A dual-targeted fluorogenic probe and microfluidic chip enables single-cell labeling and quantification of live Mycobacterium tuberculosis .

Circular RNA (circRNA) has various advantages over linear mRNA that is gaining success as a new vaccine and therapeutic agent. Thus, circRNA and its engineering methods have attracted attention recently. In this study, we developed a new in vitro circRNA engineering method by end-to-end self-targeting and splicing (STS) reaction using Tetrahymena group I intron ribozyme. We found that only the P1 helix structure of the group I intron was enough to generate circRNA by STS reaction. The efficacy of circRNA generation by STS reaction was comparable to the method using a permuted intron-exon (PIE) reaction. However, an end-to-end STS reaction does not introduce any extraneous fragments, such as an intronic scar that can be generated by PIE reaction and might trigger unwanted innate immune responses in cells, into circRNA sequences. Moreover, generated circRNA was efficiently purified by ion pair-reversed phase high-pressure liquid chromatography and used for cell-based analysis. Of note, efficient protein expression and stability with least innate immune induction by the circRNA with coxsackievirus B3 IRES were observed in cells. In conclusion, our new in vitro circRNA strategy can effectively generate highly useful circRNAs in vitro as an alternative circRNA engineering method.

Personalized cancer medicine requires measurement of therapeutic efficacy as early as possible, which is optimally achieved by three-dimensional imaging given the heterogeneity of cancer. Magnetic resonance imaging (MRI) can obtain images of both anatomy and cellular responses, if acquired with a molecular imaging contrast agent. The poor sensitivity of MRI has limited the development of activatable molecular MR contrast agents. To overcome this limitation of molecular MRI, a novel implementation of our caspase-3-sensitive nanoaggregation MRI (C-SNAM) contrast agent is reported. C-SNAM is triggered to self-assemble into nanoparticles in apoptotic tumor cells and effectively amplifies molecular level changes through nanoaggregation, enhancing tissue retention and spin-lattice relaxivity. At one-tenth the current clinical dose of contrast agent and following a single imaging session, C-SNAM MRI accurately measured the response of tumors to either metronomic chemotherapy or radiation therapy, where the degree of signal enhancement is prognostic of long-term therapeutic efficacy. Importantly, C-SNAM is inert to immune activation, permitting radiation therapy monitoring.