Construction of an Autocatalytic Hybridization Assembly Circuit for Amplified In Vivo MicroRNA Imaging

Abstract

Lowly expressed analyte in complex cytoplasmic milieu necessitates the development of non‐enzymatic autocatalytic DNA circuits with high amplification and anti‐interference performance. Herein, we engineered a versatile and robust stimuli‐responsive autocatalytic hybridization assembly (AHA) circuit for high‐performance in vivo bioanalysis. Under a moderately confined condition, the initiator motivated the autonomous and cooperative cross‐activation of cascade hybridization reaction and catalytic DNA assembly for generating an exponentially amplified readout without the parasite steric hindrance and random diffusion side effects. The AHA circuit was systematically investigated by a series of experimental studies and theoretical simulations. The successively guaranteed target recognition and synergistically accelerated signal‐amplification enabled the sensitive and selective detection of analyte, and realized the robust miRNA imaging in living cells and mice. This autocatalytic DNA circuit could substantially expand the toolbox for accurate diagnosis and programmable therapeutics.

An Autocatalytic DNA Circuit Based on Hybridization Chain Assembly for Intracellular Imaging of Polynucleotide Kinase

“…To further testify the positive-feedback amplification mechanism of the AHS circuit (Figure S7), a nonautocatalytic hybridization system (nAHS) was introduced as the control experiment, where the initiator region in hairpin H 6 was replaced with poly T to inhibit the positive feedback effect of the CDA circuit in autocatalytic module (Figure S8). As a result, the nAHS system could represent the traditional HCA circuit with linear amplification capacity . Upon the addition of initiator strand I , the AHS sensor exhibited a dramatically increased fluorescence intensity, while an almost negligible fluorescence signal was obtained for nAHS (Figures C, D, and S9), thus revealing drastically enhanced signal amplification efficiency of the positive-feedback AHS circuit.…”

“…As a result, the nAHS system could represent the traditional HCA circuit with linear amplification capacity. 48 Upon the addition of initiator strand I, the AHS sensor exhibited a dramatically increased fluorescence intensity, while an almost negligible fluorescence signal was obtained for nAHS (Figures 2C, 2D, and S9), thus revealing drastically enhanced signal amplification efficiency of the positive-feedback AHS circuit. In addition, several different hairpin-excluded experiments were also conducted in Figure S10.…”

A Rebuilding‐Free Nucleic Acid Detection Strategy Enables Ultrasensitive Genotyping, N‐in‐1 Logic Screening and Accurate Multiplex Assay of Dangerous Pathogens

“…We can get very helpful references from the methods developed for other isothermal amplifications. [24][25][26][27][28][29]…”

“…Next step, continuous efforts can be made to increase the signal‐to‐background ratio, shorten the detection time, develop portable readouts and simplify/integrate the operation procedure to realize point‐of‐care test. We can get very helpful references from the methods developed for other isothermal amplifications [24–29] …”

Monitoring and modulating a catalytic hybridization circuit for self-adaptive bioorthogonal DNA assembly

“…With its highly predictable base-pairing, low cost, ease of synthesis, and high biocompatibility, DNA has been recognized as a highly promising biomolecule for designing localized molecular biocomputing circuits for different aims. [13][14][15][16][17] A wide range of localized DNA circuits has been implemented on DNA origamis, molecular scaffolds, and spherical nucleic acids (SNA). [18][19][20][21][22] Such spatially confined arrangement plays a pivotal role in increasing the effective concentration of reactants through proximity confinement and simultaneously achieves the reduced crosstalk between these spatially separated reaction processes.…”