Methods for tracking
RNA inside living cells without perturbing
their natural interactions and functions are critical within biology
and, in particular, to facilitate studies of therapeutic RNA delivery.
We present a stealth labeling approach that can efficiently, and with
high fidelity, generate RNA transcripts, through enzymatic incorporation
of the triphosphate of tC
O
, a fluorescent tricyclic cytosine
analogue. We demonstrate this by incorporation of tC
O
in
up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding
for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization
of this mRNA shows that the incorporation rate of tC
O
is
similar to cytosine, which allows for efficient labeling and controlled
tuning of labeling ratios for different applications. Using live cell
confocal microscopy and flow cytometry, we show that the tC
O
-labeled mRNA is efficiently translated into H2B:GFP inside human
cells. Hence, we not only develop the use of fluorescent base analogue
labeling of nucleic acids in live-cell microscopy but also, importantly,
show that the resulting transcript is translated into the correct
protein. Moreover, the spectral properties of our transcripts and
their translation product allow for their straightforward, simultaneous
visualization in live cells. Finally, we find that chemically transfected
tC
O
-labeled RNA, unlike a state-of-the-art fluorescently
labeled RNA, gives rise to expression of a similar amount of protein
as its natural counterpart, hence representing a methodology for studying
natural, unperturbed processing of mRNA used in RNA therapeutics and
in vaccines, like the ones developed against SARS-CoV-2.
Recent progresses in organic chemistry and molecular biology have allowed the emergence of numerous new applications of nucleic acids that markedly deviate from their natural functions. Particularly, DNA and RNA molecules—coined aptamers—can be brought to bind to specific targets with high affinity and selectivity. While aptamers are mainly applied as biosensors, diagnostic agents, tools in proteomics and biotechnology, and as targeted therapeutics, these chemical antibodies slowly begin to be used in other fields. Herein, we review recent progress on the use of aptamers in the construction of smart DNA origami objects and MRI and PET imaging agents. We also describe advances in the use of aptamers in the field of neurosciences (with a particular emphasis on the treatment of neurodegenerative diseases) and as drug delivery systems. Lastly, the use of chemical modifications, modified nucleoside triphosphate particularly, to enhance the binding and stability of aptamers is highlighted.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.