Utilization of chromic polydiacetylene assemblies as a platform to probe specific binding between drug and RNA

Article abstract of DOI:10.1039/c7ra07178g

Recognition of nucleic acids remains an important endeavor in biology. Nucleic acids adopt shapes ranging from A-form (RNA and GC rich DNA) to B-form (AT rich DNA). We show, in this contribution, shape-specific recognition of A-U rich RNA duplex by a neomycin (Neo)-polydiacetylene (PDA) complex. PDA assemblies are fabricated by using a well-known diacetylene (DA) monomer, 10,12-pentacosadiynoic acid (PCDA). The response of poly(PCDA) assemblies is generated by mixing with a modified neomycin-PCDA monomer (Neo-PCDA). The functionalization by neomycin moiety provides specific binding with homopolyribonucleotide poly(rA)-poly(rU) stimulus. Various types of alcohols are utilized as additives to enhance the sensitivity of poly(PCDA)/Neo-PCDA assemblies. A change of absorption spectra is clearly observed when a relatively low concentration of poly(rA)-poly(rU) is added into the system. Furthermore, poly(PCDA)/Neo-PCDA shows a clear specificity for poly(rA)-poly(rU) over the corresponding DNA duplex. The variation of linker between neomycin moiety and conjugated PDA backbone is found to significantly affect its sensitivity. We also investigate other parameters including the concentration of Neo-PCDA and the DA monomer structure. Our results provide here preliminary data for an alternative approach to improve the sensitivity of PDA utilized in biosensing and diagnostic applications.

Full text of DOI:10.1039/c7ra07178g

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Utilization of chromic polydiacetylene assemblies
as a platform to probe specific binding between
drug and RNA†
Cite this: RSC Adv., 2017, 7, 41435
Anothai Kamphan,ab Changjun Gong,c Krishnagopal Maiti,c Souvik Sur,c
abd
c
*
*
Rakchart Traiphol
and Dev P. Arya
Recognition of nucleic acids remains an important endeavor in biology. Nucleic acids adopt shapes ranging
from A-form (RNA and GC rich DNA) to B-form (AT rich DNA). We show, in this contribution, shape-
specific recognition of A–U rich RNA duplex by a neomycin (Neo)–polydiacetylene (PDA) complex. PDA
assemblies are fabricated by using a well-known diacetylene (DA) monomer, 10,12-pentacosadiynoic acid
(PCDA). The response of poly(PCDA) assemblies is generated by mixing with a modified neomycin–PCDA
monomer (Neo–PCDA). The functionalization by neomycin moiety provides specific binding with
homopolyribonucleotide poly(rA)–poly(rU) stimulus. Various types of alcohols are utilized as additives to
enhance the sensitivity of poly(PCDA)/Neo–PCDA assemblies. A change of absorption spectra is clearly
observed when a relatively low concentration of poly(rA)–poly(rU) is added into the system. Furthermore,
poly(PCDA)/Neo–PCDA shows a clear specificity for poly(rA)–poly(rU) over the corresponding DNA duplex.
The variation of linker between neomycin moiety and conjugated PDA backbone is found to significantly
affect its sensitivity. We also investigate other parameters including the concentration of Neo–PCDA and
the DA monomer structure. Our results provide here preliminary data for an alternative approach to
improve the sensitivity of PDA utilized in biosensing and diagnostic applications.
Received 28th June 2017
Accepted 2nd August 2017
DOI: 10.1039/c7ra07178g
rsc.li/rsc-advances
triplex20–23 and quadruplex24–26 structures of therapeutic
interest.27
1. Introduction
Conventional methods for the study of neomycin drug–RNA
interactions utilize established analytical tools. For example,
circular dichroism (CD) spectroscopy is used to study confor-
mational changes of RNA during ligand–RNA binding.11,28
Isothermal titration calorimetry (ITC), differential scanning
calorimetry (DSC) and UV-vis spectroscopy are used to elucidate
the thermodynamic parameters of ligand–RNA interactions.11,28
Fluorescence intercalator displacement (FID) assays can be
used for the study of selectivity, affinity, stoichiometry and
binding site size of RNA and ligand.11,28 New and simple
approaches that can help us study small molecule's interaction
with biopolymers would be welcome addition to these estab-
lished rigorous methods of ligand–nucleic acids interactions.
Polydiacetylene (PDA) is a conjugated polymer, which has
received tremendous attention as a chromatic sensor. PDA can
be prepared via 1,4-addition of highly organized assemblies of
diacetylene monomers, normally initiated by UV light irradia-
tion.29,30 PDA possesses color transition, typically from blue to
red, upon the exposure to various external stimuli such as
heat,31–42 acids and bases,40,43–46 chemicals47–56 and biomole-
cules.57–66 The perturbation by these stimuli generally weakens
segmental interactions within the PDA assemblies. This results
in the reorientation of conjugated backbone and alkyl chain.
The decrease of conjugation length of PDA provides the shi of
A number of antibiotics that stop the growth of foreign patho-
gens, such as bacteria and viruses, are known to bind nucleic
acids such as DNA and RNA. One such class of antibiotics are
aminoglycosides, well known to bind bacterial ribosomal RNA.1
Neomycin is one such antibiotic, belonging to aminoglycosides
containing the deoxystreptamine core. The binding of
neomycin, and other aminoglycosides drugs, to bacterial RNA
affects the production of proteins through miscoding in trans-
lation, ultimately leading to bacterial death.2–5 In addition to
binding to ribosomal RNA, aminoglycosides have been shown
to bind numerous other RNA,6–10 DNA,11–17 hybrid duplex,18,19
aDepartment of Chemistry, Faculty of Science, Naresuan University, Phitsanulok
65000, Thailand
bLaboratory of Advanced Polymers and Nanomaterials, School of Materials Science
and Engineering, Center of Excellence for Innovation in Chemistry, Faculty of
Science, Mahidol University at Salaya, Phuttamonthon 4 Road, Salaya, Nakhon
Pathom 73170, Thailand
cLaboratories of Medicinal Chemistry, Department of Chemistry, Clemson University,
Clemson, South Carolina 29634, USA. E-mail: dparya@clemson.edu
dNANOTEC-MU Excellence Center on Intelligent Materials and Systems, Faculty of
Science, Mahidol University, Rama 6 Road, Ratchathewi, Bangkok 10400, Thailand.
E-mail: rakchart.tra@mahidol.ac.th
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra07178g
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absorption spectrum to lower energy region and hence induces
color transition.30,67–70 Many prior reports demonstrate the
ability of PDA-based biosensors for the detection of viral RNA
with quick and reliable testing without requiring advanced
instruments. Amano et al. reviewed the construction of PDA
with desirable physical, optical and electrical properties that
responds to the inuenza virus's RNA. The exposure of inu-
enza virus to modied-PDA solution causes blue shi in the
absorption spectra, corresponding to blue-to-red color transi-
tion of PDA.71 Park et al. demonstrates the detection of RNA by
using PDA conjugated with DNA complementary to RNA of
pathogenic bacteria. The hybridization of a specic DNA to RNA
enhances uorescent intensity of PDA-based sensor, leading to
the application as a biochip in the detection of pathogenic
microorganisms.72 In this report, we demonstrate the use of
PDA in a modied approach for the investigation of RNA–ligand
interactions.
Scheme 1 Reagents and conditions: (a) (Boc)2O, H2O, methanol,
Na2CO3, r.t., 18 h, 65%; (b) 2,4,6-triisopropylbenzenesulfonyl chloride,
pyridine, r.t., 40 h, 50%; (c) NaN3, DMF/H2O, 90 ꢃC, 12 h, 95%; (d) H2,
Pd/C, ethanol, r.t., 12 h, quantitative; (e) HSCH2CH2NH2, ethanol,
Na(metal), r.t, 12 h, 60%.
Here, PCDA modied neomycin head groups (Neo–PCDA) is
synthesized to generate a specic binding site for RNA. Previous
calorimetry and FID studies have shown that the binding of
poly(rA)–poly(rU), a homopolymeric dsRNA, with neomycin has
high Ka values (ꢀ107 Mꢁ1), and low AC50 (0.02 ꢂ 0.01 mM)
respectively, representing high affinities.11,14 These studies are
in concert with other data showing nanomolar binding of
neomycin to RNA targets. Therefore, poly(rA)–poly(rU) was used
here as a model RNA for investigating the neomycin–RNA
interaction. We also demonstrate here that the sensitivity of
poly(PCDA)/Neo–PCDA assemblies can be signicantly
improved by using alcohol additives. An addition of alcohols
weakens the strength of interactions within the poly(PCDA)/
Neo–PCDA assemblies resulting in the increase of colorimetric
response compared to its pure system.73 The developments of
specic and rapid color transition of PDA to biomolecules could
Scheme 2 Reagents and conditions: (a) 10,12-pentacosadiynoic acid
(PCDA), TBTU, DIPEA, DMF, r.t., 18 h, 80% (for 5) and 74% (for 6); (b)
TFA, CH2Cl2, r.t., 3 h, 76% (for Neo–PCDA1) and 71% (for Neo–PCDA2).
open wide-range of possibilities for biosensor, drug detection, neomycin amine derivatives 3 and 4 were prepared from
and diagnostic applications.
commercially available neomycin B (1) according to published
schemes (Scheme 1).11,14,22,74–76 All the compounds were charac-
terized and spectral data was matched with the reported values
from the literatures. Amine functionalized neomycin derivatives
3 and 4 were coupled with 10,12-pentacosadiynoic acid (PCDA)
monomer (Aldrich) in the presence of coupling reagent
2. Experiment
2.1 The synthesis of Neo–PCDA1 and Neo–PCDA2
The chemical structures of neomycin Neo–PCDA1 and Neo–
PCDA2 are shown in Fig. 1. Neo–PCDA1 and Neo–PCDA2 were
synthesized as shown in Schemes 1 and 2. Boc-protected
O-(Benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetra-
uoroborate (TBTU) in DMF solution, followed by deprotection
with triuoroacetic acid in dichloromethane, leading to
neomycin-PCDA conjugates, Neo–PCDA1 and Neo–PCDA2
(Scheme 2). All the four new compounds 5, 6, Neo–PCDA1 and
Neo–PCDA2 were characterized by nuclear magnetic resonance
spectroscopy (NMR, Bruker Advance-500 and 300) and MALDI-
TOF mass spectrometry (Bruker Microex) and the spectra are
provided in the ESI.† One of the a-anomeric protons of two
pyranoside units in 5, appeared as a singlet at 5.38 ppm whereas
the b-anomeric proton of furanoside unit resonates at 5.15 ppm
1
as a singlet, in H NMR spectrum of 5 (Fig. S2†). The peak at
99.09 and 100.52 ppm corresponded to a-anomeric carbon of
two pyranoside moieties whereas b-anomeric carbon of furan-
oside moiety appeared at 111.08 in 13C NMR spectrum of 5
(Fig. S3†). Further, the formation of the compound 5 was
conrmed by the appearance of molecular ion peak at 1653.268
Fig. 1 Chemical structures of diacetylene monomer, 10,12-pentaco-
sadiynoic acid (PCDA) and 10,12-tricosadiynoic acid (TCDA); and
synthesized neomycin–PCDA conjugates, Neo–PCDA1 and Neo–
PCDA2.
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[M + Na]+, as the base peak in MALDI-TOF mass spectrum of 5 solvent was removed under vacuum and the residue was dis-
(Fig. S1†). In 1H NMR spectrum of 6 (Fig. S8†), b-anomeric solved in deionized water (2 mL) and washed with diethylether
proton of the furanoside moiety appeared as a singlet at (3 ꢄ 10 mL). Lyophilization of the aqueous solution yielded
5.08 ppm whereas the corresponding anomeric carbon reso- a pale yellow powder of Neo–PCDA1 (0.034 g, 76%); 1H NMR
nated at 111.48 ppm, in 13C NMR spectrum of 6 (Fig. S9†). The (500 MHz, D2O) d 5.93 (d, J ¼ 4.0 Hz, 1H), 5.32 (d, J ¼ 1.8 Hz,
anomeric peaks of compound 6 were assigned by the help of 1H), 5.19 (s, 1H), 4.45–4.37 (m, 1H), 4.31 (m, 1H), 4.21 (t, J ¼
HMQC spectrum (Fig. S10†). The formation of compound 6 was 4.9 Hz, 1H), 4.13 (m, 2H), 4.00 (t, J ¼ 9.7 Hz, 1H), 3.94–3.76 (m,
further conrmed by MALDI-TOF mass spectrum (Fig. S7†), 5H), 3.72 (s, 1H), 3.69–3.55 (m, 5H), 3.52–3.41 (m, 3H), 3.40–3.21
where the molecular ion peak appeared as the base peak at (m, 8H), 3.18 (m, 1H), 3.08 (d, J ¼ 7.4 Hz, 2H), 2.42–2.37 (m, 1H),
1593.065 [M + Na]+.
2.16 (s, 2H), 1.79 (m, 1H), 1.44 (s, 2H), 1.21 (m, 30H); 13C NMR
The details of the synthetic procedures for compounds 5, 6, (75 MHz, D2O) d 163.55, 163.09, 162.61, 162.14, 122.10, 118.24,
Neo–PCDA1 and Neo–PCDA2 are given below. 114.37, 110.51, 110.21, 96.05, 95.46, 95.33, 84.79, 81.50, 77.71,
2.1.1. Compound 5. To a solution of compound 4 (0.1 g, 75.21, 73.57, 72.46, 70.57, 70.07, 69.41, 69.17, 68.09, 67.93,
0.078 mmol) in DMF (1 mL), the mixture of 10,12-pentacosa- 67.60, 67.33, 60.18, 54.34, 53.45, 50.83, 49.75, 49.64, 48.38,
diynoic acid (0.146 g, 0.39 mmol) and TBTU (0.126 g, 0.39 42.52, 40.42, 40.08, 32.04, 29.85, 29.16, 28.09, 22.72, 17.68,
mmol) in DMF (0.5 mL) was added. DIPEA (0.136 mL, 0.78 16.21, 13.96, 12.09; MALDI-TOF m/z calculated for
mmol) was added to the reaction mixture and it was stirred at
room temperature for 18 h. The solvent was removed and the
C
50H91N7O13S [M]+ 1029.64, found 1030.75.
2.1.4. Neo–PCDA2. To a solution of compound 6 (0.07 g,
crude reaction mixture was puried by column chromatography 0.045 mmol) in dichloromethane (0.5 mL) triuoroacetic acid
(silica gel) to furnish compound 5 (0.102 g, 80%); 1H NMR (0.5 mL) was added and stirred at room temperature for 3 h. The
(CD3OD, 300 MHz) d 5.38 (s, 1H), 5.15 (s, 1H), 4.26 (s, 2H), 3.91 solvent was removed under vacuum and the residue was dis-
(s, 1H), 3.77–3.71 (m, 2H), 3.60–3.39 (m, 13H), 3.22–3.16 (m, solved in deionized water (2 mL) and washed with diethylether
3H), 2.78–2.74 (m, 2H), 2.36 (t, J ¼ 7.9 Hz, 2H), 2.27–2.20 (m, (3 ꢄ 10 mL). Lyophilization of the aqueous solution yielded
6H), 1.99–1.94 (m, 1H), 1.65–1.61 (m, 3H), 1.47–1.44 (m, 57H), a pale yellow powder of Neo–PCDA2 (0.031 g, 71%); 1H NMR
1.34–1.30 (m, 27H), 0.90 (t, J ¼ 6.6 Hz, 3H); 13C NMR (CD3OD, 75 (500 MHz, D2O) d 5.93 (d, J ¼ 3.9 Hz, 1H), 5.33 (s, 1H), 5.19 (s,
MHz) d 176.36, 159.08, 158.88, 158.48, 158.21, 158.18, 157.92, 1H), 4.46–4.37 (m, 1H), 4.32 (s, 1H), 4.22 (t, J ¼ 4.9 Hz, 1H), 4.13
111.08, 100.52, 99.09, 87.02, 82.67, 80.73, 80.59, 80.40, 80.01, (m, 2H), 4.00 (t, J ¼ 9.7 Hz, 1H), 3.93–3.80 (m, 4H), 3.73 (s, 1H),
77.86, 75.71, 75.65, 74.54, 74.48, 73.25, 72.87, 71.57, 68.89, 3.68–3.55 (m, 5H), 3.50–3.44 (m, 2H), 3.42–3.23 (m, 7H), 3.19
66.44, 58.09, 53.60, 50.81, 41.69, 40.09, 37.18, 36.95, 33.06, (m, 1H), 3.08 (m, 2H), 2.42–2.37 (m, 1H), 2.14 (s, 2H), 1.85–1.77
31.68, 31.64, 30.74, 30.66, 30.58, 30.45, 30.31, 30.26, 30.12, (m, 1H), 1.39 (s, 2H), 1.22 (m, 30H); 13C NMR (126 MHz, D2O)
30.05, 29.84, 29.78, 29.52, 29.02, 28.88, 28.85, 28.76, 26.99, d 163.30, 163.02, 162.74, 162.45, 119.82, 117.50, 115.18, 112.86,
23.72, 19.69, 18.53, 14.45; MALDI-TOF m/z calcd for C80H139
-
110.20, 96.06, 95.47, 95.33, 84.78, 81.49, 77.72, 75.21, 75.15,
73.56, 72.46, 70.61, 70.56, 70.08, 69.40, 69.17, 68.19, 67.99,
N7O25SNa [M + Na]+: 1652.94, found 1653.268.
2.1.2. Compound 6. To a solution of compound 3 (0.1 g, 67.61, 67.33, 62.50, 60.17, 54.34, 53.44, 50.83, 49.76, 49.64,
0.082 mmol) in DMF (1 mL), the mixture of 10,12-pentacosa- 48.38, 42.52, 40.43, 40.06, 32.01, 29.81, 28.23, 27.97, 22.71,
diynoic acid (0.154 g, 0.41 mmol) and TBTU (0.132 g, 0.41 17.69, 16.22, 13.97, 12.10; MALDI-TOF m/z calculated for
mmol) in DMF (0.5 mL) was added. DIPEA (0.142 mL, 0.82
mmol) was added to the reaction mixture and it was stirred at
room temperature for 18 h. The solvent was removed and the
crude reaction mixture was puried by column chromatography
C
48H87N7O13 [M]+ 970.27, found 970.71.
2.2 The preparation of PDA/Neo–PCDA assemblies
(silica gel) to furnish compound 6 (0.96 g, 74%); 1H NMR The assemblies of PDA/Neo–PCDA were prepared by using two
(CD3OD, 500 MHz) d 5.48 (s, 1H), 5.08 (s, 1H), 4.30 (s, 1H), 4.06 DA monomers, including 10,12-pentacosadiynoic acid (PCDA)
(s, 1H), 3.93–3.86 (m, 4H), 3.75 (s, 2H), 3.56–3.50 (m, 11H), 3.25– and 10,12-tricosadiynoic acid (TCDA). DA monomer and Neo–
3.11 (m, 3H), 2.34–2.30 (m, 3H), 2.24 (t, J ¼ 6.8 Hz, 3H), 1.95– PCDA powder were dissolved in ethanol and then ltered by
1.93 (m, 1H), 1.66–1.63 (m, 2H), 1.47–1.42 (m, 65H), 1.34–1.30 using a 0.45 mm nylon lter to remove polymerized impurities.
(m, 20H), 0.90 (t, J ¼ 7.0 Hz, 3H); 13C NMR (CD3OD, 125 MHz) The ratios of Neo–PCDA were 0, 5, 10, 15 and 20 mol%. The DA/
d 176.58, 159.06, 158.87, 158.55, 158.49, 158.25, 157.89, 111.48, Neo–PCDA solutions in ethanol were slowly dried at 60 ꢃC in
100.42, 98.80, 88.47, 80.94, 80.74, 80.66, 80.36, 80.31, 79.47, a water bath. Phosphate-buffered saline solutions (PBS buffer)
77.83, 76.20, 75.51, 74.42, 73.35, 72.69, 72.61, 72.55, 71.65, at various pH values (6, 7, and 8) were added into the DA/Neo–
68.99, 66.46, 66.44, 56.80, 53.53, 52.50, 51.30, 49.85, 43.91, PCDA lms to provide 0.25 mM aqueous suspension. The pH of
42.64, 41.95, 36.96, 35.69, 33.05, 30.73, 30.66, 30.57, 30.44, PBS buffer was adjusted by using HCl and NaOH solutions. The
30.39, 30.35, 30.12, 29.86, 29.83, 29.54, 29.49, 29.02, 28.89, samples were sonicated at 70 ꢃC for 90 min to disperse DA/Neo–
28.84, 28.80, 28.74, 27.19, 23.72, 19.68, 14.46; MALDI-TOF m/z PCDA lms into aqueous medium. The suspensions were
calcd for C78H135N7O25Na [M + Na]+: 1592.94, found 1593.065.
allowed to equilibrate at room temperature and then stored at
4
ꢃC overnight to induce self-assembly process. The cloudy
2.1.3. Neo–PCDA1. To a solution of compound 5 (0.07 g,
0.043 mmol) in dichloromethane (0.5 mL) triuoroacetic acid suspensions of DA/Neo–PCDA were irradiated under UV light
(0.5 mL) was added and stirred at room temperature for 3 h. The (10 W, l ꢀ 254 nm) for 5 min, resulting in blue PDA/Neo–PCDA
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vesicles. Effect of Neo–PCDA structure was studied by using
Neo–PCDA1 and Neo–PCDA2 prepared by the same method.
2.3 The study of colorimetric response of PDA/Neo–PCDA to
poly(rA)–poly(rU)
Polyadenylic–uridylic acid (Poly(rA)–poly(rU)) was purchased
from Midland Certied Company. Tuning colorimetric
response of PDA/Neo–PCDA to poly(rA)–poly(rU) was induced by
using two alcohols, butanol and hexanol. The alcohols were
added into PDA/Neo–PCDA solution at various concentrations
by using a micropipette. Aer the addition of alcohols, the
solution was shaken by using vortex for a few seconds. Poly(rA)–
poly(rU) solution in PBS buffer (pH 8) was added into PDA/Neo–
PCDA solution at various concentrations by using a micropi-
pette. The mixture was shaken for a few seconds prior to each
addition. The color transition of PDA/Neo–PCDA assemblies
was followed by measuring the absorption spectra (UV-vis
spectrophotometer, Agilent technologies, Cary series). The
concentration of PDA/Neo–PCDA assemblies in each experi-
ment was controlled to be the same by adjusting the absorbance
value. To quantify the extent of blue-to-red color transition of
PDA/Neo–PCDA assemblies, the colorimetric response (% CR)
was dened and calculated as follows: % CR ¼ [(PB0 ꢁ PB)/PB0]
Fig. 2 In silico models (above) and TEM images (below) of poly
(PCDA) prepared by mixing with 10 mol% of (a) Neo–PCDA1 and (b)
Neo–PCDA2.
ꢄ 100. The PB is the percent blue calculated from A640/(A540
+
A640), where A540 and A640 are the absorbance of red (l ¼ 540)
and blue (l ¼ 640) phases of vesicles, respectively. The
initial PB0 value was determined before the addition of poly
(rA)–poly(rU). Morphology of these solutions was also studied
by using transmission electron microscope (TEM, Hitachi
H7600). The samples were prepared by dropping diluted PDA/
Neo–PCDA solutions onto copper grids, followed by air-drying
at room temperature.
The specicity of PDA/Neo–PCDA was study by testing with
a control DNA sequence containing only adenine and thymine
bases. The poly(PCDA)/Neo–PCDA1 containing 0.5% v/v hexanol
was used. The solution of control DNA in PBS buffer (pH 8) was
added into poly(PCDA)/Neo–PCDA1 solution by using the same
testing process as poly(rA)–poly(rU) system. The colorimetric
response of poly(PCDA)/Neo–PCDA1 vesicles was followed by
measuring the absorption spectra.
exhibit relatively large rod-liked and sheet-liked shapes as
shown in Fig. 2b. This is attributed to the difference of chemical
linker between PCDA and neomycin head group (see Fig. 1). The
Neo–PCDA2 constitutes much shorter linker compared to that
of the Neo–PCDA1, which in turn signicantly affects their
packing structure. Neo–PCDA2 has a shorter linker compared to
Neo–PCDA1 (see Fig. 1), resulting in a larger head group
repulsion. This provides the increase of head group area and the
decrease in the packing parameter, leading to the differences in
molecular organization and ultimately different shape and size
of poly(PCDA)/Neo–PCDA assembly.39,77,78 Jung,66,79 Cheng80 and
Ye81 also demonstrate that the variation of head group structure
provides different shape and size of PDA assemblies. Flower-like
morphology, rectangular shape, ber, ribbon with various size
in the range of nanometer to micrometer are observed. It is
important to note that the differences of shape and size of PDA
signicantly affect its colorimetric response as well. This will be
discussed in the following section.
3. Results and discussion
To achieve the highest response of poly(PCDA)/Neo–PCDA
assemblies to poly(rA)–poly(rU), concentration of Neo–PCDA
and pH of PBS buffer were optimized. We observed that the
poly(PCDA)/Neo–PCDA1 assemblies containing 20 mol% of
Neo–PCDA1 at pH 8 provided the highest response to poly(rA)–
poly(rU) (see Fig. S14 in ESI†). RNA-binding affinity of an ami-
noglycoside is known to vary with the change in pH, as the
protonated states of the amino groups are affected.82 However,
this system exhibits large precipitation, which is clearly unde-
sirable for sensing applications. Therefore, the highest possible
concentration of Neo–PCDA, 10 mol%, providing clear blue
solution and no precipitation of the assemblies, was used for
the entire study.
3.1 Morphologies of poly(PCDA)/Neo–PCDA assemblies and
the optimization
Pure poly(PCDA), poly(PCDA)/Neo–PCDA1 and poly(PCDA)/
Neo–PCDA2 solutions show a blue color, indicating proper
packing of PCDA monomers required for the top-
opolymerization process. Morphologies of resultant poly
(PCDA)/Neo–PCDA assemblies revealed by TEM are shown in
Fig. 2 (bottom). A model of the Neo–PCDA compounds bound to
RNA is also included in Fig. 2 (top). It has been observed that
pure poly(PCDA) shows spherical shape with featureless
internal pattern.21 The TEM image of poly(PCDA)/Neo–PCDA1
also shows spherical particles with diameter of about 100 nm
(see Fig. 2a). In contrast, poly(PCDA)/Neo–PCDA2 assemblies
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3.2 Colorimetric response of poly(PCDA)/Neo–PCDA1 to
poly(rA)–poly(rU)
Poly(PCDA)/Neo–PCDA1 assemblies were prepared by mixing of
PCDA and 10 mol% Neo–PCDA1 in PBS buffer at pH 8. Initial
absorption spectrum of poly(PCDA)/Neo–PCDA1 exhibits lmax at
ꢀ640 nm. The spectrum hardly changes aer the addition of
poly(rA)–poly(rU) up to ꢀ13 mM (see Fig. 3a). Interestingly, the
colorimetric response of poly(PCDA)/Neo–PCDA1 drastically
increases when a small amount of hexanol, 0.5% v/v, is added
into the system. Absorption peak at ꢀ540 nm grows signi-
cantly upon the addition of poly(rA)–poly(rU) (see Fig. 3b),
indicating the formation of red poly(PCDA)/Neo–PCDA1. A blue-
to-purple color transition of poly(PCDA)/Neo–PCDA1 can be
observed as shown in Fig. 3d. According to our previous studies,
the added hexanol can penetrate into the poly(PCDA)/Neo–
PCDA1 layers which in turn weakens inter-/intrachain interac-
tions within the assemblies.51,52,73 This perturbation enhances
colorimetric response of poly(PCDA)/Neo–PCDA1 to poly(rA)–
poly(rU).
Fig. 4 TEM images of poly(PCDA)/Neo–PCDA1 (a) with the addition of
0.5% v/v hexanol and then (b) was disturbed by the addition of 9.52 mM
poly(rA)–poly(rU).
poly(rA)–poly(rU). We also observe that the perturbation by
poly(rA)–poly(rU) further induces the agglomeration of poly
(PCDA)/Neo–PCDA1 (see Fig. 4b). It is worthwhile to note that
the morphologies revealed by TEM could be different from
those of PDA assemblies in the solution state due to the sample
preparation process. However, the change of morphology in
dried state still reects the properties of PDA assemblies in
solution.
The plots of % CR clearly show that the increase of hexanol
concentration systematically enhances poly(PCDA)/Neo–PCDA1
response to poly(rA)–poly(rU) (see Fig. 3c). The higher amounts
of hexanol causes increased perturbation of poly(PCDA)/Neo–
PCDA1 assemblies,51,52,73 resulting in more blue-to-purple color
transition as shown in Fig. 3d. However, the further increase of
hexanol concentration results in a cloudy suspension arising
from its limited solubility in water.83 A control set of poly
(PCDA)/Neo–PCDA1 assemblies containing 0.5% v/v hexanol
was also investigated. The absorption spectra of this solution
were measured at the same time interval with the measurement
of other solutions without the addition of poly(rA)–poly(rU)
solution. This system shows a slight increase of CR value to
ꢀ8% while the addition of poly(rA)–poly(rU) provides the CR
value of ꢀ20% as shown in Fig. 3c. The difference of these two
sets indicates the color transition of poly(PCDA)/Neo–PCDA1
induced by interaction between Neo–PCDA1 and poly(rA)–
poly(rU).
The study of its morphology by TEM supports our hypoth-
esis. The morphology of poly(PCDA)/Neo–PCDA1 changes from
spherical shape to sheet-like structure aer the addition of 0.5%
v/v hexanol as shown in Fig. 4a. This observation suggests that
the presence of hexanol induces segmental reorganization
within the assemblies, enhancing the colorimetric response to
Schematic illustration in Fig. 5a concludes our study in this
rst part. The self-assembling of PCDA and Neo–PCDA mono-
mers takes place in a solution state. A blue phase of poly(PCDA)/
Neo–PCDA assemblies is obtained via photopolymerization
process. The insertion of alcohols into poly(PCDA)/Neo–PCDA
layers weakens the interactions within the system, which in turn
enhances the sensitivity. The specic binding between
neomycin moiety and poly(rA)–poly(rU) induces the color tran-
sition. In this study, butanol was also used as an additive in
order to investigate the effect of alcohol alkyl tail on the color-
imetric response of poly(PCDA)/Neo–PCDA1. The highest
concentration of butanol, 5% v/v, was added into poly(PCDA)/
Neo–PCDA1 while the suspension still remained blue. We
observe that the addition of butanol provides lower response of
poly(PCDA)/Neo–PCDA1 compared to the use of hexanol (see
Fig. 5b). Although the added concentration of butanol is 10
times higher than that of hexanol, the % CR reaches plateau at
Fig. 3 Absorption spectra of (a) pure poly(PCDA)/Neo–PCDA1 and (b)
with the addition of 0.5% v/v hexanol upon testing with poly(rA)–
poly(rU). (c) The plots of % CR and (d) color photographs show color
transition of poly(PCDA)/Neo–PCDA1 upon increasing poly(rA)–pol-
y(rU) concentration. Error bars are obtained from 3 experiments
(control: poly(PCDA)/Neo–PCDA1 with the addition of only 0.5% v/v
hexanol, no addition of poly(rA)–poly(rU)).
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Fig. 6 The % CR curves of poly(PCDA)/Neo–PCDA1 containing 0.5%
v/v hexanol upon testing with each stimulus. The inset shows the % CR
of this system upon the addition of DNA at extremely high concen-
tration (control: poly(PCDA)/Neo–PCDA1 with the addition of 0.5% v/v
hexanol, no addition of poly(rA)–poly(rU)).
Fig. 5 (a) Model illustrates the formation of poly(PCDA)/Neo–PCDA,
the insertion of alcohols and the binding between neomycin moiety
with poly(rA)–poly(rU). (b) The plot shows the effect of alcohol addi-
tives on sensitivity of poly(PCDA)/Neo–PCDA1 to poly(rA)–poly(rU).
Concentrations of the added alcohols are 5% v/v for butanol and 0.5%
v/v for hexanol.
neomycin moiety. This corresponds to low AC50 for the binding
of neomycin moiety and poly(rA)–poly(rU).11,28
about 12% upon increasing concentration of poly(rA)–poly(rU)
to ꢀ13 mM. Our results suggests that hexanol with relatively
long chain can penetrate into deeper region of poly(PCDA) layer.
This induces larger extent of perturbation (see model in Fig. 5a),
providing higher colorimetric response.51,73 It is possible to use
other types of alcohols as additives to enhance the sensitivity of
PDA. The effects of alcohol structures on colorimetric response
of PDAs are previously reported by our group.51,73 It is worthwhile
to note that a phospholipid (dimyristoylphosphatidylcholine,
DMPC) is normally used for improving sensitivity of PDA
assemblies to biomolecules in previous studies.53,60,61 We
demonstrate in this study that alcohol is an interesting additive
for enhancing the sensitivity of PDA to biomolecules. The
alcohol is also much cheaper compared to the DMPC.
3.4 The effect of Neo–PCDA structure on colorimetric
response
Specic binding site of Neo–PCDA is an important key for the
recognition of poly(rA)–poly(rU). Here, we investigate the effect
of Neo–PCDA structure on the binding ability of poly(PCDA)/
Neo–PCDA to poly(rA)–poly(rU). Our result shows that the
change of linker between neomycin moiety and PCDA signi-
cantly inuences the colorimetric response of their assemblies.
The Neo–PCDA2 constitutes a shorter linker compared to that of
the Neo–PCDA1 (see Fig. 1). Fig. 7a shows that the addition of
poly(rA)–poly(rU) into poly(PCDA)/Neo–PCDA2 assemblies cau-
ses slight growth of absorption peak at ꢀ540 nm, corresponding
to the formation of some red phase. The plot of % CR clearly
3.3 The specicity of poly(PCDA)/Neo–PCDA1 to poly(rA)–
poly(rU)
High specicity of PDA to its target biomolecules is necessary
for the utilization as a biosensor. Here, we explore the specicity
of poly(PCDA)/Neo–PCDA1 to poly(rA)–poly(rU) by comparing
the colorimetric response to an AT rich DNA (50-ATATATATA-
TATATAT-30). The results are illustrated in Fig. 6. As discussed
earlier, the addition of 13.06 mM of poly(rA)–poly(rU) induces
the increase of % CR to ꢀ22%. In contrast, the addition of DNA
hardly induces any colorimetric response. The plot of % CR is
similar to that of the control set. No signicantly change is
observed in this system. The further increase of DNA concen-
tration to 800 mM still shows similar behavior. This result
indicates that the poly(PCDA)/Neo–PCDA1 has high specicity
Fig. 7 (a) The absorption spectra of poly(PCDA)/Neo–PCDA2 con-
taining 0.5% v/v hexanol upon testing with poly(rA)–poly(rU). (b) The
plot of % CR shows different responses of poly(PCDA)/Neo–PCDA1
and poly(PCDA)/Neo–PCDA2 to poly(rA)–poly(rU) (control: poly
(PCDA)/Neo–PCDA1 with the addition of 0.5% v/v hexanol, no addition
to poly(rA)–poly(rU) attributed to the specic binding with of poly(rA)–poly(rU)).
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shows that the color response of poly(PCDA)/Neo–PCDA2 is
much lower than that of poly(PCDA)/Neo–PCDA1 (see Fig. 7b).
The CR value of poly(PCDA)/Neo–PCDA2 system is slightly
higher than that of the control set of poly(PCDA)/Neo–PCDA1
system. The study of their morphologies by TEM (see Fig. 2)
reveals that the poly(PCDA)/Neo–PCDA1 and poly(PCDA)/Neo–
PCDA2 assemblies exhibit a different shape and size. The poly
(PCDA)/Neo–PCDA2 assemblies exhibit much larger size
compared to the system of poly(PCDA)/Neo–PCDA1. It has been
known that the smaller PDA has higher response to external
stimuli.84 Therefore, the larger size of poly(PCDA)/Neo–PCDA2
assemblies likely causes the lower response to poly(rA)–
poly(rU).
We further investigate the color-transition behaviors of poly
(PCDA)/Neo–PCDA1 and poly(PCDA)/Neo–PCDA2 upon
increasing temperature (see ESI Fig. S15†). Signicant drop of
absorbance in the system of poly(PCDA)/Neo–PCDA2 is
observed upon increasing temperature attributed to the
formation of large aggregates and then precipitation. In addi-
tion, this system also shows lower % CR compared to pure
poly(PCDA) and poly(PCDA)/Neo–PCDA1, corresponding to
higher stability. This result is consistent with the lower
response of poly(PCDA)/Neo–PCDA2 to poly(rA)–poly(rU).
4. Conclusions
Sensitive and specic PDA-based biosensor for the detection of
homopolyribonucleotide poly(rA)–poly(rU) has been developed in
this study. The poly(PCDA)/Neo–PCDA1 assemblies provide blue-
to-purple color transition while pure poly(PCDA) vesicles show no
change aer the addition of poly(rA)–poly(rU). The addition of
control DNA to poly(PCDA)/Neo–PCDA1 assemblies does not
induce the color transition, indicating high specicity of poly
(PCDA)/Neo–PCDA1 to poly(rA)–poly(rU). Sensitivity of poly
(PCDA)/Neo–PCDA1 to poly(rA)–poly(rU) is enhanced by using
alcohols as additives and the variation of DA monomer structure.
The addition of small amount of hexanol results in signicant
increase of poly(PCDA)/Neo–PCDA1 response to poly(rA)–poly
(rU). The penetration of alcohol molecules weakens interactions
within the poly(PCDA)/Neo–PCDA1 assemblies, leading to the
increase of colorimetric response. The variation of Neo–PCDA
structure also affects its sensitivity. The use of Neo–PCDA1 with
longer linker provides a higher response compared to that of the
Neo–PCDA2. The PDA/Neo–PCDA assemblies prepared by using
TCDA monomer exhibit a larger change compared to that of
PCDA system. The development of sensitive and specic PDA to
target biomolecules expands our toolkit for the utilization of PDA
in biosensing, drug detection, and diagnostic applications.
3.5 The effect of DA monomer structure on colorimetric
response
Conflicts of interest
There are no conicts to declare.
The variation of DA monomer structure is an approach for
enhancing colorimetric response of PDA to its stimuli. In this
part, the PDA/Neo–PCDA1 assemblies were fabricated by using
TCDA monomer which has slightly shorter alkyl tail than PCDA
monomer. Fig. 8a shows the absorption spectra of poly(TCDA)/
Neo–PCDA1 containing 0.5% v/v hexanol upon the addition of
poly(rA)–poly(rU) at various concentrations. We observe signif-
icant growth of absorption peak at ꢀ540 nm. The plot of % CR
clearly shows that poly(TCDA)/Neo–PCDA1 has higher CR values
compared to the system of poly(PCDA)/Neo–PCDA1 (see Fig. 8b)
attributed to the shorter alkyl tail of poly(TCDA). The decrease
of alkyl chain length results in weaker dispersion interaction
along the PDA layers.51,52,73 Therefore, we observe higher
response of poly(TCDA)/Neo–PCDA1 to poly(rA)–poly(rU)
compared to poly(PCDA)/Neo–PCDA1.
List of abbreviations
AC50
CD
DA
Concentration at 50% of maximum activity
Circular dichroism spectroscopy
Diacetylene (monomer)
DMF
DMPC
DNA
FID
ITC
MALDI
N,N-Dimethylformamide
Dimyristoylphosphatidylcholine
Deoxyribonucleic acid
Fluorescence intercalator displacement assays
Isothermal titration calorimetry
Matrix assisted laser desorption/ionization-time
of ight mass spectrometry
Neo
Neomycin
Neo-PCDA
NMR
PBS buffer
PCDA
Conjugate of neomycin and PCDA
Nuclear magnetic resonance spectrophotometer
Phosphate-buffered saline solutions
10,12-Pentacosadiynoic acid
PDA
Polydiacetylene
Poly(rA)–
poly(rU):
RNA
Polyadenylic–uridylic acid
Ribonucleic acid
TBTU
O-(Benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium tetrauoroborate
10,12-Tricosadiynoic acid
Transmission electron microscope
Triuoroacetic acid
TCDA
TEM
TFA
TPS
Fig. 8 Absorption spectra of (a) poly(TCDA)/Neo–PCDA1 and (b) the
plot of % CR shows the effect of DA monomer structure on the
response to poly(rA)–poly(rU).
2,4,6-Triisopropylbenzenesulfonyl chloride
Ultraviolet
UV
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Acknowledgements
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We thank National Institutes for Health for nancial support
(grants R42GM097917, AI114114) to DPA. The research was also
supported by the Thailand Research Fund (Grant BRG6080008).
A. Kamphan thanks the Royal Golden Jubilee Ph.D. Program for
supporting her Ph.D. scholarship. This work has partially been
supported by the Nanotechnology Center (NANOTEC), Ministry
of Science and Technology, Thailand, through its program of
Center of Excellence Network.
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