Target-Triggered Polymerization of Branched DNA Enables Enzyme-free and Fast Discrimination of Single-Base Changes

Abstract

Single-base changes lead to important biological and biomedical implications; however, the discrimination of single-base changes from normal DNA always remains a grand challenge. Herein we developed a DNA recognition and amplification system based on artificial branched DNA, namely, target-triggered polymerization (TTP), to realize enzyme-free and fast discrimination of single-base changes. Branched DNA as monomers rapidly polymerized into DNA nanospheres only with the trigger of specific DNA. Our TTP system worked reliably over a wide range of conditions. Remarkably, our TTP system was capable of discriminating base-changing DNA from normal DNA, including distinguishing 1-4 nucleotide changes and positions of single base, which was attributed to the significant amplification of small differences in hybridization thermodynamics and kinetics. We further proposed a theoretical method for calculating the hybridization probability of nucleic acids, which performed highly consistent with experimental results.

Keywords: Nanostructure; Nanotechnology; Thermodynamics.

Graphical abstract

Scheme 1

Schematic Illustration of Enzyme-free and Fast Target-Triggered Polymerization (TTP) for Simultaneous Target Recognition and Signal Amplification (A) Synthesis of X-shaped branched DNA (X-DNA) composed by four single-stranded DNA (ssDNA) through DNA hybridization and annealing procedure. (B) Polymeric DNA nanosphere triggered by the self-assembly of X-DNA (X₁ and X₂) and target DNA (T-DNA).

Figure 1

Real-Time Monitoring of Layer-by-Layer TTP System for Step-by-Step Signal Amplification (A) Frequency and dissipation changes of the self-assembled DNA nanospheres in the TTP system. Stepped frequency and dissipation change signals clearly showed the feature of layer-by-layer TTP system to form DNA nanospheres via four cycles. In the first cycle, a dramatic change reflected the efficient hybridization, and the accumulated introduction of T-DNA and X-DNA obviously increased the signals. (B) Corresponding Δm of the self-assembled DNA nanospheres in the TTP system calculated by Sauerbrey equation.

Figure 2

The TTP System Exhibited Excellent Robustness and Reliability in Relatively Harsh Environments (A) The relative ΔF signals demonstrated the robustness of the TTP system under different temperatures (n = 3, mean ± SD). (B) The relative ΔF signals demonstrated the robustness of the TTP system under different pH (n = 3, mean ± SD). (C) The relative ΔF signals demonstrated the robustness of the TTP system under different water environments (n = 3, mean ± SD). (D) The relative ΔF signals demonstrated the robustness of the TTP system under different simulated biological fluids (n = 3, mean ± SD).

Figure 3

TTP System Showed Enhanced Hybridization Thermodynamic and Kinetic Performance (A) The Δf curves changing with time. (B) The -ΔF curves after the introduction of T-DNA (marked with blue) and X₁ (marked with red) changing with T-DNA concentrations. (C) ρ_i reflecting DNA hybridization thermodynamics, which remained constant within a concentration range of 2×10⁻⁷ to 2×10⁻¹² M. (D) The dΔf/dt curves changing with time, reflecting instantaneous hybridization rates of T-DNA with different concentrations. (E) The -dΔf/dt curves after the introduction of T-DNA (marked with blue) and X₁ (marked with red) changing with T-DNA concentrations. (F) ε_i increasing first and then decreasing within a concentration range of 2×10⁻⁷ to 2×10⁻¹² M, which was limited by probe density and steric hindrance.

Figure 4

TTP System Achieved the Discrimination of Multi-base Changes and Single-Base Changes at Different Locations from Normal DNA (A) The model of hybrid-DNA based on the hybridization process of N-DNA or M-DNA (non-complementary DNA with altered bases) with two types of X-DNA. (B) The theoretical values of binding energy and equilibrium yields. (C) The frequency curves of base-changing DNA with different number of altered bases. (D) The relative ΔF of base-changing DNA with different number of altered bases (n = 3, mean ± SD). (E) The theoretical σ_m of base-changing DNA with different number of altered bases. The experimental results were in good agreement with the theoretical analysis. (F) The frequency curves of base-changing DNA with single-base changes at different positions. (G) The relative ΔF of base-changing DNA with single-base changes at different positions (n = 3, mean ± SD). (H) The theoretical σ_s of base-changing DNA with single-base changes at different positions. The experimental results were good agreement with the theoretical analysis.

Figure 5

Improved Thermodynamic and Kinetic Performance by TTP System for the Discrimination of M-DNA and N-DNA (A) The frequency curves of M-DNA and N-DNA changing with time. (B) The –ΔF values after the introduction of T-DNA (marked with blue) and X₁ (marked with red) changing with the number of altered bases, reflecting hybridization efficiency with different number altered bases. (C) The dΔf/dt curves changing with time, reflecting instantaneous hybridization rates of M-DNA with different number of altered bases. (D) The -dΔf/dt values varying with the number of altered bases, reflecting the hybridization kinetics properties of M-DNA in TTP system.