Fluorescence Detection of KRAS2 mRNA Hybridization in Lung Cancer Cells with PNA-Peptides Containing an Internal Thiazole Orange

Article abstract of DOI:10.1021/bc500304m

We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5′ end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5′ terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5-6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ～10 °C higher T_m for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA. (Figure Presented).

Full text of DOI:10.1021/bc500304m

Article
pubs.acs.org/bc
Open Access on 08/19/2015
Fluorescence Detection of KRAS2 mRNA Hybridization in Lung
Cancer Cells with PNA-Peptides Containing an Internal Thiazole
Orange
Mahesh V. Sonar,† Matthew E. Wampole,† Yuan-Yuan Jin,† Chang-Po Chen,†,‡ Mathew L. Thakur,§,∥
and Eric Wickstrom*,†,∥
§
∥
†Biochemistry & Molecular Biology, Radiology, and Kimmel Cancer Center Thomas Jefferson University, Philadelphia,
Pennsylvania 19107, United States
‡School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, China
S
* Supporting Information
ABSTRACT: We previously developed reporter-peptide nucleic acid (PNA)-peptides for
sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and
tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be
desirable. Thiazole orange (TO) dye at the 5′ end of a hybridization agent shows a strong increase
in fluorescence quantum yield when stacked upon a 5′ terminal base pair, in solution and in cells.
We hypothesized that hybridization agents with an internal TO could distinguish a single base
mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO
adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our
molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting
from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents
with an internal TO showed low fluorescence, but fluorescence escalated 5−6-fold upon
hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for
fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA.
Fluorescence measurements of treated human lung cancer cells similarly showed elevated
cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents.
Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of
detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the
corresponding mutant mRNA.
result in proteins with altered function.7 An example of this
INTRODUCTION
■
phenomenon is oncogene activation in cancers.7 Nucleic acid
Lung cancer kills more people than any other form of cancer.
probes that are designed to target oncogenes are usually only
Cancer cell growth depends on high expression of cancer
genes.1 As lung cancer progresses, more and more characteristic
capable of recognizing one distinct sequence. In the case where
several different single base substitutions occur in one location
on a particular gene, single mismatches can lead to severe
reduction in oligonucleotide targeting efficacy.
cancer genes turn on in transformed lung cells, driving
malignant cancer cell growth, such as EGFR and KRAS2.2
Overexpression of epithelial growth factor receptor (EGFR)
stimulates lung cancer cell growth. However, 70% of lung
cancer patients fail to respond to tyrosine kinase inhibitors
(TKIs) directed against EGFR.3 Similarly, 76% of lung cancer
patients fail to respond to anti-EGFR antibodies.4 KRAS2
mutation, particularly in the 12th codon, makes cancer cell
proliferation through the Ras-Raf-MEK pathway independent
of EGFR activity.2 As a result, treating lung cancer with anti-
EGFR antibodies or TKIs fails when KRAS2 has been
mutated.5 PCR of biopsy tissue is the current gold standard
for determination of KRAS2 mutation.6 A noninvasive imaging
method would reduce patient discomfort and stress, as well as
reduce healthcare costs.
Noninvasive radioimaging of gene expression by detection of
specific mutant or wild-type mRNAs in cells to diagnose cancer
and other diseases in living systems has been achieved with
antisense oligonucleotides.8 Specific imaging of target gene
mRNA requires multiple steps: probe permeation into tissue,
endocytosis into the cells, probe hybridization with the target
mRNA, probe:mRNA accumulation in the specific cells, and
effluxing of unbound probes. Nonhybridized probes must efflux
from cells with little or no expression of the target mRNA in
order to permit a specific image in the targeted cells.
Single base mutations in DNA occur frequently in nature and
variations in the genome can affect the development and
progression of certain diseases. Mutations in specific genes can
Received: July 10, 2014
Revised: August 14, 2014
Published: August 19, 2014
© 2014 American Chemical Society
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Peptide nucleic acids (PNA) are a useful nuclease-resistant
DNA derivative for the purpose of mRNA imaging. PNA
includes a N-(2-aminoethyl)glycine backbone with DNA bases
attached to the α-N.9 With such a structure, the bases are
spaced appropriately to hybridize with DNA/RNA via
Watson−Crick hydrogen bonding.10 PNA is electrically neutral,
resulting in rapid and stronger hybridization compared to
DNA:DNA or DNA:RNA duplexes. The stability of PNAs in
biological fluids and their low cytotoxicity make them attractive
for use in human diagnostic and therapeutic applications.11
Previously, we pioneered reporter-peptide nucleic acid
(PNA)-spacer-insulin-like growth factor 1 (IGF1) tetrapeptides
to enable IGF1R-mediated cellular uptake,8 followed by single-
mismatch specific PET imaging of CCND1 mRNA12 and HER2
mRNA13 in breast cancer xenografts, and mutant KRAS2
mRNA in pancreatic cancer xenografts14 and spontaneous
transgenic lung cancer.15 Mismatches lowered tumor signals to
background. Incubation with recombinant IGF1 also lowered
tumor signals to background.12 But a direct test for PNA
hybridization with RNA in the cytoplasm is desirable.
distinguish a single base mutation in RNA. Thus, we designed
KRAS2 PNA-peptides with an internal TO adjacent to the
middle base of the 12th codon, a frequent site of cancer-
initiating mutations, such as G12D (Table 1; Figure 2). We
further hypothesized that the introduction of a separate thiazole
orange PNA residue after the initial N-GCCA of the original
12mer would provide greater stability than the 12mer with TO
substituting for A. Hence, 13mer PNA-IGF1 tetrapeptides were
also designed (Table 1).
One of the primary requirements for an oligonucleotide
analogue to be successful as an antigene/antisense agent is for it
to be taken up by the cells in reasonable quantity so that it can
reach its target in sufficient concentration. Since PNAs suffer
from poor cellular uptake they have not been well developed as
an antigene/antisense therapeutic agents.28,29 To improve
cellular uptake, PNAs have been conjugated with cell-
penetrating positively charged peptides with homology to
nuclear localization sequences such as transportan, penetratin,
and TAR-binding peptides,30 or the SV40 nuclear localization
sequence itself.31,32
Seitz and co-workers utilize thiazole orange (TO) dye as a
surrogate base for detection of DNA/RNA hybridization in
cells.16−19 Free thiazole orange has a low fluorescence quantum
yield, but intercalation into DNA induces a high quantum
yield.20 The single-stranded TO probe shows low fluorescence
because the lack of planarity around the central methine bridge
minimizes delocalization and directs the excited state to
nonfluorescent decay modes. Upon TO stacking with
neighboring nucleobases in the formed probe-target duplex,
however, the TO rings adopt a planar conformation, favoring
delocalization and maximizing fluorescence quantum yield.21
TO-PNA fluorescence enhancement occurs when TO at one
end of a hybridization agent is stacked upon a matched base
pair. This has facilitated applications in real-time PCR
genotyping and RNA detection.22 In live cells, TO-PNA probes
have been used to detect H1N1 mRNA,18 miRNA-122,23 and
mutant KRAS2 mRNA.24
To apply a rigorous RNA hybridization test to our
fluorophore-PNA-IGF1 tetrapeptide agents that enter cells by
receptor-mediated endocytosis,25,26 traffic to the cytoplasm via
early endosomes,27 and accumulate in cells with single
mismatch specificity,27 we designed, modeled, synthesized,
and tested a variety of KRAS2 TO-PNA-IGF1 tetrapeptide
agents with the TO embedded within the PNA sequence,
adjacent to the site of mutation (Figure 1).
Cell penetration in these cases is not receptor-dependent and
thus not cell-specific. To elevate cellular uptake of PNA by
cancer cells that overexpress insulin-like growth factor receptor
(IGF1R), we introduced a disulfide-cyclized D-peptide IGF1
analogue, D-(CysSerLysCys),33 at the C-terminus. The
introduction of disulfide bridges into peptides allows the
creation of conformational constraints that can improve the
recognition between a ligand and its receptor, therefore
improving biological activity.34 The flexible, hydrophilic
aminoethoxyethoxyacetic acid (AEEA) spacer was introduced
between the IGF1 tetrapeptide ligand and the PNA to reduce
steric hindrance.8
Molecular Dynamics Predictions of PNA:RNA Duplex
Stabilities. Prior to initiating chemical synthesis, the energetic
effects of including a TO PNA residue in the PNA:RNA duplex
were measured computationally (Figure 3). The MM-PBSA
method was used to calculate the free energy of the PNA:RNA
duplexes from the molecular dynamics simulations. We used
normal mode approximations to calculate the entropy change
of each trajectory. Typically nonlinear solvation methods can
lead to overestimation of the free energy changes, complicating
the calculation of absolute free energy changes.35 However, this
overestimation does not impede the determination of relative
free energies.36 MM-PBSA calculations were done on all the
duplexes using the last 8 ns of each simulation (Supporting
Information Table 2). Figure 3 illustrates the predicted
destabilization induced by a mismatch just adjacent to the
TO residue in a PNA:RNA duplex.
RESULTS
■
Design of TO-PNA-Peptide Hybridization Agents. We
previously found that 12 PNA residues provided sufficient
hybridization strength and specificity for KRAS2 mRNA PET
imaging in tumors in mice with single mismatch specificity.14
We hypothesized that PNA 12mers with an internal TO could
The theoretical Tm’s were calculated from the MM-PBSA
results in order to compare them with the experimental Tm’s
from CD temperature ramps (see below). Previously we found
that using aMD to increase sampling efficiency was effective in
improving the theoretical Tm’s.37 After 25 ns of cMD were
carried out, an additional 50 ns of aMD for each duplex was
done. Post-processing of the binding energies was done using
MM-PBSA over the entire aMD run.
Synthesis and cCharacterization of TO-PNA-Peptides.
The syntheses of 12 mer/13mer TO-PNA-peptide chimeras
(Table 1) were carried out (Scheme 1) by Fmoc solid phase
peptide synthesis on TG Sieber resin on a Protein
Technologies PS3 synthesizer at the 10 μmol scale. Cys-Cys
cyclization was executed with 10 equiv of I2 in Me2NCHO for 4
h at room temperature before final cleavage.38 The PNA-
Figure 1. Schematic of KRAS2 PNA with internal TO hybridizing with
KRAS2 RNA, resulting in TO intercalation.
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Table 1
Figure 2. Structure of TO1, KRAS2 PNA-IGF1 tetrapeptide with internal TO specific for KRAS2 G12D mRNA.
peptide chimeras after cleavage from the resin were purified by
preparative HPLC and characterized by MALDI-TOF
(Supporting Information). The purity of TO-PNA-peptide
chimeras was >95% (Supporting Information).
Thermodynamics of TO-PNA-Peptide:RNA Duplexes
Measured by CD Temperature Ramps. CD spectra were
obtained for TO-PNA-peptide:RNA duplexes in a JASCO J-
810 spectropolarimeter with Peltier temperature control.37,39
Spectra were recorded over 320−220 nm at 25 °C. The CD
spectra were recorded by using fully complementary RNA
(Figure 4A) versus single base mismatch RNA (Figure 4B). CD
spectra revealed that all TO-PNA-peptide:RNA duplex spectra
showed a negative absorbance band around 290 nm and a
strong positive CD signal around 265 nm (Figure 4) which is a
distinct hallmark feature of A-like helices.40
Figure 3. MM-PBSA calculated structure of TO1, KRAS2 G12D TO-
PNA hybridized to KRAS2 G12WT RNA in 0.1 M NaCl, pH 7.0. The
TO residue was predicted to flip out of the helix at the site of
PNA:RNA mismatch. 50 ns of simulated motion of KRAS2 G12D TO-
PNA hybridized to KRAS2 G12WT RNA are shown in an mpg file
(Supporting Information).
In the case of activated KRAS2 G12D mRNA overexpressed
in multiple cancers, the Tm of the complementary KRAS2
G12D TO-PNA-peptide 12mer with a KRAS2 G12D RNA
20mer was 80
2 °C, independent of the peptide ligand
Scheme 1
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Figure 4. CD spectra of TO-PNA-peptide:RNA duplexes. 320−220 nm scans of annealed 1 μM TO-PNA-peptide:1 μM RNA duplexes in 100 mM
Na2HPO4, 1.0 M NaCl, 5.0 mM EDTA, pH 7.2, were measured at 25 °C. (A) TO-PNA-peptide with fully complementary RNA. (B) TO-PNA-
peptide with single mismatch RNA.
Figure 5. CD thermal denaturation plots of normalized ellipticity at 265 nm versus temperature at pH 7.2 for TO-PNA-peptide:RNA duplexes (fully
complementary versus single mismatch) (A, C, E). Sigmoid decreases correlate with a duplex to single strand transition upon melting. First derivative
plots determined Tm, the midpoint of the duplex to single strand transition (B, D, F).
sequence,14 following the typical behavior of PNA:RNA
duplexes.38 The CD melting studies for TO-PNA-peptides
were carried out with fully complementary as well as single
mismatch RNA (Figure 5 and Table 2).
°C.14 The Tm for TO2 (13mer) KRAS2 G12D duplex was
70.14 0.12 °C, which is 2 °C less than Tm for TO1 (12mer)
KRAS2 G12D duplex.
Figure 6 shows the average CD Tm values for fully
complementary versus single mismatch TO-PNA-peptide:RNA
duplexes. The observed average Tm for fully complementary
The Tm for TO1 (12mer) KRAS2 G12D duplex was 72.27
0.25 °C, 8 °C less than the Tm of the TO-free duplex, 80
2
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1.6 °C. A single mismatch, corresponding to the difference
between the KRAS2 G12D sequence, vs KRAS2 G12WT or
Table 2. Melting Temperatures of TO-PNA-Peptide:RNA
Duplexes
a
KRAS2 G12 V sequences, lowered the Tm by 9.6
2.3 °C.
complementary TO-PNA-
peptide:RNA duplex
single mismatch TO-PNA-
peptide:RNA duplex
These values revealed that introduction of TO into the PNA
backbone slightly destabilized TO-PNA-peptide:RNA duplexes,
with a significant difference between fully complementary
versus single mismatch sequences.
duplex
Tm (°C)
duplex
Tm (°C)
TO1 (12mer):
KRAS2 G12D
72.27 0.25 TO1 (12mer):
64.30 0.06
63.4 0.47
60.10 0.41
63.12 0.36
61.1 0.56
62.2 0.36
KRAS2 G12WT
To test the validity of molecular dynamics predictions of TO-
PNA-peptide:RNA stability, we compared n = 6 calculated Tm
values vs measured CD Tm values (Figure 7). For the 12mer
TO-PNA-peptides, good agreement (r2 = 0.87) between the
measured CD Tm and the calculated Tm was obtained. For the
13mer TO-PNA-peptides, the correlation between the
experimental and calculated Tm was poor (r2 = 0.46). This
poor correlation could be derived from limitations in the MM-
PBSA approximations, further sampling inefficiencies, or the
accuracy of the force fields. Apparently, the strain created in the
RNA backbone to accommodate the intercalated TO was not
reflected in the molecular dynamics calculations.
TO2 (13mer):
KRAS2 G12D
70.14 0.12 TO2 (13mer):
KRAS2 G12WT
TO3 (12mer):
KRAS2 G12WT
73.23 0.25 TO3 (12mer):
KRAS2 G12D
TO4 (13mer):
KRAS2 G12WT
72.33 0.12 TO4 (13mer):
KRAS2 G12D
TO5 (12mer):
KRAS2 G12V
72.3 0.25 TO5 (12mer):
KRAS2 G12D
TO6 (13mer):
KRAS2 G12V
71.7 0.21 TO6 (13mer):
KRAS2 G12D
a
CD thermal ramps from 25 to 95 °C were monitored at the peak
wavelength, 265 nm. Tm of each duplex was determined from first
derivative peaks
standard deviation from n = 3 independent
measurements.
Fluorescence Measurements of Hybridization of TO-
PNA-Peptides with Synthetic RNA. We tested the ability of
12mer and 13mer TO-PNA-peptides with internal TO residues
to hybridize in solution and fluoresce in the presence of
complementary KRAS2 G12D RNA versus single mismatch
KRAS2 G12WT RNA 20mers (Table 1). Synthetic RNAs were
hybridized with TO-PNA-peptides at 37 °C and the resulting
fluorescence intensity was compared between match and
mismatch duplexes for 12mer and 13mer TO-PNA-peptides
(Figure 8). TO-PNA-peptides alone in buffer only showed
background fluorescence intensity.
In the presence of fully complementary KRAS2 G12D RNA,
the G12D TO-PNA-peptide 12mer showed a significant 7-fold
increase in fluorescence intensity compared to the TO-PNA-
peptide alone (Figure 8A). With the single mismatch KRAS2
G12WT RNA, the G12D TO-PNA-peptide 12mer showed
only a 4-fold increase in fluorescence intensity (Figure 8A).
Similar results were observed for G12WT TO-PNA-peptide
12mer. In the presence of fully complementary KRAS2 G12WT
RNA, the G12WT TO-PNA-peptide 12mer showed a
significant 5-fold increase in fluorescence intensity compared
to the TO-PNA-peptide alone (Figure 8B). With the single
mismatch KRAS2 G12D RNA, the G12WT TO-PNA-peptide
Figure 6. Average CD melting temperatures of TO-PNA-peptide:RNA
duplexes from 25 to 95 °C were monitored at the peak wavelength 265
nm. (Violet) 12mer TO-PNA-peptide with fully complementary RNA,
(Red) 12mer TO-PNA-peptide with single mismatch RNA, (Blue)
13mer TO-PNA-peptide with fully complementary RNA, (Magenta)
13mer TO-PNA-peptide with single mismatch RNA.
TO-PNA-peptide:RNA duplexes was 72.0 1.0 °C, while for
single mismatch TO-PNA-peptide:RNA duplexes, Tm was 62.4
Figure 7. Correlation between calculated and experimental Tm. Calculated Tm from MM-PBSA for the 12mer TO-PNA-peptides (A) and 13mer
TO-PNA-peptides (B) hybridized with RNA 12mers were compared with experimental Tm from CD. N = 6 values were then correlated for the
12mer (r2 = 0.87) and 13mer (r2 = 0.46).
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Figure 8. In vitro hybridization of 50 nM 12mer or 13mer TO-PNA-peptides with 50 nM synthetic fully complementary RNA (blue) or single
mismatch RNA (red) or with no RNA (green) at 37 °C. (A) G12D TO-PNA-peptide. (B) G12WT TO-PNA-peptide. Mean values are shown for n
= 3 independent measurements SD. ** p < 0.01, *** p < 0.005.
12mer showed only a 3-fold increase in fluorescence intensity
(Figure 8B).
PNA-peptides per cell, relative to the thousands of copies of
overexpressed oncogene mRNAs in cancer cells.26 Excess
unbound PNA-peptides efflux from cells, resulting in a dynamic
equilibrium of hybridized PNA-peptides accumulated in treated
cells after suitable incubation time.27
However, G12D TO-PNA-peptide 13mers and G12WT TO-
PNA-peptide 13mers showed no significant discrimination
when hybridized with match versus single mismatch KRAS2
RNA (Figure 8). The above results indicate that as candidates
for fluorescence hybridization probes, TO-PNA-peptide
12mers are more promising than TO-PNA-peptide 13mers,
since 12mers can detect RNAs with single mismatch sensitivity
while 13mers fail. Comparable results for 5′-terminal TO-
DNA/TO-PNA:DNA were reported earlier.16
Confocal Fluorescence Microscopy of Hybridization
of TO-PNA-Peptides with KRAS2 mRNA in Human Lung
Cancer Cells. 200 nM G12D and WT TO-PNA-peptide
12mers were introduced into the culture medium of SK-LU-1
KRAS2 G12D mutant lung cancer cells and H460 KRAS G12
wild type lung cancer cells. The cells were incubated 4 h at 37
°C. SK-LU-141 and H46042 cells both overexpress IGF1R. We
previously observed that receptor-mediated endocytosis under
these conditions results in the internalization of millions of
Confocal fluorescence imaging of fixed cells showed that SK-
LU-1 cells treated with G12D TO-PNA-peptide showed higher
cytoplasmic fluorescence intensity vs G12WT TO-PNA-
peptide, while in H460 cells, G12D TO-PNA-peptide showed
negligible cytoplasmic fluorescence signal compared to G12WT
TO-PNA-peptide (Figure 9A). We have previously observed
comparable cytoplasmic accumulation in both fixed25−27 and
live27 cells, over the concentration range 4 nM − 1 μM.
Therefore, TO-PNA-peptide 12mers detected complementary
RNA targets in cells with single mismatch discrimination.
Quantification of corrected total cell fluorescence (CTCF) of n
= 9 confocal cell images agreed with the above observation that
in two different cell lines, TO-PNA-peptides with matched
sequences displayed higher CTCF compared to TO-PNA-
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Figure 9. Confocal fluorescent microscopy of KRAS2 G12D SK-LU-1 and KRAS2 G12WT H460 human lung cancer cells incubated with 200 nM
TO-PNA-peptide 12mers after 4 h at 37 °C. (A) Fluorescent images recorded after TO excitation at 488 nm. Emission was observed at 535/40 nm.
(B) Quantification of corrected total cell fluorescence (CTCF) intensity in SK-LU-1 and H460 cells treated with 200 nM G12D TO-PNA-peptide or
G12WT TO-PNA-peptide 12mers. N = 9 cells were selected, three from each of three separate images. Fluorescence intensities were quantified using
ImageJ. Fluorescence values were expressed as integrated density after background subtraction SE. * p < 0.05, *** p < 0.00001 (Student’s t test).
peptides with one mismatched base (Figure 9B), in agreement
with the lower stability of mismatched duplexes (Figure 6).
13mers, however, displayed no significant discrimination.
The indistinguishable cytoplasmic signals agreed with the
indistinguishable solution fluorescence results.
TO-PNA:RNA duplexes versus single mismatch duplexes. We
observed that the single-stranded PNA-peptide 12mer agent
with an internal TO residue in place of adenine 4 showed low
fluorescence, but when it hybridized with KRAS2 RNA,
fluorescence escalated 5−6-fold at 37 °C. We ascribe the
reduced fluorescence in the presence of a mismatched KRAS2
RNA to partial dissociation of the mismatched duplex at 37 °C.
However, the PNA-peptide 13mers with TO inserted
between adenine 4 and thymine 5 showed insufficient mutant
discrimination upon RNA binding. Perhaps intercalation of TO
upon hybridization of the 13mer to KRAS2 RNA created excess
strain on the ribose-phosphodiester backbone, limiting the
achievable fluorescence quantum yield upon hybridization to
DISCUSSION
■
We designed and modeled a variety of internally intercalating
TO-PNA-IGF1 tetrapeptide agents that hybridized with KRAS2
RNA. Molecular dynamics calculations predicted slightly
reduced thermal stability compared with normal base PNAs.
In agreement with our molecular dynamics calculations, large
CD Tm differences were observed between fully complementary
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RNA. Apparently, less strain was experienced by the RNA upon
hybridizing with the 12mer, which required the displaced uracil
to flip out of the PNA:RNA helix.
Production runs were performed using a canonical ensemble
(NVT) scheme. Langevin dynamics with a collision frequency
of 2.0 were used for temperature regulation, (ii) the SHAKE
algorithm was used for all hydrogen atoms, and (iii) the particle
mesh Ewald method was employed to treat long-range
electrostatics and van der Waals forces (cutoff of 8 Å) with
an integration step of 2.0 fs. All Amber 12 equilibration and
production runs were performed using double precision.45 All
production simulations were repeated in triplicate with random
seeding for initial velocities and extended to 25 ns. Structural
features were determined using the Curves+ software pack-
age,46 while visualization of trajectories was performed in
VMD.47
Accelerated Molecular Dynamics Simulations. Accel-
erated MD (aMD) is one method to increase the sampling of a
molecular system to overcome the computational cost of long
time scales. aMD modifies the system’s original potential
energy surface (V(r)) by adding a boost potential (ΔV(r))
when V(r) falls below a certain threshold energy (E).48 The
boost potential is defined as
Fluorescence measurements of treated human lung cancer
cells similarly showed elevated cytoplasmic accumulation of
fully complementary vs single base mismatch 12mer agents. We
ascribe the reduced cytoplasmic fluorescence in the presence of
a mismatched KRAS2 TO-PNA-peptide to partial dissociation
of the mismatched duplex at 37 °C. Sequence-specific elevation
of TO fluorescence is consistent with the model of cytoplasmic
TO-PNA-peptide:RNA hybridization. These studies gave direct
evidence that PNA conjugated with an IGF1 receptor peptide
trafficks to the cytoplasm and hybridizes specifically with a
target mRNA.
For reliable clinical application of PNA-peptide agents with
internal TO residues, more study is needed on the mechanism
of endosome trafficking, release of agents in the cytoplasm, and
efflux of unbound agent from cells. Brighter TO derivatives are
desirable, ideally a near-infrared analogue, with an even greater
reduction in quantum yield adjacent to a mismatch in the RNA.
⎧
⎪
⎨
⎪
⎩
0
V(r) ≥ E
CONCLUSION
■
(E − V(r))2
α + (E − V(r))
ΔV(r) =
We observed that single-stranded PNA-IGF1 tetrapeptide
12mers with an internal TO showed low fluorescence, but
fluorescence escalated 5−6-fold upon hybridization with
KRAS2 RNA. Circular dichroism melting curves showed ∼10
°C higher Tm for fully complementary vs single base mismatch
TO-PNA agent duplexes with KRAS2 RNA. Fluorescence
measurements of treated human lung cancer cells similarly
showed elevated cytoplasmic fluorescence intensity with fully
complementary vs single base mismatch 12mer TO-PNA
agents, in agreement with the lower Tm of a mismatched
duplex. Sequence-specific elevation of internal TO fluorescence
is consistent with our hypothesis of detecting cytoplasmic
PNA:RNA hybridization if a mutant agent encounters the
corresponding mutant mRNA.
V(r) < E
where α modulates the depth and roughness of the energy
basins on the modified potential. In order to enhance sampling
of internal and diffusive degrees of freedom, a dual boosting
potential was applied to the backbone torsion angles and the
overall boost potential as
V(r) = V (r) + V(r)
0
t
*
V (r) = {V (r) + [V(r) + ΔV(r)]} + ΔVT(r)
0
t
t
where Vt(r) is the total potential of the torsional terms, Vt(r)
and ΔVT(r) are the boost potentials applied to the torsional
terms, and the total potential energy, respectively. The
parameters were set as follows: Et = 1.2{⟨ΔVt(r)⟩}, αt = 0.20
kcal/mol, ET = 0.2 kcal/mol·number atoms, plus the ensemble
averaged potential energy from the conventional MD
simulations, and αT = 0.2 kcal/mol·number atoms.49,50 aMD
runs simulated 50 ns.
MM-PBSA Calculations. The binding energies for each
RNA:PNA duplex were calculated using the molecular
mechanics-Poisson−Boltzmann surface area (MM-PBSA)
method in Amber 12.50,51 The MM-PBSA method calculates
the binding free energy by the free energies of solvation for the
complex (ΔGduplex), PNA (ΔGPNA), and RNA (ΔGRNA).
METHODS
■
Molecular Dynamics Simulations. System equilibration
and production MD simulations were performed using the
Amber 12 suite of programs.43 The LeaP module of Amber 12
was used to create parameter and topology files for the MD
simulations using the ff99SB force field.43,44 PNA force fields
were created in our earlier study of hypoxanthine-containing
wobble base PNAs.37 RNA molecules were set to predicted
protonation states at pH 7.0. Na+ and Cl− counterions were
added to each system to achieve neutrality and a salt
concentration of 0.1 M. TIP3P water molecules were added
with a minimum spacing of 30 Å (3.0 nm) from the box edges
to the PNA:RNA molecules. Energy minimization on each
system was performed in a two-step process. First the solute
atoms were restrained while the water and ion molecules were
allowed to relax over 1000 steps. The entire system was then
subjected to energy minimization using the steepest descent
method for the first 1000 steps, followed by the full conjugate
gradient method for an additional 24 000 steps. Each molecular
system was then heated to 300 K for 100 ps followed by a 50 ps
constant pressure simulation to adjust the density to 1 g/mL.
An additional 500 ps simulation was run prior to production
simulations to allow for further temperature and pressure
equilibration.
ΔGbind = ΔGduplex − ΔGPNA − ΔGRNA
Each term is calculated by determining the enthalpic energy
of the solute using molecular mechanics (EMM), the polar
solvation free energy (ΔGSOLV), the nonpolar solvation free
energy (ΔGnp), and the entropic contribution (ΔS):
ΔG = ⟨EMM⟩ + ⟨ΔGsolv⟩ + ΔGnp − T⟨ΔS⟩
The enthalpic term is taken as the average over the molecular
mechanics force field terms for the solute. The solute
vibrational entropy is estimated using either normal-mode
analysis or quasi-harmonic approximation. ΔGsolv is calculated
using the Poisson−Boltzmann (PB) equation. The nonpolar
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Scheme 2
Scheme 3
term (ΔGnp) is estimated from the solvent accessible surface
ct is the concentration (M) of each individual RNA/PNA strand
in the CD experiments.
area (SASA).52
For each RNA:PNA system, MM-PBSA calculations were
performed using the last 8.0 ns of the simulation with 100 ps
intervals. The entropy was determined using normal-mode
analysis using 50 frames over the 8.0 ns. All energy values
represent 3 independent MD simulation runs for each
RNA:PNA duplex. aMD MM-PBSA calculations were
performed over the entire 50 ns simulation with 50 ps intervals.
aMD entropy values were determined using the same method
as for conventional MD (cMD) simulations. All calculations
were performed with the MMPBSA.py.MPI module in Amber
11 with an ionic strength equal to 1.0 M. For MM-PBSA
calculations the PB equation was solved numerically by the
PBSA program included with in the AmberTools 12 module.
ΔGnp = γSASA + β
where γ is the surface tension, set to 0.0072 kcal/Å2. β is an
offset value used to correct for the nonpolar contribution to the
solvation free energy term.53 Free energy values were converted
to theoretical melting temperatures using the following
equation:54
1
Tm
R
ΔH
ΔS − R ln[2]
ΔH
=
ln[ct] +
where ΔH is calculated from the sum of the enthalpic terms in
the MM-PBSA calculation, ΔS is the entropy value from
normal-mode analysis, R is the gas constant in kcal/mol·K, and
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The hydrophobic contribution was approximated by the LCPO
method implemented within the Sander module.55
Synthesis of TO-PNA Monomer. The synthesis of TO-
Aeg-PNA monomer (8) was achieved in seven steps (Scheme
2) by using an adaptation of previously developed meth-
ods.56−58
was excited at 495 nm and the fluorescence intensities were
collected at 531 nm.
Confocal Fluorescence Microscopy of TO-PNA-Pepti-
de:RNA Duplexes in Cells. Fluorescent imaging was carried
out of TO-PNA-peptides in KRAS wild type and mutant cells.
SK-LU-1 cells (ATCC) were cultured in EMEM medium with
10% fetal bovine serum (FBS). H460 cells (ATCC) were
cultured in RPMI 1640 medium with 10% FBS. 20 000 cells
were seeded in an 8-chamber well slide (Millipore) in 10%
growth medium and incubated overnight. 200 nM TO-PNA-
peptides were added to each well in serum-free medium and
were incubated for 4 h at 37 °C to allow equilibration of bound
TO-PNA-peptide. At the end of incubation, cells were washed
3× with PBS containing Ca2+ and Mg2+ (Fisher) followed by
fixation with 4% paraformaldehyde in PBS. Cells were then
washed one more time with PBS containing Ca2+ and Mg2+.
Chambers were removed and the slides were mounted with
Prolong Gold Antifade reagent with DAPI (Life Technologies).
All images were taken on a Nikon C1 Plus two point-scanning
laser confocal microscope using 40× oil objective. Excitation
was at 488 nm, and emission was observed at 535/40 nm.
Quantification of Confocal Images Using ImageJ. Nine
cells were selected individually using drawing tools in ImageJ,
followed by measurement of the area and the integrated density
of each cell. Three different background areas were selected for
each image and were measured for mean pixel values. The
corrected total cell fluorescence for each cell = integrated
density of the cell − area of the cell × mean pixel values of
three background areas. Standard errors were calculated, and
significance was assessed by Student’s t test.
The synthesis began with commercially available 2-
(methylthio)benzothiazole (1) which was treated with iodo-
methane to yield the N-alkyl compound 3. The quinoline
derivative 4 was obtained by reaction of 4-methylquinoline (2)
with bromoacetic acid. The condensation of N-alkyl compound
3 and the quinoline derivative 4 in the presence of Et3N yielded
TO carboxylic acid 5. The TO carboxylic acid 5 was coupled
with commercially available Fmoc-Aeg-OtBu (6) in the
presence of PyBOP59 and HOBt gave tBu ester compound 7
with moderate yield. When compound 7 was treated with 20%
CF3CO2H in CH2Cl2 to deprotect the tBu-ester group, a
complex reaction mixture was observed that was difficult to
purify. Hence, a new synthetic route (Scheme 3) was adopted.
The tBu group of compound 6 was deprotected by
CF3CO2H followed by treatment with allyl alcohol in the
presence of thionyl chloride to yield the allyl ester 9. This was
coupled with TO carboxylic acid 5 in the presence of PyBOP
and HOBt to give the TO allyl ester 10. Finally, the allyl
protecting group in compound 10 was removed by Pd0-
catalyzed allyl transfer to N-methyl aniline60 to yield TO-Aeg-
PNA monomer 8 which was used in solid phase peptide
synthesis.
Circular Dichroism (CD) Spectra and Thermal Melting
of PNA:RNA Duplexes. CD spectra were obtained for TO-
PNA-peptide:RNA duplexes using a JASCO J-810 spectropo-
larimeter with Peltier temperature control.37,39 Spectra were
recorded over 320−220 nm at 25 °C. Gel-purified RNA
icosamers were purchased from Thermo Scientific (Dharma-
con). Prior to measurement, samples were heated to 95 °C for
10 min and then cooled for 10 min. All spectra were subjected
to baseline correction. Final spectra were calculated as the
average of 3 independent measurements. Stock RNA samples
were suspended in H2O and TO-PNA-peptide molecules in 20
mM sodium phosphate buffer (pH 7.2), TO-PNA-peptide:R-
NA solutions were diluted in 20 mM Na2HPO4, 1.0 M NaCl, 5
mM EDTA, pH 7.2, to reach a final concentration of 1 μM:1
μM. Thermal denaturation experiments were carried out by
increasing the temperature from 25 to 95 °C at a rate of 0.5
°C/min while monitoring the CD signal at 265 nm. Tm values
were determined based on the assumption of a two-state model.
Melting curves were normalized by subtracting baseline
slopes.54 The first derivative of the melting curves was
computed with respect to the temperature (dθ/dT), and the
maximum was used to determine the Tm. We have previously
reported no perturbation of Tm due to peptide moieties on the
PNAs.38
ASSOCIATED CONTENT
* Supporting Information
■
S
Methods and analytical data for synthesis of thiazole orange-
PNA monomer and solid phase assembly of PNA-AEEA-IGF1
tetrapeptide sequences containing thiazole orange are shown.
50 ns of simulated motion of KRAS2 G12D TO-PNA
hybridized to KRAS2 G12WT RNA are shown in an mpg
file, which illustrates the TO residue flipping out of the helix at
the site of PNA:RNA mismatch. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: eric@wickstromlab.org. Phone: 215-955-4578. Fax:
215-955-4580.
■
Present Addresses
Mahesh V. Sonar, Department of Chemistry, State University
of New York, Binghamton NY 13902
Matthew E. Wampole, Discovery Science, Thomson-Reuters,
Boston MA 02210
Notes
Fluorescence Intensity Measurements of TO-PNA-
Peptide:RNA Duplexes. Hybridization was carried out of
TO-PNA-peptides with synthetic RNAs. 50 nM G12D TO-
PNA-peptide and G12WT TO-PNA-peptide were incubated
with 50 nM RNAs (Thermo Scientific Dharmacon) in 3 mM
Na2HPO4 buffer (Molecular Research Center) for 1 h at 37 °C.
Single stranded TO-PNA-peptides were incubated with buffer
only. At the end of 1 h incubation, fluorescent intensities were
measured with a plate reader (infiniteM200PRO, TECAN).
Background intensity values consisting of only buffer were
subtracted from every fluorescence measurement. Each well
The authors declare the following competing financial
interest(s): We wish to inform the Editor of potential conflicts
of interest. Prof. Eric Wickstrom and Prof. Mathew L. Thakur
hold shares in GeneSeen LLC, which might ultimately benefit
from the results of this investigation, but did not support the
work.
ACKNOWLEDGMENTS
We thank Dr. Yolanda Covarrubias for assistance with confocal
microscopy in the Kimmel Cancer Center Molecular Imaging
■
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Facility, supported in part by NCI P30 CA56036. This work
was supported by NIH 1 R01 CA148565 to E.W. IP owned by
EW/MLT, licensed to MTTI.
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