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.
Gene therapy is often limited by gene vectors. To reduce the impact of intracellular reactive oxygen species and improve transfection efficiency, thioether bonds were introduced in poly(β-amino ester) (PBAE), a well-known nonviral gene vector, to synthesize PBAE-poly(β-thioether ester) copolymer (PBAE-TE). By varying the monomer feeding ratio and reaction time, the optimized copolymer (PBAE-TE2–5) showed the highest transfection efficiency in SiHa and HeLa cells (75.00 and 78.70%) with a 75:1 copolymer/GFP ratio. PBAE-TE2–5 also demonstrated superior transfection performance compared to PBAE in multiple cell lines (HeLa, Me180, C666–1, and HEK 293T) and better penetration capability in 3D spheroid cell models (SiHa and HEK 293T). The PBAE-TE2–5-CRISPR/Cas9 plasmid polyplexes could effectively edit target genes in HEK 293T (MUC2) and SiHa (HPV16 E7) cells, and inhibit cancer cell proliferation both in vitro and in vivo. These findings highlight the promising potential of PBAE-TE as an effective gene delivery system.
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