Department of Biochemistry and Molecular Biology. Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Dong Dan San Tiao #5, Beijing, 100005, PR China.
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, 610041, PR China.
Biosens Bioelectron. 2021 Nov 15;192:113507. doi: 10.1016/j.bios.2021.113507. Epub 2021 Jul 21.
Rolling circle amplification (RCA) had the prospect of assisting clinic diagnosis with advantage in in situ mRNA detection at single cell level. However, for direct mRNA detection, RCA had relatively low detection specificity and efficiency. Here, we introduced 4-(10, 15, 20-Triphenylporphyrin-5-yl)phenylamine (TPP) modified Au nanoparticle (Au-TPP) to improve the specificity of in-situ RCA. Through photothermal effect, Au-TPP acted as the specific heat source upon irradiation of 635 nm laser. The photothermal mediated RCA would be initiated only when the Au-TPP as well as the padlock anchored adjacently on the same target mRNA. Furthermore, we introduced 'C' form target-specific oligonucleotide linker probes to make generic padlock and Au-TPP for different mRNA targets, so that for a new mRNA target one does not have to redesign the padlock and the Au-TPP probe. By these strategies, we successfully developed a specific and photothermal mediated hyperbranched rolling circle amplification for direct in situ mRNA detection, suitable for both formalin-fixed paraffin-embedded (FFPE) tissue section and frozen tissue section.
滚环扩增 (RCA) 具有在单细胞水平原位检测 mRNA 的优势，有望辅助临床诊断。然而，对于直接检测 mRNA，RCA 的检测特异性和效率相对较低。在这里，我们引入了 4-(10,15,20-三苯基卟啉-5-基)苯胺(TPP)修饰的金纳米颗粒(Au-TPP)来提高原位 RCA 的特异性。通过光热效应，Au-TPP 在 635nm 激光照射下充当特定的热源。只有当 Au-TPP 与相邻锚定在同一靶 mRNA 上的发夹探针结合时，才会引发光热介导的 RCA。此外，我们引入了“C”型靶标特异性寡核苷酸连接探针，以使通用发夹和 Au-TPP 适用于不同的 mRNA 靶标，因此对于新的 mRNA 靶标，无需重新设计发夹和 Au-TPP 探针。通过这些策略，我们成功开发了一种用于直接原位 mRNA 检测的特异性和光热介导的超支滚环扩增，适用于福尔马林固定石蜡包埋（FFPE）组织切片和冷冻组织切片。
