The terminal deoxynucleotidyl transferase (TdT) belongs to the X family of DNA polymerases. This unusual polymerase catalyzes the template‐independent addition of random nucleotides on 3′‐overhangs during V(D)J recombination. The biological function and intrinsic biochemical properties of the TdT have spurred the development of numerous oligonucleotide‐based tools and methods, especially if combined with modified nucleoside triphosphates. Herein, we summarize the different applications stemming from the incorporation of modified nucleotides by the TdT. The structural, mechanistic, and biochemical properties of this polymerase are also discussed.
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.
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