5 Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing,
translation initiation and stability. Here we describe
a biosensor designed to detect the presence of cap
structures on mRNAs that is also sensitive to mRNA
degradation, so uncapped or degraded mRNAs can
be detected in a single step. The biosensor is based
on a chimeric protein that combines the recognition
and transduction roles in a single molecule. The main
feature of this sensor is its simplicity, enabling semiquantitative analyses of capping levels with minimal
instrumentation. The biosensor was demonstrated
to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range
remained constant with RNAs ranging in size from
250 nt to approximately 2700 nt and the biosensor
was able to detect variations in the capping level in
increments of at least 20%, with a limit of detection of
2.4 pmol. Remarkably, it also can be applied to more
complex analytes, such mRNA vaccines and mRNAs
transcribed in vivo. This biosensor is an innovative
example of a technology able to detect analytically
challenging structures such as mRNA caps. It could
find application in a variety of scenarios, from quality
analysis of mRNA-based products such as vaccines
to optimization of in vitro capping reactions.