Hyebin Na1,Jae Chul Park1,Yoon Sung Nam1
Korea Advanced Institute of Science and Technology1
Hyebin Na1,Jae Chul Park1,Yoon Sung Nam1
Korea Advanced Institute of Science and Technology1
MicroRNA (miRNA) is a small non-coding RNA that plays an important role in regulating the expression of various target genes. miRNA has emerged as a key biomarker for the diagnosis of diseases, including point-of-care diagnosis. Target-catalyzed toehold-mediated strand displacement (TMSD) cascade has been investigated as an enzyme-free isothermal amplification. However, the TMSD-based detection of RNA targets is still very challenging compared to DNA targets because the reaction rate constant of RNA-DNA strand displacement is ~35 times lower than that of DNA-DNA strand displacement. Herein, we present a new method based on DNA-streptavidin (sAv) hydrogel to increase the sensitivity of TMSD-based miRNA detection. Our key idea is to introduce a single internal base-pairing mismatch to the stem of hairpin DNA. The mismatch near the toehold locally destabilizes the hairpin DNA to enhance the kinetics of opening the hairpin by miRNA without increasing non-specific reactions. A single miRNA target catalyzes the opening of the hairpin DNAs through a series of TMSD reactions and forms multiple Y-shaped DNA (Y-DNA) tethered with quencher and biotin at the end. The resulting Y-DNA are self-assembled into nanoscale DNA-Alexa Fluor<sup>TM</sup> 594-labeled sAv hydrogel. The three-dimensional hydrogel network enables efficient Förster resonance energy transfer (FRET) from Alexa 594 to the quencher. The mismatch-induced thermodynamic driving method dramatically lowered the limit of detection (LOD) from 1.5 nM to 60 pM increasing the strand displacement-based DNA-sAv hydrogel formation. The hydrogel-based platform exhibits high specificity in discriminating single-base mismatch. The DNA-sAv hydrogel system also applies to serum samples and different target sequences with a LOD value of 60 pM. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020R1A2C2004168).<br/>[1] Cheol Am Hong, Jae Chul Park, Hyebin Na, Huiju Jeon, Yoon Sung Nam, <i>Biosensors and Bioelectronics</i> (2021) 182, 113110<br/>[2] Jae Chul Park, Se Yeon Choi, Moon Young Yang, Lin Nan, Hyebin Na, Ha Neul Lee, Hyun Jung Chung, Cheol Am Hong, and Yoon Sung Nam, <i>ACS Applied Materials & Interfaces</i> (2019) 11, 37, 33525-33534