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. 2020 Oct 21;56(82):12319-12322.
doi: 10.1039/d0cc05092j. Epub 2020 Sep 17.

Responsive fluorescent nucleotides serve as efficient substrates to probe terminal uridylyl transferase

Jerrin Thomas George  1 , Seergazhi G Srivatsan
Affiliations

Responsive fluorescent nucleotides serve as efficient substrates to probe terminal uridylyl transferase

Jerrin Thomas George et al. Chem Commun (Camb). .

Abstract

We repurposed a terminal uridylyl transferase enzyme to site-specifically label RNA with microenvironment sensing fluorescent nucleotide mimics, which in turn provided direct read-outs to estimate the binding affinities of the enzyme to RNA and nucleotide substrates. This enzyme-probe system provides insights into the catalytic cycle, and can facilitate the development of discovery platforms to identify robust enzyme inhibitors.

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Conflict of interest statement

Conflicts of interest

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
(a) TUTase efficiently incorporates UTP analogs (1 and 2, see Fig. 2) to generate 3′-end labeled RNA ONs exhibiting high fluorescence. Labeled RNA upon titration with the enzyme results in progressive quenching in fluorescence, which enables the determination of the binding constant. (b) The analog (e.g., 2) bound to the enzyme shows low fluorescence and the addition of a small molecule (e.g., UTP) displaces the analog resulting in an enhancement in fluorescence, which constitutes a simple turn-on fluorescent platform for the identification of TUTase binders/inhibitors.
Fig. 2
Fig. 2
(a) Crystal structure of SpCID1 bound to UTP showing uracil stacking with Tyr212 (PDB: 4FH5) (b) Crystal structure of DmTailor bound to RNA showing the last nucleobase, uracil stacking with Tyr390 (PDB: 6I0V). Vacant space around the C5-position of uracil can been seen in the structures. (c) Microenvironment sensing UTP analogs used in terminal uridylation reactions.
Fig. 3
Fig. 3
3′ -Terminal uridylation of 5′-FAM-labeled RNA ON 3 with (a) SeUTP 1 and (b) BFUTP 2 using SpCID1. Arrows indicate the number of insertions at the 3’-end.
Fig. 4
Fig. 4
Emission spectra of (a) BFU-labeled RNA ON 4BFU and (b) BFUTP 2 (200 nM) as a function of increasing concentration of SpCID1 (0–2 μM). (c) A plot of normalized fluorescence intensity at λem = 435 nm versus [SpCID1] showing the curve fit for the binding of 4BFU and 2 to SpCID1. See ESI† for details.
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