Charge-elimination strategy for constructing RNA-selective fluorescent probe undisturbed by mitochondria

Observing RNA behavior in living cells remains a major challenge in life sciences. Most current RNA probes rely on cationic groups to bind RNA, but mitochondria, which have a strong negative membrane potential, can interfere with these probes. This interference limits the accuracy of RNA visualization. Prior research has shown that cationic probes often accumulate in mitochondria, leading to false signals. However, the exact mechanism by which mitochondria disrupt RNA imaging has not been fully resolved. This gap motivated the development of new strategies to avoid mitochondrial interference. No prior work had resolved how to design RNA probes that remain selective while avoiding mitochondrial accumulation. The need for RNA-specific imaging tools that function in live cells without mitochondrial interference is clear. Researchers have proposed various modifications to probe structures, but none have effectively eliminated mitochondrial binding. This study addresses the need for RNA imaging that is both selective and free from mitochondrial artifacts.

This study aimed to develop a new RNA imaging strategy that avoids mitochondrial interference. The goal was to design a fluorescent probe that binds RNA but does not accumulate in mitochondria. The motivation came from the limitations of existing cationic RNA probes. These probes often bind to mitochondria due to their negative membrane potential. The researchers proposed a charge-elimination strategy to address this issue. The new probe, H-SMBT, was designed to switch from a cationic to a neutral state in mitochondria. This shift would allow the probe to detach from mitochondria and bind only to RNA. The study also aimed to test the effectiveness of this strategy in live cells and zebrafish. The researchers sought to confirm that the phenol group in H-SMBT was responsible for RNA selectivity.

The researchers designed a fluorescent probe called H-SMBT to target RNA in live cells. The probe was modified with a phenol group to reduce mitochondrial interference. A comparative molecule, M-SMBT, was used to test the role of the phenol group. Both probes were tested in live cells to assess their RNA selectivity. The study evaluated how quickly the probes stained RNA and how well they avoided mitochondria. The researchers used fluorescence microscopy to observe probe behavior in cells. They also tested the probes in zebrafish to assess tissue permeability. The study compared the performance of H-SMBT and M-SMBT in different conditions. The researchers analyzed the charge state of the probes in mitochondria and RNA. The experiments focused on confirming the role of the phenol group in RNA specificity.

H-SMBT stained RNA in live cells within 5 minutes with high selectivity. The probe avoided mitochondrial accumulation by switching to a neutral state. This charge-elimination strategy reduced interference from mitochondria. The phenol group in H-SMBT was shown to be critical for RNA specificity. M-SMBT, which lacked the phenol group, did not exhibit the same RNA selectivity. The probe successfully imaged RNA in live zebrafish due to its tissue permeability. The study demonstrated that H-SMBT could monitor cellular damage processes. The researchers confirmed that the phenol group enabled the charge shift in mitochondria. The probe's ability to detach from mitochondria was verified through fluorescence imaging. The results showed that H-SMBT outperformed existing RNA probes in selectivity. The study also showed that the probe could be used for RNA-related life science research. The charge-elimination strategy proved effective in avoiding mitochondrial interference.

The charge-elimination strategy in H-SMBT effectively reduced mitochondrial interference. The phenol group was essential for the probe's RNA selectivity. The probe's ability to switch from cationic to neutral in mitochondria was confirmed. H-SMBT stained RNA in live cells with high specificity and speed. The study showed that the probe could be used in zebrafish for tissue imaging. The researchers demonstrated that the probe could monitor cellular damage processes. The charge-elimination approach provides a new design strategy for RNA probes. The study supports the use of H-SMBT for RNA-related life science research. The findings suggest that the phenol group plays a key role in RNA specificity. The probe's performance in live cells and zebrafish was validated. The study highlights the importance of avoiding mitochondrial interference in RNA imaging. The results align with the authors' stated goal of improving RNA visualization accuracy.