Study of a 1,8-naphtylimide derivative as uridine diphosphate selective probe: Synthesis, optical properties and in?vivo imaging application

Article abstract of DOI:10.1016/j.dyepig.2017.04.006

A 1, 8-naphtylimide-based probe 1 was developed for fluorescence sensing of uridine diphosphate (UDP). Probe 1 selectively recognized UDP in solvant system (DMSO-H₂O) over other structurally similar nucleotides. Among all the tested targets, only the addition of UDP resulted in fluorescence enhancement of about 13-fold. Probe 1, the first receptor moiety without metal center, could recognize UDP only by multi H-bonds formed between the organic ligand and the base group in nucleotide. Moreover, probe 1 exhibited excellent cell permeability. The fluorescence imaging of UDP by probe 1 in Hela cells and Caenorhabditis elegans (C.?elegans) demonstrated its potential as a fluorescent sensor for in?vivo imaging of UDP.

Full text of DOI:10.1016/j.dyepig.2017.04.006

Accepted Manuscript
Study of a 1,8-naphtylimide derivative as uridine diphosphate selective probe:
Synthesis, optical properties and in vivo imaging application
Xue Wei Lv, Guang Ke Wang, Xiao Ju Zou, Xiao Guang Xie, Ying Zhou
PII:
S0143-7208(17)30555-7
10.1016/j.dyepig.2017.04.006
DYPI 5900
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 18 March 2017
Revised Date: 1 April 2017
Accepted Date: 2 April 2017
Please cite this article as: Lv XW, Wang GK, Zou XJ, Xie XG, Zhou Y, Study of a 1,8-naphtylimide
derivative as uridine diphosphate selective probe: Synthesis, optical properties and in vivo imaging
application, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.04.006.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Study of a 1,8-naphtylimide derivative as uridine diphosphate selective probe:
synthesis, optical properties and in vivo imaging application
a, ‡
a, ‡ ,*
a
a
a,*
Xue Wei Lv , Guang Ke Wang
, Xiao Ju Zou , Xiao Guang Xie , Ying Zhou
a
College of Chemical Science and Engineering, Yunnan University, Kunming 650091, China
Corresponding author. e-mail: gkwang@ynu.edu.cn; yingzhou@ynu.edu.cn
‡
These authors contributed equally to this work.
Abstract
A 1, 8-naphtylimide-based probe 1 was developed for fluorescence sensing of uridine diphosphate
(
UDP). Probe 1 selectively recognized UDP in solvant system (DMSO-H O) over other structurally similar
2
nucleotides. Among all the tested targets, only the addition of UDP resulted in fluorescence enhancement
of about 13-fold. Probe 1, the first receptor moiety without metal center, could recognize UDP only by
multi H-bonds formed between the organic ligand and the base group in nucleotide. Moreover, probe 1
exhibited excellent cell permeability. The fluorescence imaging of UDP by probe 1 in Hela cells and
Caenorhabditis elegans (C. elegans) demonstrated its potential as a fluorescent sensor for in vivo imaging
of UDP.
Keywords: Probe, 1,8-naphtylimide, Fluorescence, UDP, in vivo imaging
1. Introduction
Developing efficient methods for sensing biologically important anions has drawn significant attention in
recent years. [1-4] Especially, nucleotides recognition has become an active research area due to their
biological significance. Compared with the well-investigated adenosine-triphosphate (ATP) sensors, [5-9]
sensors for recognizing other nucleotides have rarely been reported. Importantly, other nucleoside
polyphosphates also play pivotal roles in various physiological events. For example, uridine triphosphate
(UTP) and uridine diphosphate (UDP) are widespread in living cells. These nucleoside polyphosphates are
not only the key building blocks for RNA synthesis, but are also active in glycotransfer pathways.[10]

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Compared with the popular conventional detection methods, fluorescent and colorimetric chemosensors
are more convenient and faster in detecting targets. [11-14] However, the design and synthesis of sensors
for selective recognition of one nucleotide among various structurally similar nucleotides and their
derivatives remain a fundamental challenge due to the structural similarities among the base groups in the
nucleotides. Hence, UDP-sensing has rarely been reported. In 2009, Yoon introduced a water-soluble,
“off-on” type fluorescence sensor based on a perylene-bis(2-pyridylmethyl) amine-Zn platform, and this
sensor exhibited high selectivity towards UTP and UDP relative to other phosphate derivatives at
2
+
physiological pH. [15] In 2010, König et al. reported two pyrene labelled Zn -cyclen and
2
+
bis-Zn -bis-cyclen complexes, which showed different selectivity patterns and increased excimer emission
in the presence of UTP, UDP, TDP (deoxythymidine diphosphate), or TTP (thymidine triphosphate). [16]
In 2015, Chen designed a perylene diimide derivative-based zinc complex, which demonstrated high
selectivity for UDP over other phosphor-containing anion under physiological conditions. [17] Literature
2
+
study shows that most of the UDP recognition work used Zn complexes as the coordination sites. Other
kinds of metal complexes and ligands based nucleoside sensors have rarely been reported.
Recently, we have reported a series of fluorescent sensors exhibiting selective colorimetric and
fluorescence changes toward phosphate (Pi) over ATP and PPi [18-22]. Meanwhile, we have also studied
1,8-naphtylimide based sensor for the detection of GMP (guanosine monophosphat), TMP
(deoxythymidine monophosphate) and UMP (uridine monophosphate) and its application in in vivo
imaging. [23] Based on our recent research findings, it is believed that a reasonable design of organic
recognition group in a sensor can lead to anticipative multi H-bonds formation and ensure successful
interaction between the ligand and the base group in the tested nucleotides, without using metal centers.
In this study, a 1,8-naphtylimide derivative 1 was designed and synthesized as the UDP sensor. The
reason behind choosing 1,8-naphtylimide fluorophore for probe 1 was its ability to emit strong fluorescence
in the presence of UDP compared with control conditions. A 2-(propylamino)acetamide moiety was
introduced in 1, anticipating that the moiety would provide good binding sites to form stable H-bonds with

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the base group uracil in UDP. When pro 1 and UDP are organized through appropriate H-bonds links,
b eE P
selectivity for UDP over other nucleotide is expected.
Probe 1 could sense the binding process of UDP, showing distinct fluorescent changes. The optical tests
revealed that compared with the other structurally similar nucleotides, only UDP generated a “turn-on”
fluorescence response of 1 with a fluorescence enhancement of about 13-fold. Multi-hydrogen bonds
formed between the acetamide of 1 and the related uracil group in UDP, thus demonstrating the unique
response of 1 towards PPi among all the tested molecules. In the presence of Hela cells and C. elegans,
obvious blue fluorescence changes were clearly observed, demonstrating the probe’s (1) excellent cell
permeability. This also suggests that it can combine with UDP to trace their enrichment and distribution in
in vivo imaging. Probe 1 is the first 1, 8-naphtylimide derivative with no metal center that can be
successfully applied in UDP bioimaging.
2. Experimental section
2.1. General procedures and materials
Unless otherwise specified, all materials were purchased from commercial suppliers and were used
1
without further purification. Flash chromatography was carried out on silica gel (230-400 mesh). The H
1
3
NMR and C NMR spectra were measured on a Bruker 500 MHz magnetic resonance spectrometer.
Chemical shifts were expressed in ppm and coupling constants (J) in Hz. Mass spectrometry was recorded
with a Agilent LC/MSD TOF mass spectrometer. The UV-Vis spectra were obtained using UV-240 IPC
spectrophotometer. The fluorescence measurements were performed on a F-4500 FL spectrophotometer
with a 1 cm standard quartz cell. Excitation and emission slit widths were 5 nm/5 nm.
2.2. Synthesis of compound 2
Compound 3 was synthesized according to a published method. [24-25] Compound 3 (101 mg, 0.4 mmol)
was dissolved in CH Cl (20 mL) under nitrogen. 4-dimethylamino pyridine (122 mg, 1.0 mmol) was added,
2
2
followed by a dropwise addition of chloroacetyl chloride (40 μL, 0.53 mmol) in 3 mL CH Cl . After stirred
2
2
for 4 h at room temperature, the solvent was removed under reduced pressure to obtain a pale-yellow solid,
which was purified by silica gel column chromatography using dichloromethane as eluent to afford

1
compound 2. Yield: 99 mg (75 %) as
a pale-yellow solid. H NMR (500 MHz, DMSO-d6): δ (ppm) 1.022
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(t, J=12.5 Hz, 3H), 1.786 (m, J=12.5 Hz, 2H), 4.150 (dd, J=12.5, 2.5 Hz, 2H), 4.400 (s, 2H), 7.847 (dd,
J=12.5, 2.5Hz, 1H), 8.217 (d, J=13.5 Hz, 1H), 8.504 (d, J=13.5 Hz, 1H), 8.654 (t, J=13.5 Hz, 2H), 9.152 (s,
-
1
H). ESI MS: calcd for C H ClN O : m/z 329.1, found: m/z 329.1.
1
7
15
2
3
2
.3. Synthesis of compound 1
Compound 1 were prepared by stirring 2 (110 mg, 0.33 mmol) and n-propylamine (0.36 mmol) in
acetonitrile (15 mL), using K CO (48 mg,0.35 mmol) as a catalyst. After 8 h, the solvent was removed
2
3
under reduced pressure to obtain a orange solid. Chromatography of the crude product on silica gel, using
1
CH Cl : MeOH (100:1) as an eluent, resulted in a yellow solid product of 1 (44 mg, 38 %). H NMR (500
2
2
MHz, CDCl ) δ (ppm) 1.01 (t, 3H), 1.03 (t, 3H), 1.67 (m, J=12.0 Hz, 2H), 1.77 (dd, J=12.5, 9.5 Hz, 2H),
3
2
1
1
1
3
.80 (t, J=12.0 Hz, 2H), 3.60 (s, 2H), 4.12 (t, J=12.5 Hz, 2H), 7.75 (t, J=12.5 Hz, 1H), 8.22 (d, J=14.0 Hz,
13
H), 8.58 (dd, J=12.0, 5.0 Hz, 2H), 8.6 (d, J=13.5 Hz, 1H). C NMR (500 MHz, CDCl ) δ (ppm) 11.53,
3
1.66, 21.39, 23.41, 41.91, 52.36, 53.31, 116.74, 117.96, 122.93, 123.05, 125.71, 126.60, 131.05, 132.78,
+
38.53, 145.4, 163.66, 164.21, 170.41. ESI MS: calcd for C H N O : m/z 354.1638, found: m/z
2
0
23
3
3
54.1816.
2.4. UV-Vis and fluorescence emission spectra
-3
The probe 1 stock solution (1×10 mol/L) was prepared in analytical grade DMSO. The concentration of
-5
probe 1 in test solution was 1×10 mol/L. For selectivity studies, various analytes, including ATP, ADP,
AMP, GTP, GDP, GMP, TTP, TDP, UTP, UDP and UMP were prepared in deionized water.
2.5. Culture of Hela cells and fluorescent imaging
Hela cells was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with
1
0 % FBS (fetal bovine serum) in an atmosphere of 5 % CO and 95 % air at 37 ℃. The cells were seeded
2
in 24-well flat-bottomed plates and then incubated for 24 h at 37 ℃ under 5 % CO . In this experiment,
2
some groups of Hela cells were incubated with 250 µM (25 equiv.) of UDP for 2 h. Then the cells were
incubated with 10 µM of 1 for 30 minutes at 37 °C. The cells were imaged using an Olympus IX71 inverted
fluorescence microscopy.

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2
.6. Culture of C. elegans and Fluorescent Imaging
The C. elegans wild type strain N2 was acquired from the Kunming Institute of Zoology. C. elegans in
the larval stage 4 (L4) were incubated at 20 ℃ for 1 h in centrifuge tube filled with M9 buffer, containing
µM or 250 µM of UDP. After incubation, the exposed nematodes were washed three times with M9
0
buffer, and centrifuged at 3000 r/min for 2 minutes. Then the nematodes were incubated at 20 °C for 1 h in
centrifuge tube filled with 1 mL of M9 buffer, containing 10 µM of 1. The pretreated nematodes were
imaged using an Olympus BX51 fluorescence microscopy.
3. Results and discussion
3.1. Synthesis and characterizations
Scheme 1 shows the synthetic route to obtain the probe 1 in moderate yield. Compound 3 was
synthesized according to published methods. [24-25] Following is the brief description of the synthesis of 1:
A solution of 3 (101 mg, 0.4 mmol) and 4-dimethylamino pyridine (122 mg, 1.0 mmol) was prepared, and
then chloroacetyl chloride (40 µL, 0.53 mmol) in 3 mL CH Cl was added dropwise. The reaction mixture
2
2
was stirred at room temperature for 4 h. The solvent was removed under reduced pressure to obtain a
pale-yellow solid, which was purified by column chromatography to get 2 (99 mg, 75%). The crude
product 2 (110 mg, 0.33 mmol) and n-propylamine (0.36 mmol) were taken in acetonitrile (15 mL), and
K CO (48 mg, 0.35 mmol) was added as a catalyst. The reaction mixture was then stirred for 8 h. The
2
3
target orange compound 1 (44 mg, 38%) was obtained after the purification by column chromatography.
for characterization data, see ESI).
(

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Scheme 1. Synthetic route of probe 1.
3.2 UV-Vis absorption and fluorescent emission spectra
To investigate the interaction of probe 1 with nucleotides, the UV-Vis absorption and fluorescence
-5
spectra of 1 in DMSO-H O (V/V = 99:1) were recorded. In the absence of nucleotides, probe 1 (1.0×10
2
M) exhibited a major absorption band centered at 357 nm in DMSO-H O (V/V = 99:1). Fig. S6 showed that
2
the addition of different equivalents of UDP to the solution of probe 1 led to no significant change.
Meanwhile, no obvious spectrum change was observed in the presence of all tested molecules (Fig. S7).
To obtain insight into the binding of nucleotides with 1, the emission changes of 1 with ADP, AMP,
ATP, GDP, GMP, GTP, TDP, TTP, UDP, UMP, UTP were further recorded (Fig. 1). The addition of UDP
to compound 1 generated a significant “turn-on” fluorescence response of the peak at 458 nm,
corresponding to a fluorescent enhancement of up to 13-fold from dark to bright blue (Fig. 2). Even a
3-fold enhancement for UMP and a 2-fold enhancement for UTP were observed. The high selectivity of 1
towards UDP could not be interfered by its analogues. The results revealed that the emission selectivity of
compound 1 towards UDP was higher than that of other above targets that were used to measure the
selectivity of probes 1. Based on these results, further investigations and tests were performed on 1 with
UDP.

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-5
-5
Figure 1. (a) Fluorescence responses of
1 (1.0 ×10 M) to 25 ×10 M of AMP, ADP, ATP, GMP,
GDP, GTP, TDP, TTP, UMP and UTP in DMSO-H O (V/V = 99:1); (b) Fluorescence intensity of
2
-5
1
(1.0 ×10 M) at 458 nm after addition of 25 equiv of selected ions; (c) Fluorescent change of
1 in
presence of different nucleotides.
-5
Figure 2. (a) Fluorescence emission titration spectra of 1 (1.0 ×10 M) in the presence of varying
-5
concentrations of UDP in DMSO-H O (V/V = 99:1); (b) A plot of fluorescence intensity of 1 (1.0 ×10 M)
2
at 458 nm vs. the concentration of UDP; (c) Fluorescent change of 1 in presence of UDP (right).
3.3. The reaction mechanism of compound 1 with UDP
In our previous work, a GMP, TMP and UMP-targeted fluorescent probe was developed for visual
detection as well as for tracing the accumulation of these three nucleotides in C. elegans [23]. In the present
study, we used the similar strategy by introducing an amido link to 1, 8-naphthalimide fluorophore to build
a highly selective sensor for UDP. Multi-hydrogen bonds, formed during the interaction of probe 1 with

AC PTED MANUSCR T
UDP, restrained photoinduced electro nC Et ransfer (PET) of th eI Pfluorophore, leading to fluorescence
enhancement. Figure 3 depicts the proposed reaction mechanism. As expected, when probe 1 interacted
with UDP, significant changes in the fluorescence signal were detected. Our previous study confirmed that
the target three nucleotides XMP (X = G, T, U)) and the binding moiety of the sensor interacted in 1:1
binding mode [23]. In this study, acylamide was used as the substituent group. Acylamide has the similar
steric structural feature and binding sites as the reported XMP sensor [23]; therefore, a 1:1 binding mode
was expected for probe 1 during the design of the molecule. Job’s method was used to determine the
stoichiometry of the formed complex (Fig. S8). Further, theoretical calculations were performed to confirm
the binding patterns of 1 with UDP.
Figure 3. The proposed binding mechanism of 1 with UDP (top) and the calculated structure of 1 binding
with UDP (down).

CE ED MANUSCR T
To describe the binding patternA Ca nd HP T- bond interactions,I Pt he density functional theory (DFT)
(B3LYP/6-31G ⁄ level) [26] was used. The results showed formation of multi-hydrogen bonds between the
N and O atoms, respectively, in amido group of 1 and uracil group of UDP. Each molecule of 1 is
…
…
connected to one molecule of UDP via two O H-N bonds. Distances of O H-N bonds are 2.866 and 2.883
Å in 1-UMP with the bond angle of 172.0° and 176.7°, respectively. All the distances and angles of
…
O H-N in this case are in good agreement with the description of intermolecular hydrogen bonds, thus
strongly supporting the H-bond interactions in present recognition process. In previous work, we calculated
the stable form of UDP and found that two intramolecular hydrogen bonds were formed inside one UDP
molecule. Based on this finding, we introduced propylamine to make the recognition unit more flexible in
probe 1 and examined the properties in DMSO with little water. (DMSO-H OV/V = 99:1). The results
2
showed that the flexible recognition unit, and the nonpolar test condition contributed to the formation of
hydrogen bonds between 1 and UDP.
To obtain further evidence for the binding interactions of 1-UDP, NMR titration studies (Fig. S9) were
carried out. Figure S8 shows that downfield shifts of protons (Hd, He, and Hf) with the addition of varying
concentrations of UDP to 1 (31 mM) in DMSO-d , indicating the hydrogen bond interactions between 1
6
4
-1
and UDP. The corresponding binding constants of 1 with UDP were estimated to be 2.1×10 M (Fig. S10).
The fluorescence intensity of 1 (1 µM) was linearly proportional to the concentrations (0–25 µM) of UDP,
-7
and a detection limits as low as 1.61×10 M concentration of UDP was achieved using 1, with a
signal-to-noise ratio of 3 (Fig. S11).
3.4. Imaging UDP in Hela cells
To demonstrate the feasibility of 1 for its application in vivo imaging, fluorescence imaging tests were
performed in Hela cells. As shown in Fig. 4, no fluorescence was observed in the control test. However, the
blue fluorescence intensity of the probe in the cultured cells increased with increasing UDP, as expected.

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Figure 4. Fluorescent imaging (right) and phase contrast (left) for UDP with probe 1 in Hela cells: (a) and
-5
-5
-4
(
b): probe 1 (1.0 ×10 M) only; (c) and (d): probe 1 (1.0 ×10 M) and UDP (2.5 ×10 M)
3.5 Imaging UDP in C. elegans
In the previous study, we used C. elegans, as the biological model, to assess the distribution of GMP,
TMP, and UMP in the nematode. To check the feasibility of the probe to be used in in vivo imaging, the
present study also used C. elegans, as the biological model, to assess the distribution of UDP visually in
nematode. C. elegans larvae at developmental stage 4 (L4) were used. Fig 5 shows that the addition of UDP
leads to a significant change in the color of the fluorescence from dark to blue, demonstrating the probe’s
ability to trace the enrichment and distribution of UDP in biological specimens. This result also confirms
excellent cell permeability 1. Therefore, the probe 1 is an potential candidate for visual monitoring of UDP
in living cells.

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Figure 5. Fluorescent imaging (right) and phase contrast (left) for UDP with probe 1 in C. elegans: (a) and
-5
-5
-4
(
b) with 1.0 ×10 M of probe 1 only; (c) and (d) with 1.0 ×10 M of probe 1 and 2.5×10 M UDP
4. Conclusions
This study introduced a 1,8-naphtylimide based probe (1) without metal center for UDP detection and
examined the binding between the probe and the target. The study findings have revealed 1 as a new
turn-on” fluorescent probe for highly selective detection of UDP over other structurally similar
“
nucleotides in solvant system (DMSO-H O (V/V = 99:1). Remarkable enhancement of up to 13-fold in
2
fluorescence intensity was observed in the presence of UDP. Probe 1 exhibited excellent cell permeability
and visually detected UDP accumulation in Hela cells and C. elegans through fluorescence imaging. Based
on the above findings, it can be concluded that the probe 1 is a new UDP selective stain with potential to be
applied in fluorescence in vivo imaging.
Acknowledgments
This work was supported by the Natural Science Foundation of China (21462050 and 21672185), the
Foundation of the Department of Science and Technology of Yunnan Province of China (2013HB062,
2016FB020), the Foundation of the Department of Education of Yunnan Province (2015Y012), and the
Program for Excellent Youth Talents, Yunnan University.

Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://
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Supporting Information for
Study of a 1,8-naphtylimide derivative as uridine diphosphateselective
probe: synthesis, optical properties and in vivo imagingapplication
a, ‡
a, ‡,*
a
a
Xue Wei Lv , Guang Ke Wang
, Xiao Ju Zou , Xiao Guang Xie , Ying
a,*
Zhou
a
College of Chemical Science and Engineering, Yunnan University,
Kunming 650091, China
Corresponding author. e-mail: gkwang@ynu.edu.cn;
yingzhou@ynu.edu.cn
‡
These authors contributed equally to this work.

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1
Figure S1. H NMR (DMSO-d6, 500MHz) spectra of 2

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Figure S2. Mass of compound 2.
1
Figure S3. H NMR (CDCl ,500 MHz) spectra of compound 1.
3

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Figure S4. C NMR (CDCl ,500 MHz) spectra of compound 1.
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Figure S5. FAB mass of compound 1.

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Figure S6. Absorbance titration spectra of 1 (10 µM) in the presence of varying
concentrations of UDP in DMSO-H O (V/V = 99:1).
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Figure S7. Absorbance selectivity spectra of 1 (1.0 ×10 M) to 25 ×10 M of ADP,
AMP, ATP, GMP, GDP, GTP, TDP, TTP, UMP and UTP in DMSO-H O (V/V = 99:1).
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Figure S8. The Job Plot using fluorescent intensity at 458 nm of 1 (1×10 M) and UDP
in DMSO-H O (V/V = 99:1). The total concentration of 1 and UDP was 100 μM.
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Figure S9. H NMR titration spectra (500 MHz)of compound 1 (31 mM) with UDP in
DMSO-d6.

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Figure S10. The equation plot of 1 with UDP.The binding constant is 2.1 × 10 M .
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Figure S11. The Change in the fluorescence intensity of 1 (1.0×10 M) at 458 nm
against varied concentrations of UDP from 0 to 25 μM in DMSO-H O (V/V = 99:1).The
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limit of detection is 1.61×10 M.
Reference:
The details for [26]:Gaussian 09, Revision A.02, Frisch MJ, Trucks GW, Schlegel HB,
Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson
GA, Nakatsuji H, Caricato M, Li X, Hratch HP, Izmaylov AF, Bloino J, Zheng G,
Sonnenberg JL, Hada M, Toyota K, Fukda R, Hasegawa J, Ishida M, Nakajima T, Honda
Y, Kitao O, Nakai H, Vreven T, Montgomery JA, Peralta JJE, Ogliaro F, Bearpark M,
Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Norman, J, Raghavachari
K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M,
Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev
O, Austin AJ, Cammi R, Pomelli, C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski
VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkbs O, Foresman
JB, Ortiz JV, Cioslowski J, Fox DJ. Gaussian Inc., Wallingford CT, 2009.

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Highlights
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A 1, 8-naphtylimide-based probe for UDP sensing is studied.
The sensor displays remarkable optical response towards UDP.
The sensor is applied in UDP fluorescence in vivo imaging in Hela cells and C. elegans.