SUPPLEMENTARY INFORMATION

A Metabolic Engineering Approach to Incorporate Modified Pyrimidine

Nucleosides into Cellular RNA

Yu Zhang and Ralph E. Kleiner*

Department of Chemistry, Princeton University, Princeton, NJ 08540 USA

Table of Contents

i. Supplementary Discussion
ii. Experimental Methods
iii. Supplementary Table 1
iv. Supplementary Figure 1
v. Supplementary Figure 2
vi. Supplementary Figure 3
vii. Supplementary Figure 4
viii. Supplementary Figure 5
ix. Supplementary Figure 6
x. Supplementary Figure 7
xi. Supplementary Figure 8
xii. Supplementary Figure 9
xiii. Supplementary Figure 10
xiv. Supplementary Figure 11
xv. Supplementary Figure 12
xvi. Supplementary Figure 13
xvii. Supplementary Figure 14
xviii. Supplementary References

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Supplementary Discussion

We applied UCK2-mediated RNA labeling to visualize transcription and turnover

with 5-AmU and compared it to the properties of 5-EU RNA. While both nucleosides are

substrates for RNA polymerase I and II, and show similar overall turnover kinetics, we do

observe differences in their subcellular localization: 5-EU is highly concentrated in

nucleoli, while 5-AmU exhibits staining in the nucleus and cytosol without strong

enrichment in the nucleoli. These differences may arise from several factors including

differences in nucleoside metabolism, polymerase utilization, as well as effects of the C5

modification on interactions with RNA transport and turnover machinery.

Similarly, we observe several key differences in how 5-EU-labeled and 5-AmU-

labeled RNA behave during arsenite stress. During arsenite stress, the localization of 5-

AmU-labeled RNA is restricted to the nucleus. We speculate that this may result from

inhibition of nuclear export – thereby explaining why there is little effect on 5-EU RNA,

which we do not observe in the cytosol during normal conditions. Alternatively, the

turnover of 5-AmU RNA in the cytosol (but not the nucleus) could be accelerated during

arsenite stress. Again, this effect would not be apparent in our 5-EU labeling experiments

since little of this modification is detected in the cytosol to begin with. Finally, transcription

with 5-EU is strongly inhibited during arsenite stress, but not during heat shock. This effect

is not apparent on 5-AmU labeling which is largely unaffected during heat shock and

arsenite stress. While this may be a result of differences in metabolism or polymerase

utilization, we also cannot exclude the possibility that CuAAC functionalization of 5-EU is

affected by arsenite stress – that is, the nucleoside is incorporated into RNA but

chemically or enzymatically modified in a manner that no longer allows click chemistry

S2
detection. To test this possibility, we incubated 100 µM 5-EU with 0.5 mM sodium arsenite

in 1X PBS for 4 hr at 37 °C, but could not detect any change in composition, arguing

against direct chemical modification of the nucleoside.

Experimental Methods

Chemicals

5-azidomethyl uridine (5-AmU) was purchased from Jena Bioscience. 5-ethynyl uridine (5-

EU), THPTA ligand, Cy3-alkyne, Cy3-azide, and Cy3-DBCO were obtained from Click

Chemistry Tools. 5-fluorouridine (5-FUrd) was purchased from Alfa Aesar. BODIPY-BCN

was a gift from Jeremy Baskin1. All other chemicals were purchased from Sigma-Aldrich or

Fisher Scientific unless otherwise indicated.

Plasmids

A plasmid encoding cDNA for human UCK2 was a gift from Willian Hahn and David Root

(Addgene plasmid #23863). For protein expression, wild-type and mutant UCK2 genes were

cloned into a modified pET28a vector. Mutations at residue 65 were introduced into UCK2

using overlap extension PCR with mutagenic primers. For transient transfection and

construction of Flp-In cell lines, UCK2 genes were cloned into a modified pCDNA5/FRT/TO

(Life Technologies) vector containing an N-terminal 3x-FLAG affinity tag.

Protein expression and purification

UCK2 enzymes were expressed and purified from E.coli BL21 Rosetta cells. When OD600

reached 0.9, expression was induced by 1 mM IPTG at 37 °C for 4 hours, and cells were

harvested by centrifugation at 5000 g for 30 minutes at 4 °C. The pellet was resuspended in

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lysis buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF)

supplemented with cOmplete, mini EDTA-free protease inhibitor tablets (Sigma) and

sonicated. Clarified lysate was then purified by HisPur Cobalt Resin (Thermo Scientific) and

eluted with 250 mM imidazole in 50 mM HEPES, pH 7.5, 150 mM NaCl. Proteins were

further purified by passage over MonoQ (GE Healthcare) and MonoS (GE Healthcare)

columns using buffer containing 50 mM HEPES, pH 8.0, 1 mM DTT with a gradient of 0 to

500 mM NaCl. Since UCK2 mutants do not bind strongly to ion exchange columns, the flow-

through was collected, dialyzed into storage buffer, and flash frozen in liquid N2.

In vitro enzyme assay

The catalytic activity of wild-type and mutant UCK2 enzymes was tested by preparing

reactions containing enzyme (50 nM), nucleoside (2 mM), KCl (100 mM), MgCl2 (5 mM),

NaF (15 mM), and ATP (5 mM) in Tris buffer (50 mM, pH 7.6). To study the catalytic

efficiency of mutant UCK2 enzymes, reactions were incubated at 37 °C for 2-20 hours and

quenched by heating at 95 °C for 5 minutes. To explore the kinetics of reactions catalyzed

by the wild-type enzyme and the Y65G mutant, the reactions of uridine, 5-EU and 5-AmU in

the presence of the wild-type or Y65G UCK2 enzyme were quenched at 1, 2, 4, 8, and 20

hours, with the exception of the reaction of uridine catalyzed by wild-type UCK2, which was

quenched at 5 min, 10 min, 30 min, 1 h, 2 h, and 20 h. After centrifugation (17,000g for 10

minutes), reaction products were analyzed by HPLC on an Agilent Poroshell 120 EC-C18 (4

μm, 4.6×150 mm) column using a 0.1 M TEAA/MeOH gradient (0% to 1.5% methanol over

10 minutes, 1.5% to 2.0% methanol over 10 minutes, followed by 2.0% to 4.5% methanol in

10 minutes), except the analysis in Supplementary Figure 1a, where a 0.1 M

TEAA/acetonitrile gradient was used (isocratic 0% acetonitrile over 6 minutes followed by

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isocratic 2% acetonitrile over 24 minutes) at a flow rate of 1.2 mL/min. Product identities

were confirmed by comparing retention times with available standards as well as HRMS

analysis on an Agilent 6220 ESI-TOF.

General cell culture

HeLa, U2OS, and Flp-In TRex HeLa cells were cultured at 37 °C in a humidified

atmosphere with 5% CO2 in DMEM (Life Technologies) supplemented with 10% fetal

bovine serum (Atlanta), 1x penicillin-streptomycin (Life Technologies) and 2 mM L-

glutamine (Life Technologies). For nucleoside labeling experiments in Figures 2 and 3, and

Supplementary Figures 6, 7, and 10, cells were seeded at 0.4 × 106 (for U2OS) or 0.6 x 106

(for HeLa) cells per well in six-well plates with 12 mm glass coverslips. 24 hours later, cells

were transfected with pcDNA5-UCK2 plasmid (2 μg) and Lipofectamine 2000 reagent

(Invitrogen, 5 μL) according to the manufacturer’s instructions. 20 hours after transfection,

fresh media containing 5-azidomethyl uridine (5-AmU) or 5-ethynyl uridine (5-EU) was

added for the indicated time. To test DNA incorporation of 5-azidomethyl uridine,

transfected cells were labelled with 100 μM 5-azidomethyl uridine in the presence or

absence of 10 mM hydroxyurea for 4 hours. To test the effect of RNA synthesis inhibition on

5-AmU labeling, Flp-In Hela cells containing Y65G UCK2 were induced for 24 hours with 1

μg/mL tetracycline. The cells were treated with 100 μM 5-AmU and 100 nM or 2 μM

actinomycin D (Sigma) for 6 hours. The control experiment without any actinomycin

treatment was performed in parallel. To test polymerase dependence of incorporation of 5-

AmU, the induced Flp-in Hela cells were pre-treated with α-amanitin (Sigma, 50 μg/mL) for

9 hours and immediately treated with 100 μM 5-AmU and α-amanitin (50 μg/mL) for 1 hour.

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The control experiment was performed by treating the cells with 100 μM 5-AmU for 1 hour

without any α-amanitin treatment.

Generation of stable cell lines

Flp-In TRex Hela cells were grown in media containing 15 μg/mL blasticidin and 100 μg/mL

zeocin. To generate stable cell lines expressing wild-type UCK2 or mutant UCK2, the Flp-In

Hela cells were seeded at 0.4 × 106 cells per well in six-well plates, and co-transfected with

pOG44 (2 μg), the plasmid expressing flp recombinase, and pCDNA5/FRT/TO plasmid

containing WT UCK2, Y65G UCK2 or Y65A UCK2 gene (0.2 μg). Following selection in 350

μg/mL hydromycin B and 15 μg/mL blasticidin, colonies were expanded. To confirm

expression of the UCK2 gene, cells were grown in the presence or absence of 1 μg/mL

tetracycline for 24 hours. Cells were harvested and lysed in NP-40 lysis buffer. Protein

concentration in the lysate was quantified by BCA assay and lysate containing same

quantities of protein was loaded onto gel for SDS-PAGE. For western blot analysis, the

membrane was blocked in 5% BSA for 1 hour after transfer. The membrane was incubated

in anti-FLAG M2 antibody (Sigma, 1 μg/mL) for 2 hours, washed with TBST three times for

5 minutes, and stained with IRDye-conjugated donkey anti-mouse 680LT antibody (LI-COR

Biosciences, 0.05 μg/mL).

Nucleoside labeling during stress conditions

Flp-In Hela cells expressing Y65G UCK2 were seeded at 0.15 × 106 cells per well in six-

well plates with 12 mm glass coverslips. After induction with 1 μg/mL tetracycline for 24

hours, the cells were labeled with 100 μM 5-AmU or 100 μM 5-EU under stress conditions

for 1 hour. For oxidative stress, cells were treated with 0.5 mM sodium arsenite. For heat

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shock, media containing the nucleosides were prewarmed in 42 °C water bath and added to

the cells. The cells were subsequently cultured at 42 °C for 1 hour.

Pulse chase experiments

Flp-In Hela cells expressing Y65G UCK2 were seeded and induced as described above.

The cells were labeled with 100 μM 5-AmU for 2 hours, washed three times in DPBS, and

incubated in fresh media for various time periods (0-24 h). For EU labeling, wild-type Hela

cells were seeded at 0.3 × 106 cells per well in six-well plates with 12 mm glass coverslips.

After 24 hours, Hela cells were labeled with 1 mM EU for 2 hours, washed three times in

DPBS, and incubated in fresh media for various time periods (0-24 h). To study the effect of

arsenite treatment on the fate of labeled RNA, Flp-in Hela cells expressing Y65G UCK2

were labeled with 100 μM 5-AmU for 2 hours, and wild-type Hela cells were labeled with 1

mM EU for 2 hours. The cells were washed three times in DPBS, and immediately

incubated in media containing 0.5 mM arsenite for 1, 2, 3, or 6 hours.

Cell viability assay

Cells were plated at 1000 cells per well in 96-well plates. After 16 hours, 1 μg/mL

tetracycline was used to induce expression of UCK2 gene. Following 24 hours of induction,

cells were treated with various concentrations of 5-fluorouridine (0.01-65.6 μM) and 1 μg/mL

tetracycline for 72 hours. CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS)

(Promega) was performed according to the manufacturer’s instructions. Absorbance was

measured at 490 nm, and cell viability was calculated as the ratio of absorbance of 5-

fluorouridine treated cells to that of untreated cells. To test cytotoxicity of tetracycline,

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parental Flp-In Hela cells were assayed with various concentrations of 5-fluorouridine (0.37

nM-65.6 μM) in the presence or absence of 1 μg/mL tetracycline.

Fluorescence microscopy

For immunofluorescence and CuAAC imaging, cells were fixed for 20 minutes at RT with

PBS containing 3% paraformaldehyde and 2% sucrose adjusted to pH 7.3 and then washed

3 times for 5 minutes each with PBS. Next, cells were permeablized with PBST (0.1% Triton

X-100 in PBS) for 20 minutes and washed with PBS once. For CuAAC labeling of cellular

RNA, coverslips were incubated upside down with drops (100 μL) of freshly prepared

reaction mixture. The reaction mixture was prepared by combining Cy3-alkyne (5 μM) or

Cy3-azide (5 μM), CuSO4 (1 mM), THPTA ligand (2 mM), and sodium ascorbate (10 mM) in

PBS. The reaction was allowed to proceed for 2 hours at room temperature in the dark.

Cells were washed three times with PBST for 30 minutes each to remove free Cy3 dye. To

stain for 3xFLAG-UCK2, cells were blocked with 5% goat serum in PBST for 1 hour. The

coverslips were incubated with anti-FLAG M2 antibody (Sigma, 0.667 μg/mL) for 2 hours,

and washed with PBST (0.1% Triton X-100 in PBS) 3 times for 5 minutes each. Goat anti-

mouse Alexa 488 antibody (Jackson ImmunoResearch, 1.875 μg/mL) for 1 hour was used

for secondary antibody staining. The cells were washed twice with PBST for 5 minutes,

stained with Hoechst 33342 (Thermo Scientific, 1 μg/mL) for 5 minutes, and washed with

PBS twice for 5 minutes. The coverslips were mounted in ProLong Gold AntiFade Reagent

(Life Technologies) and sealed with nail polish. Images of fixed cells were acquired using

NIS Elements AR software and a Nikon Eclipse Ti microscope equipped with 100x objective

and CMOS camera. Images used for direct comparison were acquired using standardized

illumination and exposure settings and displayed with identical lookup table (LUT) settings.

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For live-cell imaging, U2OS cells transfected with pcDNA5-UCK2-Y65G plasmid were

labeled with 0.5 mM 5-AmU for 2 hr, and then washed 3 times with 1X PBS before reaction

with 1 µM BODIPY-BCN1-2 in HEPES-buffered Tyrode’s solution for 10 min at 37 °C. After 3

more washes with 1X PBS, cells were incubated in HEPES-buffered Tyrode’s solution for

15 min at 37 °C and then directly imaged.

In-gel fluorescence

For gel electrophoresis analysis of labeled RNA, HeLa TRex Flp-In cells containing UCK2

Y65G were induced for 24 hr with 1 µg/mL tetracycline and then treated with 100 µM 5-

AmU, 5-EU, or no nucleoside for 2 hrs. Cells were harvested by scraping in 1X PBS and

total RNA was extracted using Trizol LS (Invitrogen) according to the manufacturer’s

instructions. RNA samples were then reacted with Cy3 fluorophore using CuAAC or SPAAC

conditions. For CuAAC labeling, 10 µg of total RNA was combined in a 20 µL reaction

volume containing 1X PBS, 200 µM Cy3-azide or Cy3-alkyne (Click Chemistry Tools), 0.2

mM CuSO4, 1 mM THPTA, and 1.77 mM sodium ascorbate and incubated for 30 min at

room temperature. For SPAAC, 10 µg of total RNA was combined with 50 µM Cy3-DBCO

(Click Chemistry Tools) and 1X PBS in a 20 µL reaction volume and incubated for 2 hr at

37 °C. All reactions were purified using Zymo RNA clean and concentrator-5 spin columns

according to the manufacturer’s instructions and separated by native gel electrophoresis

using 1% TAE-agarose. Labeled RNA was visualized by in-gel fluorescence on a Typhoon

FLA 9500 Fluorescent Image Analyzer Scanner (GE Healthcare) using a Cy3 filter set. Total

RNA was visualized by staining with EtBr.

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Table S1. ESI-MS of 5-AmU-PO4 generated by UCK2 phosphorylation of 5-AmU.

Calculated [M-H]- (m/z) Detected [M-H]- (m/z)

5-AmU-PO4 (C10H14N5O9P) 378.04564 378.04297

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Supplementary Figure 1. Enzyme progress curves for WT and Y65G UCK2 enzymes
with 5-AmU, 5-EU, and uridine. Reactions containing UCK2 (50 nM), nucleoside (2
mM), and ATP (5 mM) in buffer were incubated at 37 °C for the indicated times and the
production of nucleoside monophosphate was measured by RP-HPLC.

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Supplementary Figure 2. Phosphorylation of 5-AmU by WT and mutant UCK2
enzymes. Reactions containing UCK2, 5-AmU, and ATP in buffer were run for 20 hr at
37 °C and subsequently analyzed by RP-HPLC. The retention times of nucleotide
standards are indicated.

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Supplementary Figure 2 (con’t).

S13
Supplementary Figure 2 (con’t).

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Supplementary Figure 3. Phosphorylation of 5-EU by WT and mutant UCK2 enzymes.
Reactions containing UCK2, 5-EU, and ATP in buffer were run for 20 hr at 37 °C and
subsequently analyzed by RP-HPLC. The retention times of nucleotide standards are
indicated.

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Supplementary Figure 3 (con’t).

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Supplementary Figure 4. Phosphorylation of uridine by WT and mutant UCK2
enzymes. Reactions containing UCK2, uridine, and ATP in buffer were run for 20 hr at
37 °C and subsequently analyzed by RP-HPLC. The retention times of nucleotide
standards are indicated.

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Supplementary Figure 4 (con’t).

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Supplementary Figure 4 (con’t).

S19
Supplementary Figure 5. UCK2 Y65G-mediated incorporation of 5-AmU into U2OS
cells. (a) Workflow for detection of 5-AmU incorporation into cellular RNA. (b) U2OS
cells were transfected with the indicated construct and treated with 100 µM 5-AmU for 2
hr. Click chemistry and microscopy analysis were performed as in Figure 2.

S20
Supplementary Figure 6. Live-cell imaging of 5-AmU labeled RNA. U2OS cells were
transfected with pcDNA5-UCK2-Y65G and subsequently grown in the presence or
absence of 0.5 mM 5-AmU for 2 hrs. Cells were washed, reacted with BODIPY-BCN for
10 min at 37 °C, washed again, and then imaged directly.

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Supplementary Figure 7. Labeling of cells with 5-AmU in the presence of hydroxyurea.
HeLa cells were transfected with UCK2 Y65G and then treated with 100 µM 5-AmU in
the presence or absence of 10 mM hydroxyurea for 4 hrs. Fluorescence microscopy
was performed as described in Figure 2.

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Supplementary Figure 8. Labeling of HeLa cells expressing Y65G UCK2 with 5-AmU
in the presence of the RNA polymerase inhibitor actinomycin D or a-amanitin. (a) Cells
were treated with 100 µM 5-AmU in the presence or absence of the indicated
concentrations of Actinomycin D for 6 hr. Fluorescence microscopy was performed as in
Figure 2. (b) Cells were pretreated with 50 µg/mL a-amanitin for 9 hr and then co-
treated with 100 µM 5-AmU and 50 µg/mL a-amanitin for 1 additional hr. Fluorescence
microscopy was performed as in Figure 2.

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Supplementary Figure 9. Gel electrophoresis analysis of 5-EU and 5-AmU labeled
RNA. Total RNA from cells treated with 5-EU (samples 3 and 4), 5-AmU (samples 5 and
6), or untreated cells (samples 1, 2, and 3) expressing UCK2 Y65G was labeled with a
Cy3 fluorophore using CuAAC or SPAAC conditions and separated on a TAE-agarose
gel. In-gel fluorescence was used to measure Cy3 intensity and EtBr staining to quantify
total RNA.

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Supplementary Figure 10. Western blot analysis of stable HeLa cell lines containing
tetracycline inducible 3xFLAG-UCK2 constructs.

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Supplementary Figure 11. Sensitivity of parent HeLa cells to 5-FUrd in the presence or
absence of tetracycline (1 mg/mL). Cell viability was measured using an MTS-based
assay. Data represent the mean +/- s.d. (n=3).

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Supplementary Figure 12. Labeling of cells with 5-AmU or 5-EU during stress
conditions. HeLa cells expressing Y65G UCK2 were exposed to heat shock (42 °C) or
sodium arsenite (0.5 mM) for 1 hr while being treated with 100 µM 5-EU or 5-AmU.
Fluorescence microscopy was performed as described in Figure 2.

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Supplementary Figure 13. Labeling of HeLa cells with 5-EU (1 mM) during heat shock
or arsenite induced stress. Experiments were performed as in Supplementary Figure 9.

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Martinez, E.; Lee, J. Y.; Hoppmann, C.; Pena-Cabrera, E.; Ha, H. H.; Park, H. S.; Wang,
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