at unusually high substrate concentrations using terminal hairpin strategy. Lane 1, L64_3-4,24-4 without T4 treatment; lane 2, T4 reaction at [L64_3-4,24-4]₀ = 10 μM; lane 3, [L64_3-4,24-4]₀ = 20 μM; lane 4, [L64_3-4,24-4]₀ = 40 μM; lane 5, [L64_3-4,24-4]₀ = 60 μM; lane 6, [L64_3-4,24-4]₀ = 100 μM. In lanes 8_-10, L64_16-4,37-4 having no terminal hairpin is used. Lane 8, L64_16-4,37-4 without T4 treatment; lane 9, [L64_16-4,37-4]₀ = 100 μM. Reaction conditions: [l-DNA]₀/[splint]₀ = 1/2 and 10 U T4 DNA ligase in 1 × T4 DNA ligase buffer at 25°C. In lanes 7 and 10, 0.1× T4 DNA ligase buffer was used in place of 1× T4 buffer, according to ref. (30) (see text for details).