Bubble DNA tweezer: A triple-conformation sensor responsive to concentration-ratios

Abstract

DNA tweezers, with their elegant simplicity and flexibility, have been pivotal in biosensing and DNA computing. However, conventional tweezers are confined to a binary transformation pre/post target signal recognition, limiting them to presence/absence judgments. This study introduces bubble DNA tweezers (BDT), capable of three distinct conformations based on variable target signal ratios. In contrast to traditional compact tweezers, BDT features a looser structure centered around a non-complementary bubble domain located between the tweezer arms' connecting axis and target signal recognition jaws. This bubble triggers toehold-free DNA strand displacement, leading to three conformational changes at different target signal concentrations. BDT detects presence/absence and true concentration with remarkable specificity and sensitivity. This adaptability is not confined to ideal scenarios, proving valuable in complex, noisy environments. Our method facilitates target DNA/miRNA signal quantification within a specific length range, promising applications in clinical research and environmental detection, while inspiring future biological assay innovations.

Keywords: Biophysical chemistry; Biophysics; Chemistry.

Graphical abstract

Figure 1

BDT scheme (A) Overview of BDT triple conformational transformation: as S increases, C₂ and C₃ appear sequentially, while C₁ and C₂ gradually disappear. (B) The biological mechanism underlying the formation of distinct conformations resulting from the interaction between BDT and varying proportions of S.

Figure 2

Fluorescence and electrophoretic characteristics of BDT (A) The fluorescence changes after adding 1×S twice to C₁. Here, 1× is defined as the concentration of a species in a solution (the same below, unless otherwise specified). In this context, 1× represents a concentration of 1 μM, and the concentration in solution [C₁] = 1×. Here, ΔF represents the increment of the current fluorescence value relative to the lowest, ΔF_Max represents the biggest ΔF in entire reaction process. (B) Electrophoretic results of BDT at different ratios of S. The substances in different swimming lanes: lane 1 1×S, lane 2 C₁, lane 3 C₂, lane 4 C₃, lane 5 C₁+0.5×S, lane 6 C₁+1×S, lane 7 C₁+1.5×S, lane 8 C₁+2×S, lane 9 C₁+2.5×S, lane 10 C₁+3×S. In lanes 2–10, all BDTs are at 1× concentration (C₁, C₂ or C₃).

Figure 3

Analysis of jaw length gradient in BDT (A) Research on the effect of changes in target signal length on BDT’s recognition ability. When the length of the target signal is less than 20 nt, BDT may encounter challenges in achieving closure. (B) Real time fluorescence results of target signal A with different lengths. Optimal fluorescence characteristics are achieved by BDT when the length of the target signal exceeds 24 nt. Here, F₀ represents the initial fluorescence value, while F represents the specific fluorescence value at each moment. [C₁] = [A] = 1×.

Figure 4

Analysis of bubble scale gradient in BDT (A) Research on the effect of bubble scale change on the BDT V-shaped kinetics. When the bubble scale exceeds 10 nt, BDT is expected to undergo the complete process of C₁-C₂-C₃. In contrast, it cannot be reopened after reaching the closed state of C₂. The b(e) represents b domain and e domain. (B) Real-time fluorescence results and the effect of bubble size changes on the maintenance of the BDT V-shaped characteristic. Bubbles below 10 nt, even when an excessive signal is added, do not exhibit fluorescence rebound. Here, F₀ represents the initial fluorescence value, while F represents the specific fluorescence value at each moment. [C₁] = 1×. Adding twice 1×A₁₂.

Figure 5

LOD analysis of BDT (A) The effect of different concentrations of A₁₂ on fluorescence intensity, from top to bottom, the [A₁₂] are 0 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 20 nM, 30 nM, 40 nM and 50 nM. [C₁] = 50 nM. (B) Linear regression of the BDT on different concentrations range of target signal A₁₂. F_Blank represents the blank group ([A₁₂] = 0 nM), while F_Input represents the specific fluorescence value at different [A₁₂] (0.01 nM–50 nM), F_Blank-F_Input is the fluorescence variation.

Figure 6

Specificity and application of the BDT in miRNA measurement (A) Specificity of BDT, BDT:miRNA = 1:0.9. BDT is the conformation C₁, [C₁] = 1×. (B) The V-shaped fluorescence method of measuring the true strength of miR-29b using BDT ([C₁] = 1×) in a multi-noise environment. Fluorescence rebound occurred during the 6th and 7th rounds of Mix titration. Here, ΔF represents the increment of the current fluorescence value relative to the lowest, ΔF_Max represents the biggest ΔF in entire reaction process.