A microRNA-initiated DNAzyme motor operating in living cells

Abstract

Synthetic DNA motors have great potential to mimic natural protein motors in cells but the operation of synthetic DNA motors in living cells remains challenging and has not been demonstrated. Here we report a DNAzyme motor that operates in living cells in response to a specific intracellular target. The whole motor system is constructed on a 20 nm gold nanoparticle (AuNP) decorated with hundreds of substrate strands serving as DNA tracks and dozens of DNAzyme molecules each silenced by a locking strand. Intracellular interaction of a target molecule with the motor system initiates the autonomous walking of the motor on the AuNP. An example DNAzyme motor responsive to a specific microRNA enables amplified detection of the specific microRNA in individual cancer cells. Activated by specific intracellular targets, these self-powered DNAzyme motors will have diverse applications in the control and modulation of biological functions.

Rational Engineering of a Dynamic, Entropy‐Driven DNA Nanomachine for Intracellular MicroRNA Imaging

“…This is attributed to slow target-recycling kinetics,w hich hinders potential bioanalytical applications for low-abundance biomolecules in living cells.Recently,extensive attention has been focused on designing intelligent, processive DNAwalking machines with AuNP-constructed, three-dimensional landscapes,m ost of which in principle convert nuclease-mediated hydrolysis to mechanical motions. [9] Themotor employs DNAzyme-mediated hydrolysis of DNA-RNAc himeric substrates in the presence of cofactor Mn 2+ , which must be supplied to cells due to the insufficient native cellular Mn levels.Their work provides agood example of the operation of aDNA nanomachine in living cells. [8b] This dynamic nanomachine accelerates the rates from the intramolecular reactions,b ecause all reaction components are co-conjugated on as ingle AuNP,l eading to high local effective concentrations and stable signal outputs due to the irreversible release of DNAs ubstrates.H owever, they require aspecific endonuclease to propel the motion of the walker, thus limiting potential application in living cells.…”

In Situ Amplification‐Based Imaging of RNA in Living Cells

“…Inspired by protein motors, scientists recently have made efforts to design and develop diverse synthetic DNAm otors (DNAzymes). [106] All motor components were integrated on as ingle AuNP,i ncluding substrate strands serving as DNA tracks and DNAzyme molecules that were each silenced by al ocking strands.Atarget miRNAd isplaced the locking strand from DNAzyme molecule through toehold-mediated strand displacement reaction. Binding with the ligand activates the DNAzyme,w hich then catalyzes the cleavage of as ubstrate strand (especially at ar ibonucleotide site).…”

“…[74,102,103] Recently,D NAzymes have been used to imitate the functions of protein motors in living cells. [43,[104][105][106][107] As the first example,P eng et al constructed am iRNAinitiated DNAzyme motor operating in living cells for in situ amplified imaging of the target miRNAi ni ndividual cancer cells ( Figure 5). [106] All motor components were integrated on as ingle AuNP,i ncluding substrate strands serving as DNA tracks and DNAzyme molecules that were each silenced by al ocking strands.Atarget miRNAd isplaced the locking strand from DNAzyme molecule through toehold-mediated strand displacement reaction.…”

“…[43,[104][105][106][107] As the first example,P eng et al constructed am iRNAinitiated DNAzyme motor operating in living cells for in situ amplified imaging of the target miRNAi ni ndividual cancer cells ( Figure 5). [106] All motor components were integrated on as ingle AuNP,i ncluding substrate strands serving as DNA tracks and DNAzyme molecules that were each silenced by al ocking strands.Atarget miRNAd isplaced the locking strand from DNAzyme molecule through toehold-mediated strand displacement reaction. Theu nlocked DNAzyme then hybridized with its substrate strand and cleaved it to release the FAM-labelled segment in the presence of manganese ion (Mn 2+ ), recovering the fluorescence signal.…”

Rational Engineering of a Dynamic, Entropy‐Driven DNA Nanomachine for Intracellular MicroRNA Imaging

“…To address this challenge,t he Le group recently reported an ongoing design of DNAzyme motor operated in living cells. [9] Themotor employs DNAzyme-mediated hydrolysis of DNA-RNAc himeric substrates in the presence of cofactor Mn 2+ , which must be supplied to cells due to the insufficient native cellular Mn levels.Their work provides agood example of the operation of aDNA nanomachine in living cells.…”