Real-time Visualization of Transcribed mRNA via Click Chemistry in a Liposomal Space.

We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.

A Self-Regenerating Artificial Cell, that is One Step Closer to Living Cells: Challenges and Perspectives.

Controllable, self-regenerating artificial cells (SRACs) can be a vital advancement in the field of synthetic biology, which seeks to create living cells by recombining various biological molecules in the lab. This represents, more importantly, the first step on a long journey toward creating reproductive cells from rather fragmentary biochemical mimics. However, it is still a difficult task to replicate the complex processes involved in cell regeneration, such as genetic material replication and cell membrane division, in artificially created spaces. This review highlights recent advances in the field of controllable, SRACs and the strategies to achieve the goal of creating such cells. Self-regenerating cells start by replicating DNA and transferring it to a location where proteins can be synthesized. Functional but essential proteins must be synthesized for sustained energy generation and survival needs and function in the same liposomal space. Finally, self-division and repeated cycling lead to autonomous, self-regenerating cells. The pursuit of controllable, SRACs will enable authors to make bold advances in understanding life at the cellular level, ultimately providing an opportunity to use this knowledge to understand the nature of life.