Malachite green‐conjugated microtubules as mobile bioprobes selective for malachite green aptamers with capturing/releasing ability

Abstract

We have developed a novel mobile bioprobe using a conjugate of a kinesin-driven microtubule (MT) and malachite green (MG) as a platform for capturing MG RNA aptamers. The fluorescence of MG increases when it is bound to an MG aptamer, allowing MT-MG conjugates to work as sensors of RNA transcripts containing the MG aptamer sequence. Kinesin motor proteins provide an effective driving force to create mobile bioprobes without any manipulation. Although the fluorescence of a small number of MG-binding aptamers is low, the self-organization of tubulins into MTs enables the microscopic observation of the bound aptamers by collecting them on MTs. We demonstrate that MT-MG conjugates can select target aptamers from a transcription mixture and transport them without losing their inherent motility. Because the MG aptamer binds MG in a reversible manner, MT-MG conjugates can conditionally load and unload the target aptamers. This is one advantage of this system over the molecular probes developed previously in which reversible unloading is impossible due to high-affinity binding, such as between avidin and biotin. Furthermore, an MT-MG conjugate can be used as a platform for other MG aptameric sensors with recognition regions for various target analytes optimized by further selection procedures. This is the first step to applying living systems to in vitro devices. This technique could provide a new paradigm of mobile bioprobes establishing high-throughput in vitro selection systems using microfluidic devices operating in parallel.