Concatemeric Broccoli reduces mRNA stability and induces aggregates

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Fluorogenic aptamers provide a novel method for real-time RNA imaging.
• Established techniques for observing RNA transport and dynamics have limitations.

Purpose of the Study:

• To develop and characterize Broccoli-aptamer concatemers for enhanced real-time RNA imaging.
• To investigate the impact of concatemer length on fluorescence and RNA stability.

Main Methods:

• Construction of Broccoli-aptamer concatemers with varying repeat numbers (4-128).
• Fusion of concatemers to an mCherry-coding mRNA for expression studies.
• Characterization in transient transfection, stable cell lines, and cytomegalovirus infection models.
• Assessment of fluorescence, RNA aggregation, and stability.

Main Results:

• Concatemerization increased Broccoli fluorescence up to 16 repeats.
• Higher concatemer lengths (beyond 16) led to decreased fluorescence due to RNA aptamer aggregation and reduced RNA stability.
• Cellular and cytomegalovirus genomes struggled to maintain high concatemer copy numbers, potentially due to recombination.
• Linker sequences partially rescued negative effects of concatemers.
• Substrate-bound Broccoli, despite photobleaching, is viable for live-cell imaging with time-lapse protocols.

Conclusions:

• Broccoli aptamer concatemers can enhance RNA imaging, but optimal length is crucial to avoid aggregation and instability.
• Challenges exist in maintaining high concatemer expression in certain genomic contexts.
• Further research into linker sequences may improve concatemer utility.
• Adapted imaging protocols enable the use of Broccoli aptamers in live-cell studies despite photobleaching.