A fuel-initiated DNA molecular machine for microRNA detection in serum via poly-adenine-mediated spherical nucleic acids.

MicroRNAs (miRNAs) have been identified as promising disease diagnostic biomarkers. However, it is challenging to sensitively detect miRNAs, especially in complex biological environments, due to their low abundance and small size. Herein, we have developed a DNA-fueled molecular machine for sensitive detection of miRNA-22 (miR-22) in undiluted serum by combining poly-adenine-mediated spherical nucleic acids (polyA-SNAs) with a toehold mediated strand displacement reaction (TMSDR). The polyA-SNAs are constructed by the assembly of diblock DNA probes on a AuNP surface through the high binding affinity of polyA to AuNPs. The surface density of the diblock DNA probe can be controlled by tuning the length of the polyA block, and the orientation of the diblock DNA probe can adopt an upright conformation, which is beneficial to target hybridization and TMSDRs. TMSDR is an enzyme-free target recycling amplification approach. Taking advantage of polyA-mediated SNAs and TMSDR, the operation of the molecular machine based on two successive TMSDRs on polyA20-SNAs is rapid and efficient, which can significantly amplify the fluorescence response for detection of miR-22 in an undiluted complex matrix. The developed sensor can detect as low as 10 pM of target miRNA/DNA in undiluted fetal bovine serum within 30 min. The synergetic effect of polyA-mediated SNAs and TMSDR presents a potential alternative tool for the detection of biomarkers in real biological samples.

Programming the dynamic range of nanobiosensors with engineering poly-adenine-mediated spherical nucleic acid.

Spherical nucleic acid (SNA) conjugates consisting of gold cores functionalized with a densely packed DNA shells are of great significance in the field of medical detection and intracellular imaging. Especially, poly adenine (polyA)-mediated SNAs can improve the controllability and reproducibility of DNA assembly on the nanointerface, showing the tunable hybridization ability. However, due to the physics of single-site binding, the biosensor based on SNA usually exhibits a dynamic range spanning a fixed 81-fold change in target concentration, which limits its application in disease monitoring. To address this problem, we report a tri-block DNA-based approach to assemble SNA for nucleic acid detection based on structure-switching mechanism with programmable dynamic range. The tri-block DNA is a FAM-labeled stem-loop structure, which contains three blocks: polyA block as an anchoring block for tunable surface density, stem block with different GC base pair content for varying the structure stability, and the fixed loop block for target recognition. We find that varying the polyA block, the reaction temperature, and the GC base pair, SNA shows different target binding affinity and detection limit but with normally 81-fold dynamic range. We can extend the dynamic range to 1000-fold by using the combination of two SNAs with different affinity, and narrow the dynamic range to 5-fold by sequestration mechanism. Furthermore, the tunable SNA enables sensitive detection of mRNA in cells. Given its tunable dynamic range, such nanobiosensor based on SNA offers new possibility for various biomedical and clinical applications.