Design, assembly, and operation of a self-regulating DNA rotaxane linear actuator

Abstract

DNA is not only the carrier of genetic information but also an excellent bottom-up material for constructing nanostructures due to its high degree of programmability. Interlocked DNA nanostructures have a potential use as nanomachines to perform certain tasks at the molecular scale, in which subcomponents can move a large amplitude with respect to each other. Additionally, DNA is also able to interact with motor-like enzymes, such as RNA polymerase. Such interactions can be utilized to generate directional forces to create biohybrid artificial nanodevices capable of autonomous active motion and produce RNA. <br /> In this thesis, T7 RNA polymerase (T7RNAP) and a DNA nanostructure – DNA rotaxane are combined to develop an automatic linear nanoactuator for the following purposes: 1) transferring the linear motion of T7RNAP along the DNA track to the periodic translational displacement of macrocycle threading in rotaxane; 2) self-controlled transcription. <br /> An interlocked macrocycle-reconstructed promoter is proposed to control the transcription of T7RNAP. In this arrangement, the split promoter sequences of the template strand are distributed over the axle and the threading macrocycle in rotaxane, which together constitute the complete promoter sequence. Transcription by T7RNAP is then utilized to control the release of the macrocycle from a single-stranded (ss) gap region in the promoter and deplete the movement range of the interlocked macrocycle on the axle. Upon reaching the terminator sequences of transcription, T7RNAP detaches from the axle, and the whole system can be reset as the macrocycle shifts back and rehybridizes with the promoter gap to reconstitute the double-stranded (ds) promoter structure, which is a prerequisite for the next transcription cycle. <br /> The transcriptional performance of the rotaxane actuator with an interlocked macrocycle-mediate promoter is studied through molecular beacon (MB) fluorescence experiments. Meanwhile, the detailed kinetics in the operation of the rotaxane actuator are also investigated. The dynamic stability of the system after transcription is then demonstrated through gel analysis, AFM, and coarse-grained modelling simulations. An updated rotaxane actuator with a spherical stopper is proposed and compared with the original design. The properties of the macrocycle-mediated promoter are also carefully investigated. <br /> This T7RNAP-DNA rotaxane hybrid nanodevice was shown to produce self-repeating linear motion without any intervention and achieve self-controlled transcription without any intermediate. Moreover, it has the potential to develop delivery systems and self-regulating RNA production automatons at the molecular scale.