Highly efficient loop cleavage for human papillomavirus detection with a novel thermophilic Argonaute from Thermus...

Argonaute proteins (Agos), endowed with the capacity to cleave DNA or RNA under the guidance of small nucleic acid guides, have emerged as versatile biotechnological tools. This study endeavored to characterize a novel thermophilic Argonaute protein from Thermus brockianus (TbAgo), revealing its proficiency as a DNA-guided DNA endonuclease. Demonstrating high catalytic efficiency and precision at 65 °C, TbAgo possessed compatibility with loop-mediated isothermal amplification (LAMP) method, whose optimal temperature is also around 65 °C. Therefore, an innovative isothermal nucleic acid detection platform named AMEND (Argonaute-mediated loop cleavage for nucleic acid detection) was developed by integrating LAMP with TbAgo's targeted cleavage. This novel detection strategy was used to detect human papillomavirus (HPV) 16 and 18 DNA simultaneously with the limit of detection (LoD) of 1 aM within 30 min. Furthermore, a two-step microfluidic chip was designed to streamline the above HPV DNA detection workflow with high sensitivity of HPV 16 (1 aM) and 18 (10 aM) within 30 min. The present work not only characterized a novel Argonaute protein with the highest cleavage efficiency among the literature, but also paved the way to coordinate and streamline the two sequential reactions (isothermal DNA amplification and Ago mediated cleavage) at the same optimal temperature for high-efficiency DNA detection.