Cycling of Rational Hybridization Chain Reaction To Enable Enzyme-Free DNA-Based Clinical Diagnosis

Abstract

In order to combat the growing threat of global infectious diseases, there is a need for rapid diagnostic technologies that are sensitive and that can provide species specific information (as might be needed to direct therapy as resistant strains of microbes emerge). Here, we present a convenient, enzyme-free amplification mechanism for a rational hybridization chain reaction, which is implemented in a simple format for isothermal amplification and sensing, applied to the DNA-based diagnosis of hepatitis B virus (HBV) in 54 patients. During the cycled amplification process, DNA monomers self-assemble in an organized and controllable way only when a specific target HBV sequence is present. This mechanism is confirmed using super-resolution stochastic optical reconstruction microscopy. The enabled format is designed in a manner analogous to an enzyme-linked immunosorbent assay, generating colored products with distinct tonality and with a limit of detection of ca. five copies/reaction. This routine assay also showed excellent sensitivity (>97%) in clinical samples demonstrating the potential of this convenient, low cost, enzyme-free method for use in low resource settings.

Keywords: DNA self-assembly; HBV; cycling; diagnostic; enzyme-free; hybridization chain reaction.

Figure 1

Reaction principle of db DNA-based rational HCR. (a) Schematic illustration of synthesis and inter-reaction of db DNA units. Two starting oligonucleotides (U1–1/U1–2 or U2–1/U2–2) possess the same sticky ends at 3′ and 5′ ends, as well as  complementary midsequence segments, as indicated. For the process starting from U1 (left), the db DNA units (U1 or U2) hybridize (step 1). Similarly, the DNA synthesis process can also begin from U2 (right). The formed db DNA units (U2) can be combined to U1 during the rational HCR process (step 2), due to their exposed complementary sticky ends. Thus, by addition of U2 and U1 successively and repeatedly, these two units can bind to each other leading to an exponential growth of a packed complex 3D DNA structure (step n); (b) Evaluation of the product of db DNA-based rational HCR using gel electrophoresis. L lane was 100 bp ladder, lane 1 and 2 were U1 and U2, Lanes 3 and 4 were the hybridized products of U1 and U2 with stoichiometry ratio at 5:1 (and 1:5, respectively), and Lanes 5 and 6 were the product of stoichiometry ratio at 20:1 (and 1:20, respectively), the unit with a low concentration was kept at 0.1 μM. Bands from 90 to 225 bp (from lane 3 to 6) illustrate the expected products ratios, which are different to those from the smallest units (lane 1/2). For this concentration of single units, larger products are not formed efficiently (evidence of limited formation is shown by smears in the large sizes), since the single units become depleted as the reaction progresses.

Figure 2

Microbead-based DNA detection: (a) Schematic drawing of db DNA-based rational HCR for signal amplification of target DNA from magnetic microbeads. Briefly, biotin-labeled capture probes were immobilized onto avidin-functionalized microbeads. Target DNA was then captured by these specific microbeads. The detection probe links to U1′ and is also complementary to a second part of the target DNA and thus enabled U1 to bind to the surface of microbeads (see Table S1 for details). Fluorescence-labeled db DNA units were successively added to amplify the signal, forming larger constructs as the reactions proceeds. A washing step is necessary after each hybridization step to remove the excess db DNA units. (b) Fluorescence signal of microbeads at different cycles, imaged by super-resolution stochastic optical reconstruction microscopy (STORM) (the images’ contrast and brightness were modified for ease of presentation) and analyzed using segmentation (see Figure S4 for an example of the process), showing the average area of a fluorescent spot (measured in pixels) for at least 10 beads for each cycle of washing and addition of reagent. All beads are 5 μm in diameter.

Figure 3

Schematic representation of db DNA-based ELISA. The target DNA is anchored to the magnetic microparticles (MMPs) by the biotin-labeled capture probes and then recognized and amplified by the biotin-labeled db DNA units. The enzyme is linked to the complex through the interaction between enzyme-decorated avidin and biotin reaction.

Figure 4

Rational enzyme-linked HCR. (a) Photograph indicates the color changing with different db DNA units addition times from 2 to 20 (cycles), respectively (left to right, target DNA concentration 5 pM), in a 96 microtiter plate. (b) Absorbance with respect to the blank was monitored at 450 nm. Black square: target concentration 5 pM. Red disk: ddH₂O as a negative control. Data are the average of at least three replicates, and error bars represent the standard deviation. The data were fitted with nonlinear curve fitting to a Boltzmann curve (R² > 0.98). (c) Analytical sensitivity. Color change with different target DNA concentration from 0.5 copies to 5 × 10⁵ copies/reaction (10× increments per well) with the db DNA units addition times at 35 (cycles). The results are quantified in (d). Absorbance at 450 nm. Data are the average of at least three replicates, and error bars represent the standard deviation. The signal increases until a plateau is reached after 5000 copies/reaction. The limit of detection is 5 copies/reaction. (e) 1 pM of different target DNA molecules was added to the reaction: perfect match DNA, single mismatch DNA with the defect located at different position (toward the 5′ end, the 3′ end and in the middle respectively), and noncognate DNA. The sequences are provided in Table S1. Data are the average of at least three replicates, and error bars represent the standard deviation.

Figure 5

Correlation between the copy number of each clinical sample and the intensity of the signal (absorbance) obtained after rational 35 cycles of HCR. Dashed red line is a linear fit (R² = 0.9). The lowest copy number sample was not detected using rational HCR (signal below threshold of 0.12).