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.

New Ideas for in Vivo Detection of RNA

“…This inspired Grate and Wilson to select an RNA aptamer to bind MG and selectively target specific RNA molecules for inactivation in vivo (Grate & Wilson, 1999). An RNA aptamer to Hoechst dye 33258 was also selected to study gene regulation while RNA aptamers for Sulforhodamine B and Fluorescein were selected for potential in vivo RNA labeling (Werstuck & Green, 1998;Holeman et al, 1998). Werstuck and Green showed that they could regulate a gene in vivo which had been modified by the insertion of the Hoechst dye by adding the dye to the medium (Werstuck & Green, 1998).…”

“…The nucleotides of the internal loop form a series of stacking and non-Watson-Crick basepairing interactions to create a unique binding pocket that restricts the flexibility of the dye and keeps it in a highly fluorescent state (Figure 2A, 2B). The high affinity of this aptamer for MG (K d =117 nM) and the high fluorescence enhancement that results from binding (2360-fold) have encouraged several groups to apply it to create programmable nucleic acid biosensing platforms (Babendure et al, 2003;Stojanovic & Kolpashchikov, 2004;Hirabayashi et al, 2006;Afonin et al, 2008). These will be described in more detail in the next section after we cover other efforts to develop new light-up pairs.…”

“…The authors call this method "blue fluorescent RNA" and reported K d =35 nM for binding of the dye to the isolated aptamer, but did not report apparent K d 's in the context of the mRNA construct. Eydeler et al reported detailed measurements of the Sulforhodamine B-binding aptamer (SRB2m), selected by Holeman et al (1998), fused to a variety of genetic constructs, including intact EGFP-mRNA produced in vivo and assayed in vitro in the presence of other cellular RNAs (Eydeler et al, 2009). They used fluorescence correlation spectroscopy (FCS), a method that allows one to obtain diffusion constants and other kinetic parameters from fluorescence fluctuations generated by molecules diffusing in and out of the focal volume.…”

An Autonomous Molecular Transport System Using DNAs and Motor Proteins in Molecular Communication

“…However, in achieving autonomous loading/unloading of cargoes, a new approach is required because existing systems require external stimuli such as UV-light exposure [8]- [9], ligand supplementation [10], restriction enzyme digestion or temperature fluctuation [11] to unload cargoes from gliding MTs; thus making those systems non-autonomous. This paper describes design and empirically study of a molecular transport system that autonomously loads/unloads specified cargoes using DNA strand exchange and that transports the loaded cargoes using the reverse geometry of MT motility on kinesins.…”

Herding Nanotransporters: Localized Activation via Release and Sequestration of Control Molecules

“…In the context of molecular shuttles, the release of cargo can be achieved by delivering a competitor for the cargo linkage. 23,24 Alternatives to control via activating molecules exist but are not superior. Localized heating in a cold environment 25,26 has been demonstrated; however, for kinesin motors a 10-fold difference in activation (chosen here as benchmark to define the activation zone) is difficult to achieve due to the limited range of protein stability.…”