Entropy-Driven Molecular Beacon Assisted Special RCA Assay with Enhanced Sensitivity for Room Temperature DNA Biosensing

Abstract

The Phi29 DNA polymerase is renowned for its processivity in synthesizing single-stranded DNA amplicons by rolling around a circularized DNA template. However, DNA synthesis rolling circle amplification (RCA) is significantly hindered by the secondary structure in the circular template. To overcome this limitation, an engineered circular template without secondary structure could be utilized to improve the sensitivity of RCA-based assays without increasing its complexity. We herein proposed an entropy-driven special RCA technology for the detection of HPV16 E7 gene at room temperature. The strategy is composed of a molecular beacon containing a loop region for nucleic acid target recognition and a stem region to initiate RCA. With the target analyte, the stem region of the molecular beacon will be exposed and then hybridized with a special circular template to initiate the DNA amplification. We tested different designs of the molecular beacon sequence and optimized the assay's working conditions. The assay achieved a sensitivity of 1 pM in 40 min at room temperature. The sensitivity of this assay, at 1 pm, is about a hundred-fold greater than that of conventional linear RCA performed in solution. Our proposed sensor can be easily reprogrammed for detecting various nucleic acid markers by altering the molecular beacon's loop. Its simplicity, rapid assay time, and low cost make it superior to RCA sensors that utilize similar strategies.

Keywords: HPV detection; minimum secondary structured RCA; molecular beacon; rolling circle amplification; sensitivity.

Scheme 1

Principle of the molecular beacon-assisted entropy-driven MSSRCA for room temperature nucleic acid detection. The (a) comparison between the MSSRCA and the SSRCA is shown, and the MSSRCA benefits from the minimum secondary structured circular template used, resulting in high-speed, unconstrained amplification using Phi29 DNA polymerase. The (b) scheme using our assay for nucleic acid detection is shown, and the special circular template is first ligated using CircLigase and purified from PAGE. In the one-tube reaction, the molecular beacon undergoes a conformational change upon target gene binding, allowing the circular template to hybridize and initiate the MSSRCA process. Phi29 DNA polymerase then extends the molecular beacon, forming a linear DNA strand. SYBR Gold dye intercalates with the DNA, providing fluorescence to monitor MSSRCA amplicon generation.

Figure 1

Feasibility of the nucleic acid sensing. (a) Hybridization of the molecular beacon and target (Lane 1: synthetic E7 DNA; Lane 2: molecular beacon 1; Lane 3: hybridization in 1× Phi29 buffer; Lane 4: hybridization in 1× NEB 2.0 buffer; and Lane 5: hybridization in 1× Phi29 (2) buffer) is shown. For each experiment, a 5 µM of concentration was used for all probes. (b) Agarose gel electrophoresis of the molecular beacon-assisted MSSRCA in the presence or absence of a 10 nM HPV16 E7 DNA is shown. (c) Optimization of the buffering conditions for the assay is shown (Buffer 1: 1× NEB 2.0 buffer; Buffer 2: 1× Phi29 buffer; and Buffer 3: 1× Phi29 (2) buffer). The fluorescence intensity is normalized within groups. (d) Calibration curve of the molecular beacon-assisted MSSRCA using molecular beacon 1 is shown. (e) Sensitivity of the molecular beacon-assisted MSSRCA using molecular beacon 1 is shown. n = 3, and error bars represent means  ±  s.d. Two-tailed t-test was performed using GraphPad prism software Version 9.5.0 with 0.1234 (ns), 0.0332 (*).

Figure 2

Optimization of the design of the molecular beacon. (a) Schematic illustration of the mismatch primed MSSRCA. (b) Comparison of the fluorescence signal using two different molecular beacons.

Figure 3

Optimization of the molecular beacon assisted MSSRCA. Optimization of (a) sodium ion concentration, (b) time intervals, (c) concentration of the molecular beacon, and (d) amount of Phi29 DNA polymerase using 1 nM of the target nucleic acid.

Figure 4

Detection of HPV16 E7 DNA using molecular beacon-assisted MSSRCA. (a) shows the target-induced fluorescence response of the system. The target concentration of 100 fM to 100 nM was tested. n = 3, and the bars represent means  ±  s.d. (b) shows fluorescence of the reaction using the nucleic acid target concentration of 100 fM and 1 pM. A two-tailed t-test was performed using GraphPad Prism software Version 9.5.0 with 0.1234 (ns), 0.0332 (*).