Effect of each guanidinium group on the RNA recognition and cellular uptake of Tat-derived peptides

Article abstract of DOI:10.1016/j.bmc.2014.03.037

The six arginine (Arg) residues in the human immunodeficiency virus transactivator of transcription protein (HIV Tat protein) basic region (residues 47-57) are crucial for two bioactivities: RNA recognition and cellular uptake. Herein, we report a systematic study to investigate the role of the guanidinium group on Arg at each position in Tat-derived peptides for the two bioactivities. Tat-derived peptides, in which each guanidinium-bearing arginine was replaced with a urea-bearing citrulline (Cit) or an ammonium-bearing Lys, were synthesized by solid phase peptide synthesis. RNA recognition of the peptides was studied by electrophoretic mobility shift assays, and cellular uptake into Jurkat cells was determined by flow cytometry. Our results showed that removing the positive charge and altering the hydrogen bonding capacity of Arg affect the two biological functions differently. Furthermore, the effects are position dependent. These findings should be useful for the development of functional molecules containing guanidinium, urea, and ammonium groups for RNA recognition to affect biological processes and for cellular uptake for drug delivery.

Full text of DOI:10.1016/j.bmc.2014.03.037

Accepted Manuscript
Effect of each guanidinium group on the RNA recognition and cellular uptake
of Tat-derived peptides
Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li, Richard
P. Cheng
PII:
S0968-0896(14)00222-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.03.037
BMC 11484
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 February 2014
19 March 2014
21 March 2014
Please cite this article as: Wu, C-H., Weng, M-H., Chang, H-C., Li, J-H., Cheng, R.P., Effect of each guanidinium
group on the RNA recognition and cellular uptake of Tat-derived peptides, Bioorganic & Medicinal Chemistry
(2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.03.037
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Graphical Abstract
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Effect of each guanidinium group on the
RNA recognition and cellular uptake of Tat-
derived peptides
Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li, and Richard P. Cheng*
Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan

Bioorganic & Medicinal Chemistry
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Effect of each guanidinium group on the RNA recognition and cellular uptake of Tat-
derived peptides
Cheng-Hsun Wu, Ming-Huei Weng, Hsien-Chen Chang, Jhe-Hao Li and Richard P. Cheng∗
dDepartment of Chemistry, National Taiwan University, Taipei 10617, Taiwan
ARTICLE INFO
ABSTRACT
Article history:
Received
The six arginine (Arg) residues in the human immunodeficiency virus transactivator of
transcription protein (HIV Tat protein) basic region (residues 47-57) are crucial for two
bioactivities: RNA recognition and cellular uptake. Herein, we report a systematic study to
investigate the role of the guanidinium group on Arg at each position in Tat-derived peptides for
the two bioactivities. Tat-derived peptides, in which each guanidinium-bearing arginine was
replaced with a urea-bearing citrulline (Cit) or an ammonium-bearing Lys, were synthesized by
solid phase peptide synthesis. RNA recognition of the peptides was studied by electrophoretic
mobility shift assays, and cellular uptake into Jurkat cells was determined by flow cytometry.
Our results showed that removing the positive charge and altering the hydrogen bonding
capacity of Arg affect the two biological functions differently. Furthermore, the effects are
position dependent. These findings should be useful for the development of functional molecules
containing guanidinium, urea, and ammonium groups for RNA recognition to affect biological
processes and for cellular uptake for drug delivery.
Received in revised form
Accepted
Available online
Keywords:
Tat-derived peptide
Arginine
Guanidinium group
RNA recognition
Cellular uptake
2009 Elsevier Ltd. All rights reserved.
1. Introduction
β-hairpin and an α-helical structure for the Tat-derived peptide,
respectively.32,
33
Nonetheless, replacing the Arg residues at
The human immunodeficiency virus transactivator of
transcription protein (HIV Tat protein) is essential for HIV
proliferation.1, 2 The Tat protein consists 101 amino acids with a
basic region containing six arginines, which are highly conserved
for RNA recognition3-5 and cell penetration.6-8 This 11-amino
acid basic region of HIV Tat protein, Tat(47-57), specifically
interacts with the bulge region of the transactivator response
element (TAR) RNA.3-5 During HIV transcription, Tat protein
binds TAR RNA to enable the mRNA transcription elongation9
and production of full-length RNA transcripts. As such, the Tat-
TAR interaction is crucial for HIV proliferation,1, 2 serving as a
target for potential development of anti-HIV therapeutics.10-20
The cell penetration capability of Tat enables the initiation of
viral proliferation in infected neighboring cells21 and apoptosis of
nearby healthy immune cells.22, 23 Exploiting this cell penetration
characteristic, Tat-derived peptides have been used as molecular
transporters to access intracellular targets for potential drug
delivery.24-29
positions 52, 53, 55, 56 in HIV Tat-derived peptides with an
uncharged Ala individually reduced the TAR RNA binding
affinity by two fold.30 However, the positive charges on the Tat-
derived peptide should facilitate the binding to the poly-anionic
RNA through electrostatic interactions. Accordingly, attenuation
of the RNA binding affinity in the alanine scanning study30 could
be attributed to just the removal of the positive charge and thus
the corresponding electrostatic interaction. Also, replacing the
Lys residues in Lys9 with Arg suggested that the Arg residues at
positions 52 and 53 may be important for TAR RNA
recognition.34 The importance of the guanidinium side chain of
Arg for cellular uptake has also been explored in homopeptides.35
The homopeptides Lys9, Cit9, and His9 all exhibited significantly
lower cellular uptake compared to Arg9.35 Furthermore,
simultaneously mono- or di-methylating all the guanidinium
groups in Arg8 resulted in significantly reduced cellular uptake
into Jurkat cells,36 suggesting the importance of the hydrogen
bond donating capacity of Arg for cellular uptake. However,
Tat(47-57) is distinctly different from homopeptides such as Arg8,
Arg9, or Lys9.37
The six Arg residues in Tat(47-57) are critical for both TAR
RNA recognition30 and cell penetration.31 The atomic resolution
structure of the HIV Tat-TAR complex remains elusive, even
though the structures of the bovine immunodeficiency virus and
The aforementioned pioneering studies were important for
identifying the crucial Arg positions for RNA binding30, 34 and for
demonstrating the importance of the Arg guanidinium group for
t—he—equ—ine infectious anemia virus Tat-TAR complexes showed a
∗ Corresponding author. Tel.: +886-2-3366-9789; fax: +886-2-3366-8671; e-mail: rpcheng@ntu.edu.tw
1

the cellular uptake. To provide further insight into the role of the
Arg guanidinium groups in Tat(47-57), herein we report the TAR
RNA binding and cellular uptake of Tat-derived peptides in
which each guanidinium-bearing arginine in Tat(47-57) was
replaced with a urea-bearing citrulline (Cit)38 or an ammonium
bearing Lys individually.
concentration of the peptides was determined by UV-vis
spectroscopy.
42-44
2.2. Investigating RNA binding by electrophoretic
mobility shift assays
Electrophoretic mobility shift assays (EMSA) were performed
to determine the binding of the Tat-derived peptide to TAR
RNA.45 The HIV TAR RNA was labeled with fluorescein at the
3’-terminus to enable these experiments. The bands
corresponding to the presence of fluorescein-label RNA were
monitored upon adding varying amounts of Tat-derived peptide
(Figs. S1 and S7). The fraction of RNA bound to peptide was
derived from the ratio of the peptide-RNA complex to the total
RNA. The binding isotherm was fit globally assuming a 1:1
2. Results
2.1. Peptide design and synthesis
The Tat-derived peptides were designed based on the sequence
of wild-type Tat protein residues 47-57 (Fig. 1). The native
Tat(47-57) sequence was capped at both termini to give peptide
ArgTat. To provide further insight into the role of the Arg
guanidinium group in the TAR RNA binding of Tat(47-57), each
Arg residue in ArgTat was replaced with citrulline (Cit) and Lys
individually to give the TatCitN and TatLysN peptides,
respectively (Fig. 1). Although both Arg and Lys are positively
charged, the charge on the guanidinium group of Arg is more
diffuse compared to the charge on the ammonium group of Lys.
Furthermore, the guanidinium group is capable of forming
46
binding stoichiometry using the full quadratic equation39, to
obtain the apparent dissociation constant (KD) (Figs. S2 and S8).
All the peptides bound TAR RNA with sub-micromolar affinity
(Fig. 2 and Table S1). All the apparent KD values for the peptide-
TAR RNA complexes increased upon replacing Arg with Cit
(Fig. 2A). For the RNA binding of the TatLysN peptides, the
apparent KD also increased upon replacing Arg with Lys except
for TatLys57, which exhibited an apparent KD similar to that for
ArgTat (Fig. 2B).
multiple hydrogen bonds in a multidentate fashion, which is
critical for various bioactivities.30,
35
Replacing one of the
terminal nitrogens of Arg with oxygen gives the neutral urea-
bearing citrulline (Fig. 1). The overall shape of the Cit side chain
is similar to the Arg side chain, however the net charge and
hydrogen bonding profiles are different. To enable the detection
of the peptides in cellular uptake experiments, the peptides were
capped with 6-carboxyfluorescein at the N-terminus with an
intervening βAla39 to give Flu-ArgTat, and the Flu-TatCitN and
Flu-TatLysN peptides (Fig. 1). The peptides were synthesized by
solid phase peptide synthesis using Fmoc-based chemistry.40, 41
Upon cleavage with concomitant side chain deprotection, the
peptides were confirmed by MALDI-TOF mass spectroscopy.
All peptides were purified by reverse-phase high performance
liquid chromatography to greater than 95% purity. The
Figure 2. Apparent dissociation constant (KD) for the TAR RNA
complexes with ArgTat, TatCitN and TatLysN peptides determined by
EMSA using 100 nM fluorescein-labeled HIV TAR RNA.
Many negatively charged entities exist in the cell, which may
bind non-specifically to positively charged peptides such as the
Tat-derived peptides. To investigate the specific binding of the
peptides to TAR RNA, EMSA was performed in the presence of
the competing negatively charged poly(dI-dC)47 to obtain the
apparent dissociation constant (KD) with attenuated nonspecific
binding, i. e., specificity (Fig. 3 and Table S1). The KD for the
binding between the peptides and poly(dI-dC) was not
determined because poly(dI-dC) is an alternating copolymer with
a range of molecular weights, multiple non-specific peptide
binding sites, and unknown binding stoichiometry to the peptides.
The binding of ArgTat to TAR RNA was not significantly
affected by the presence of poly(dI-dC) (Figs. 2 and 3; Table S1).
However, the binding affinity of the TatCitN peptides to TAR
RNA was significantly reduced upon adding poly(dI-dC) (Figs.
2A and 3A; Table S1). The apparent KD of the TatCitN-TAR
complexes in the presence of poly(dI-dC) followed the trend:
ArgTat < TatCit57 ~ TatCit56 ~ TatCit52 ~ TatCit49 ≤ TatCit55
~ TatCit53 (Fig 3A), which was the same as the apparent KD
trend in the absence of the non-specific competitor poly(dI-dC)
(Figs. 2A and 3A). However, the binding affinity of TatLysN
peptides in the presence of poly(dI-dC) followed the trend:
ArgTat ~ TatLys57 ~ TatLys52 ~ TatLys49 > TatLys56 ~
TatLys55 > TatLys53 (Fig. 3B) which was different from the
trend in the absence of the non-specific competitor poly(dI-dC)
(Figs. 2B and 3B). Furthermore, the affinity of TatLys55 and
TatLys53 for TAR RNA was significantly decreased compared to
Figure 1. Sequences of Tat(47-57) and Tat-derived peptides.
2

ArgTat (Fig. 3B). Interestingly, the affinity of TatLys57,
TatLys56, TatLys52, and TatLys49 for TAR RNA was not
affected by the presence of poly(dI-dC) (p value > 0.05).
should be particularly important for TAR RNA binding.
2.3. Cellular uptake determined by flow cytometry
Figure 3. Apparent dissociation constant (KD) for the TAR RNA
complexes with ArgTat, TatCitN and TatLysN peptides using 100 nM
fluorescein-labeled HIV TAR RNA in the presence of 10 µg/mL
poly(dI-dC).
Figure 5. Flow cytometry results for cellular uptake of Flu-ArgTat and
Flu-TatCitN Tat peptides into Jurkat cells in the presence of fetal bovine
serum at 37 °C. Mean cellular fluorescence upon incubation with 30 µM
peptide (A) and various peptide concentrations (B) for 15 minutes.
A more stringent non-specific competitor (than poly(dI-dC))
would be the bulk transfer ribonucleic acids (tRNA) from
Escherichia coli.48 This mixture of various tRNAs would contain
various types of RNA secondary structures with some sequence
variation, making the direct determination of the KD for the
complexes between the peptides and such tRNAs impractical.
Nonetheless, the binding between the peptide and TAR RNA in
the presence of bulk tRNA would be more representative of
intracellular conditions and should screen out non-specific
binding more effectively compared to poly(dI-dC). Indeed, the
binding affinity for TAR RNA was attenuated more in the
presence of bulk E.coli tRNA compared to poly(dI-dC) (Figs. 3
and 4; Table S1). Upon replacing Arg with Cit, all the apparent
KD values for the peptide-TAR RNA complexes in the presence
of E.coli tRNA increased significantly (Fig. 4A). Similarly, the
apparent KD for TatLys53 binding to TAR RNA increased
dramatically compared to ArgTat (Fig 4B). However, the
apparent KD of TatLys55 and TatLys52 for binding TAR RNA
was similar to ArgTat (Fig 4B). Surprisingly, the apparent KD for
binding peptides TatLys57, TatLys56, and TatLys49 to TAR
RNA in the presence of tRNA was lower than ArgTat (Fig 4B).
These results showed that replacing Arg with Cit at any position
decreased both the binding affinity and specificity of Tat for
TAR RNA. However, the effect of replacing Arg with Lys in
Tat-derived peptides on Tat-TAR RNA binding affinity and
specificity was position dependent. Since the apparent KD
increased dramatically upon replacing the Arg at position 53 with
either Cit or Lys, the guanidinium group of Arg at this position
Figure 6. Flow cytometry results for cellular uptake into Jurkat cells in
the presence of fetal bovine serum upon incubation with 120 µM Flu-
ArgTat, Flu-TatCitN (A) and Flu-TatLysN (B) peptides for 15 minutes at
37 °C.
Cellular uptake experiments were performed on Jurkat cells,
because these cells are a CD4+ helper T cell cancer cell line, and
CD4+ helper T cells are the target of HIV-1. Jurkat cells were
incubated separately with various concentrations (30, 60, 90, and
120 µM) of Flu-TatCitN for 15 minutes at 37°C in the presence
of fetal bovine serum, and then treated with trypsin to remove
cell-surface bound peptide.7 The amount of peptide uptake into
the cells was quantified by flow cytometry (Figs. 5 and S13-S16).
Only live cells were included in these studies. The fluorescence
intensity increased significantly upon increasing the peptide
concentration. Incubating with 30 µM peptide, all the Flu-
TatCitN peptides exhibited lower uptake compared to Flu-ArgTat
(Fig. 5A). At concentrations higher than 60 µM, peptides Flu-
TatCit52 and Flu-TatCit49 clearly exhibited higher cellular
uptake compared to Flu-ArgTat (p value < 0.05) (Fig. 5B). The
cellular uptake of peptides Flu-TatCit56, Flu-TatCit55, and Flu-
TatCit53 were similar to Flu-ArgTat at concentrations higher
than 60 µM (Fig. 5B). Interestingly, Flu-TatCit57 had the lowest
cellular uptake efficiency regardless of peptide concentration. In
contrast to the range of uptake efficiencies upon replacing Arg
with Cit, the cellular uptake efficiency was not affected upon
replacing Arg with Lys at any of the Arg positions in the Flu-
ArgTat peptide, even up to 120 µM peptide (Fig. 6B). Based on
these results, replacing the Arg at positions 52 and 49 with Cit
increased the cellular uptake of the peptides at higher peptide
concentrations (> 60 µM) and the positive charge on Arg at
position 57 was more important compared to the other positions
for cellular uptake.
Figure 4. Dissociation constant (KD) for the TAR RNA complexes
with ArgTat, TatCitN and TatLysN peptides using 100 nM fluorescein-
labeled HIV TAR RNA in the presence of 10 µg/mL bulk E.coli
tRNA.
3

constituents such as carboxylates, phosphates, and sulfates.36 The
urea group of Cit may also form such bidentate hydrogen bonds
3. Discussion
50
similar to the guanidinium group.38,
However, the cellular
Our studies showed that replacing any of the Arg residues in
the Tat-derived peptide with Cit greatly diminished the affinity
and specificity for binding TAR RNA (Fig. 2A, 3A, and 4A). For
positions 52 and 49, replacing Arg with Cit slightly increased the
cell penetration at high peptide concentration (> 60 µM) (Fig.
5B). In contrast, replacing most of the Arg residues in the Tat-
derived peptide with Lys diminished the affinity for binding TAR
RNA (Fig. 2B), however the binding specificity was position
dependent (Fig. 3B and 4B). For positions 57, 56, and 49,
replacing Arg with Lys increased the specific binding to TAR
RNA (Fig. 4B). Nonetheless, there was minimal effect on cellular
uptake upon replacing the Arg residues with Lys individually
(Fig. 6B).
uptake decreased dramatically at 30 µM for the Flu-TatCitN
peptides compared to ArgTat, demonstrating the importance of
the positive charge on Arg for cellular uptake. Interestingly, the
cellular uptake for two of the Flu-TatCitN peptides was slightly
higher than Flu-ArgTat at 120 µM (Figs. 5B and 6A). Removing
one positive charge would attenuate the electrostatic interaction
between the peptide and the negatively charged moieties on the
membrane during the association step, leading to diminished
cellular uptake. However, the decreased association could be
compensated by increasing the peptide concentration to increase
the amount of cell surface binding, leading to similar overall
cellular uptake efficiency compared to Flu-ArgTat. Furthermore,
the positive charge at position 57 of the Tat-derived peptide was
more sensitive to Cit incorporation compared to the other
positions for cellular uptake. Significantly decreased uptake was
previously shown upon simultaneously replacing all the Arg
residues with Lys in Arg9.35 In contrast, our results on Flu-
TatLysN peptides showed that replacing Arg with Lys
individually has minimal effect for cellular uptake (Fig. 6B). This
result suggests that the effect of the number and form of
hydrogen bond donors on cellular uptake was less significant
compared to removing the positive charge at any of the six
positions of Tat-derived peptide except for position 57. The
results on TAR RNA binding and cellular uptake together
suggest that Lys may be incorporated at positions 49, 56, or 57
(individually) in the Tat basic region without attenuating either
specific binding of TAR RNA or cell penetration. As such, these
three substitutions may be natural mutations in viable HIV Tat
proteins.
The side chain functional group of citrulline is a neutral urea
group, which is
a chemical denaturant like guanidinium
chloride.49 The urea group can form multiple hydrogen bonds
with the base and backbone of nucleic acids.38, 50 In contrast, the
side chain functional group of lysine is a positively charged
ammonium group, which cannot form such a high number of
hydrogen bonds compared to the guanidinium group. The
binding affinity of the Tat-derived peptide for TAR RNA
decreased upon introducing either Cit or Lys (to replace Arg)
(Fig. 2). As such, the positive charge and the ability to form
multiple hydrogen bonds in a multidentate fashion are both
important for TAR RNA binding. Arg contains two primary
amine groups (NH2) and one secondary amine group (NH), each
of which can serve as a hydrogen bond donor. For Cit, oxygen is
introduced at one of the terminal nitrogen positions (of Arg) and
serves as a hydrogen bond acceptor. Accordingly, the difference
in hydrogen bond donor/acceptor profile and charge between Arg
and Cit may give rise to the significantly decreased specific
binding of TAR RNA in the presence of negatively charged
competitors (Fig. 3A and 4A).
4. Conclusions
The guanidinium group on the six Arg residues is important
for TAR RNA recognition and cellular uptake of Tat-derived
peptides. The effect of replacing the guanidinium-bearing Arg
with either the urea-bearing Cit or ammonium-bearing Lys on
these two biological functions were investigated. Our results
showed that removing the positive charge and altering the
hydrogen bonding capacity of Arg affected the two biological
functions differently. Furthermore, the effects were position
dependent. The positive charge of Arg was important for specific
TAR RNA binding. However, for position 53, any change to the
guanidinium group resulted in significant attenuation of the
specific RNA binding. Cellular uptake was not affected
significantly so long as the positive charge was retained.
Interestingly, replacing the positively charged Arg with the
neutral Cit at positions 49 or 52 enhanced the uptake at
concentrations greater than 60 µM. The same replacement at
position 57 resulted in reduced uptake. These findings should be
useful for the development of functional molecules containing
guanidinium, urea, and ammonium groups for RNA recognition
to affect biological processes and for cell penetration for
intracellular drug delivery.
Introducing Lys at positions 57, 52, and 49 resulted in similar
TAR RNA binding specificity compared to ArgTat in the
presence of poly(dI-dC) (Fig. 3B), suggesting that charge is
essential for these positions. However, introducing Lys at
positions 56, 55, and 53 resulted in attenuated binding to TAR
RNA (Fig. 3B). This suggests that the higher hydrogen bonding
capacity of the guanidinium group on Arg at these positions is
more important for interacting with TAR RNA compared to
poly(dI-dC). Introducing Lys at positions 57, 56, and 49 resulted
in higher binding specificity compared to ArgTat in the presence
of bulk E.coli tRNA (Fig 4B). It is possible that ArgTat bound
stronger to tRNA compared to TatLys57, TatLys56, and
TatLys49, because the guanidinium group has more hydrogen
bond donors than the ammonium group for binding. This would
lower the specificity of ArgTat for binding TAR RNA, resulting
in the relative higher binding specificity for Tat-derived peptide
with Lys at positions 57, 56, and 49. Furthermore, the binding
specificity of the Tat-derived peptide for TAR RNA diminished
considerably upon introducing Cit or Lys individually at position
53, suggesting that the guanidinium group at position 53 is
essential for the specific recognition of TAR RNA by Tat,
consistent with the Ala scanning30 and Lys9 replacement
studies.34
5. Experimental section
5.1. Peptide synthesis
The first step for the cellular uptake of guanidinium rich
transporters, including the Tat-derived peptide, is the association
of the positively charged guanidinium groups with the negatively
charged entities on the membrane.51 The positively charged
guanidinium group features a rigidly planar hydrogen bond
donors which can form a bidentate hydrogen bond with
negatively charged functional groups on the membrane
The peptides were synthesized by solid phase peptide
synthesis using Fmoc-based chemistry.40, 41 After cleavage, the
peptides were purified by reverse phase HPLC and confirmed by
MALDI-TOF MS.
5.2. Electrophoretic mobility shift assay (EMSA)
4

The fluorescein-labeled RNA (100 nM) and peptide (at
various concentrations) were incubated in pH 7.4 buffer (10 µL)
containing Tris-HCl (50 mM), KCl (50 mM), 2% glycerol, and
Triton X-100 (0.05%) in the presence of poly(dI-dC) (10 µg/mL)
or bulk E.coli tRNA (10 µg/mL) at room temperature. The
samples were analyzed by loading into 12% native
polyacrylamide gels in 0.5X TB buffer and electrophoresis was
performed with 140 V at room temperature. Bands corresponding
to the free and bound RNA were used to determine to fraction
bound RNA. The fraction bound RNA data was used to globally
derive the apparent dissociation constants assuming a 1:1 binding
stoichiometry39, 46 using the full quadratic equation (see SI).
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Jurkat cells (8 x 105) were incubated with the peptides at
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5% CO2 for 15 minutes. The cells were then incubated with 0.05
% trypsin/EDTA in PBS for 5 minutes to remove the peptides
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Acknowledgments
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This work was supported by National Taiwan University
(NTU-ERP-103R891302 and NTU-CESRP-103R7621) and the
National Science Council in Taiwan (NSC-99-2113-M-002-002-
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Supplementary data
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Supplementary data associated with this article can be found,
in the online version, at
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