Supramolecular hydrogel of kanamycin selectively sequesters 16S rRNA

Article abstract of DOI:10.1039/c2cc34935c

As the first example of hydrogelator derived from aminoglycoside antibiotics, the hydrogel of kanamycin indicates that the hydrogel of aminoglycosides preserve the specific interaction with their macromolecular targets (e.g., 16S rRNA), thus illustrating a simple approach to explore and identify possible biological targets of supramolecular nanofibers/hydrogels.

Full text of DOI:10.1039/c2cc34935c

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COMMUNICATION
Supramolecular hydrogel of kanamycin selectively sequesters 16S rRNAw
Zhimou Yang,a Yi Kuang,b Xinming Li,b Ning Zhou,b Ye Zhangb and Bing Xu*b
Received 10th July 2012, Accepted 26th July 2012
DOI: 10.1039/c2cc34935c
As the first example of hydrogelator derived from aminoglycoside
antibiotics, the hydrogel of kanamycin indicates that the hydrogel
of aminoglycosides preserve the specific interaction with their
macromolecular targets (e.g., 16S rRNA), thus illustrating a
simple approach to explore and identify possible biological targets
of supramolecular nanofibers/hydrogels.
This communication reports the first hydrogelator derived
Scheme 1 Supramolecular nanofibers sequester the potential targets
from an aminoglycoside antibiotic (e.g., kanamycin1) and
(e.g., 16S rRNA (in red)) in the gel phase.
the resultant hydrogel for sequestering 16S rRNA selectively
via divalent interaction. Hydrogelators are small molecules
the formation of supramolecular nanofibers, which entangle to
imbibe water and to afford hydrogels.2 The early development of
result in a network, stop the flow of water, and afford a hydrogel.
that self-assemble in water to yield supramolecular nanofibers to
hydrogelators,3 as a curiosity of physical organic chemistry,4 has
Thus, hydrogelation can serve as a simple visual assay to indicate
the self-assembly of small molecules in water. After the hydrogel
demonstrated that carefully balanced hydrophobic interactions
reaches the steady state, it is possible to add the solution containing
and hydrogen bonding induce the aggregation of small molecules
the potential target and appropriate control molecules on the top of
in water to form nanofibers and afford hydrogels. After that, the
the hydrogel. The gel phase is in contact with the solution, but the
successful integration of biofunctional or bioactive molecules into
the hydrogelators5 has led to rapid development of supramolecular
two phases remain immiscible. While the nanofibers remain in
hydrogels6,7 as a new class of biomaterials that show precise
the gel phase, the target and control molecules, however, can enter
control on molecular structures and supramolecular organization.2
the gel phase by diffusing through the pores of the network. The
specific interaction of the nanofibers with the targets would retain
For example, the hydrogelator of vancomycin has exhibited high
activities towards vancomycin resistance enterococci (VRE).8,9
more target molecules than the control molecules in the gel phase.
Since it is easy to separate the gel phase from the solution phase by
Although a vast pool of bioactive molecules, ranging from
peptides10 to clinically used drug molecules,9,11 are able to serve
aspiration of the supernatant, the analysis of the remaining
molecules in the solution phases (i.e., the supernatant) should be
as building blocks for constructing supramolecular assemblies
able to identify the target molecules sequestered in the hydrogel due
with a superior effectiveness that is unattainable by monomeric
to the specific interaction between the target molecules and the
epitopes or individual drug molecules, there is little exploration
supramolecular nanofibers.
on the self-assembly of functional small molecules in water,
To demonstrate the concept illustrated in Scheme 1, we prepared
partially due to the lack of a general approach to promote
the hydrogelator of kanamycin (1) by attaching multiple
phenylalanine residues to the 60-N amino group of kanamycin
molecular self-assembly and to verify the interactions between
the supramolecular assemblies and their target(s).
A, which is a member of aminoglycoside antibiotics that bind
We hypothesized that the conversion of a functional molecule
16S rRNA in ribosome to inhibit protein synthesis and act as
to a hydrogelator offers a simple way to create the self-assembly of
bactericides.1 We found that 1 not only acts as an effective
the functional molecule and to verify the interactions between the
hydrogelator at neutral pH with critical gelation concentrations,
nanofibers and the potential targets. Scheme 1 illustrates
cgc, at 0.3 wt%, but the hydrogel of 1 also selectively sequesters 16S
the concept. The self-assembly of the hydrogelators leads to
rRNA—the receptor of kanamycin A. Fluorescent titration
confirms two molecules of 1 bind to the same site on the
receptor (i.e., A-site of 16S rRNA,1,12). This work not only
a Key Laboratory of Bioactive Materials, Ministry of Education,
College of Life Sciences, Nankai University, Tianjin, 300071,
indicates that the hydrogel of aminoglycosides preserve the
P. R. China
b Department of Chemistry, Brandeis University, 415 South St.,
Waltham, MA 02454, USA. E-mail: bxu@brandeis.edu;
Fax: +1-781-736-5201
specific interaction with their macromolecular targets (e.g.,
16S rRNA1), but also illustrates a feasible approach for
exploring molecular self-assembly in water and screening the
binding of the resulting supramolecular nanofibers/hydrogels
to the potential target(s).
w Electronic supplementary information (ESI) available: Details of
materials, synthesis, characterization and measurements. See DOI:
10.1039/c2cc34935c
c
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Chem. Commun., 2012, 48, 9257–9259 9257

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Scheme 2 Structures of hydrogelators based on kanamycin A.
Fig. 2 (A) A possible molecular arrangement of 1 in the gel phase
(some hydrogen bonds shown as the dot lines). (B) A hypothetical
binding between two molecules of 1 and their receptor (A-site of 16S
rRNA) derived from the crystal structure of kanamycin A.12
Scheme 2 shows the structure of the hydrogelators of kanamycin
A, 1 and 2. The synthesis of 1 is straightforward. Being activated by
N-hydroxysuccinimide (NHS), 2-(naphthalen-2-yl)acetamido)-3-
phenylpropanamido)-3-phenylpropanoic acid (NapFF)13 reacts
with kanamycin in a mixture of acetone and water (pH B 8.0)
for 24 h to afford the crude product, which undergoes chromato-
graphic purification to give 1 as a white powder. Typically, 2 mg of
1 dissolves in 0.4 mL of water at pH 2.0 to result in a clear solution,
which turns into a transparent hydrogel (gel I) after the pH of the
solution was adjusted to 7.0. The transparent hydrogel easily
changes back to a clear solution when the pH is lowered to less
than 6.5 or the temperature is increased to higher than 46 1C. The
hydrogel of 2 (Gel II) is formed similarly. The resulting transparent
hydrogels (Fig. 1A and B) are stable at room temperature for
months without visible changes.
by the peptidic backbone and kanamycin A. The emission
spectrum of 1 (Fig. S1w) shows a peak centered at 336 nm in
solution and at 342 nm in the gel phase, indicating the monomeric
naphthalene moieties. The additional peaks at 381 and 400 nm
suggest strong interactions between the aromatic parts (naphthyl
and phenyl aromatics) of the molecules, which is consistent with
its self-assembled nanofibers observed in the TEM image. Based
on the above information and the crystal structure of kanamycin
A12 and NapFF,13 we propose a possible molecular arrangement
of 1 in the gel phase. As shown in Fig. 2, the NapFF motif plays
an important role for the formation of supramolecular structures:
the p–p interactions of aromatic rings and the intermolecular
hydrogen bonds between peptide bonds help 1 self-assemble into
a b-sheet like chain. Part of the kanamycin A sticks out to the
aqueous environment and forms hydrogen bonds with water
molecules and other kanamycin molecules to help in the formation
of nanofibers. Although other modes of molecular arrangements
may exist in the gel phase, the current evidences indicate that the
arrangement in Fig. 2A likely dominates.
To study the nanostructures or morphology of the hydrogels, we
obtained their transmission electron micrographs (TEM) (Fig. 1C
and D), which show small fibrils with the diameters from 50 nm to
60 nm. The elongated small fibrils (longer than 5 mm) tend to tangle
with each other and form a dense network, which serves as the
matrices of gels I and II. According to the dynamic frequency
sweep of gel I, the values of the storage moduli (G0) exceed the loss
moduli (G00) by a factor of about 5 (Fig. S2w), indicating that the
sample behaves like a solid.7 The value of G0 of the sample is higher
than 600 Pa, indicating that the sample is viscoelastic. Both the
values of G0 and G00 of the hydrogel exhibit weak dependence with
frequency (from 0.1 to 100 rad sꢀ1), suggesting that the matrices of
the gel have a good tolerance to external force.
It is well established that kanamycin A bound to the A-site
of 16S rRNA to lead to the misreading of mRNA and the
death of the bacteria.1,12,14 To prove further that 1 binds to
the A-site of 16S rRNA, we add a commercial oligonucleotide,
50-UUGCGUCACACCGGUGAAGUCGC-30 (i.e., A-site of
16S rRNA12), to a solution of 1 and observed the change of
fluorescence upon binding. We choose to evaluate the binding in
PBS buffer to minimize the influence of the self-quenching of the
naphthalene of 1 in the gel phase. Fig. 3A clearly shows that the
intensity of the fluorescent peak at around 336 nm decreases after
the addition of the oligonucleotide. The fluorescent peak almost
disappears after the addition of 7.33 equivalents of the oligo-
nucleotide. Based on this fluorescent titration (Fig. 3A),15 the
calculated binding number is 1.97 and the association constant
We used circular dichroism (CD) and fluorescent spectroscopy to
investigate the molecular arrangement of the small molecule in gel I
(Fig. S1w). A positive band near 196 nm (pp* transition) and a
negative peak at about 208 nm (np* transition) reveal the b-sheet
like conformation of the peptidic backbones. The broad peak
centered at 246 nm (pp* transition of phenyl groups), two small
peaks at 286 nm (pp* transition of naphthyl aromatics) and 296 nm
(np* transition of naphthyl aromatics) originate from the aromatic
parts of the molecules, which adopt helical arrangements induced
Fig. 3 (A) Fluorescence spectra of solution of 1 (1 mg mLꢀ1) and
mixtures of 1 with different equivalents of oligonucleotide (A site
of 16S rRNA) in PBS buffer solution. (B) Gel electrophoresis of
16S rRNA and 23S rRNA in the extract before and after incubation
with hydrogel of 1 for 4 h.
Fig. 1 Optical images of (A) gel I formed by 1 (0.5 wt%, pH 7.0); (B)
gel II formed by 2 (1.0 wt%, pH 7.0); TEM of (C) gel I and (D) gel II.
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is 4.0 ꢁ 109 M between 1 and the A-site of 16S rRNA. This result
indicates two molecules of 1 bind to the same A-site of 16S rRNA,
which agrees with the recently reported crystal structures of the
interaction between the ribosomal subunit and aminoglycoside
antibiotics12,14 that reveals two aminoglycosides to bind to the
same A site of 16S rRNA.12 The association constant is much
higher than the binding constant of kanamycin with 16S rRNA
(5.6 ꢁ 105 M),16 suggesting cooperative binding.
The authors acknowledge the partial financial support
from NIH (R01CA142746), HFSP, and a start-up fund from
Brandeis University and thank the assistance of Brandeis EM
facility.
Notes and references
1 L. Brunton, J. Lazo, K. Parker, I. Buxton and D. Blumenthal, The
Pharmacological Basis of Therapeutics, McGraw-Hill, New York,
2005.
To verify whether the hydrogel of 1 is able to bind selectively
to the 16S rRNA, we extracted total RNAs from E. coli and
added the solution containing total RNAs on the top of the
hydrogel of 1. Gel electrophoresis shows that 5S, 16S and 23S
rRNA are the major components of the total RNAs (Fig. S2w),
in which the initial ratio of 16S : 23S : 5S rRNA is about
1 : 1 : 8. The large quantity of 5S rRNA in the total RNA,
together with 23S rRNA, serves as a useful control. After being
incubated with the hydrogel of 1 for 4 h, the amounts of RNAs
in the supernatant decrease due to the absorption of RNA in the
hydrogel of 1, as indicated by gel electrophoresisw. Quantitative
analysis of the RNA bands in Fig. 3B confirms that 20% of 16S
rRNA and 70% of 23S rRNA remain in the supernatant.
Despite the initial amount of 5S rRNA being about eight times
of that of 16S rRNA (or 23S rRNA), 70% of 5S rRNA remains
in the supernatant. This result suggests that the hydrogel of 1
selectively sequesters 16S rRNA. Because only 30% of 5S
sRNAw undergoes uptake by the hydrogel of 1 (though the size
of 5S rRNA is much smaller and the quantity of 5S rRNA in the
total RNA much larger than those of 16S rRNA), the observed
selectivity is unlikely to arise from the smaller size of 16S rRNA
compared to 23S rRNA. Rather, the above results clearly
indicate that 1 binds to the A-site of 16S rRNA, thus leading
to the hydrogel of 1 binding selectively to 16S rRNA. If the
hydrogel of 1 bound extracted RNAs via only electrostatic
attractions, the hydrogel of 1 would bind 23S rRNA that carries
the most negative charges among the extracted RNAs. Therefore,
the hydrogel of 1 selectively binds 16S rRNA, which also indicates
that the derivatization at the N60 position is unlikely to completely
eliminate the binding of 1 to the A-site of 16S rRNA. Thus, we
suggest that the self-assembled molecules of 1 to bind with 16S
rRNA as shown in Fig. 2B, which is a possible interaction of 1 with
the A-site of 16S rRNA derived from the binding of kanamycin to
the RNA groove without involving the ammonium N60.12
Although the suggested model in Fig. 2B agrees with the 2 : 1
stoichiometry (between 1 and the A-site of 16S rRNA) obtained
from Fig. 3A, the atomistic description of the interactions between 1
and the A-site of 16S rRNA has yet to be established.
2 L. A. Estroff and A. D. Hamilton, Chem. Rev., 2004, 104,
1201.
3 L. A. Estroff and A. D. Hamilton, Angew. Chem., Int. Ed., 2000,
39, 3447; J. H. Jung, G. John, M. Masuda, K. Yoshida, S. Shinkai
and T. Shimizu, Langmuir, 2001, 17, 7229; M. Suzuki, M. Yumoto,
M. Kimura, H. Shirai and K. Hanabusa, Chem. Commun., 2002,
884.
4 P. Terech and R. G. Weiss, Chem. Rev., 1997, 97, 3133.
5 F. Zhao, M. L. Ma and B. Xu, Chem. Soc. Rev., 2009, 38, 883.
6 R. J. Williams, R. J. Mart and R. V. Ulijn, Biopolymers, 2010,
94, 107; R. V. Ulijn, B. Baragana, P. J. Halling and S. L. Flitsch,
J. Am. Chem. Soc., 2002, 124, 10988; R. J. Williams, A. M. Smith,
R. Collins, N. Hodson, A. K. Das and R. V. Ulijn, Nat. Nanotechnol.,
2008, 4, 19; S. Kiyonaka, K. Sada, I. Yoshimura, S. Shinkai, N. Kato
and I. Hamachi, Nat. Mater., 2003, 3, 58; D. J. Adams and
P. D. Topham, Soft Matter, 2010, 6, 3707; N. M. Sangeetha and
U. Maitra, Chem. Soc. Rev., 2005, 34, 821.
7 C. Q. Yan and D. J. Pochan, Chem. Soc. Rev., 2010, 39, 3528.
8 B. G. Xing, P. L. Ho, C. W. Yu, K. H. Chow, H. W. Gu and B. Xu,
Chem. Commun., 2003, 2224.
9 B. G. Xing, C. W. Yu, K. H. Chow, P. L. Ho, D. G. Fu and B. Xu,
J. Am. Chem. Soc., 2002, 124, 14846.
10 G. A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. Harrington,
J. A. Kessler and S. I. Stupp, Science, 2004, 303, 1352; S. Toledano,
R. J. Williams, V. Jayawarna and R. V. Ulijn, J. Am. Chem. Soc.,
2006, 128, 1070; J. Gao, H. M. Wang, L. Wang, J. Y. Wang,
D. L. Kong and Z. M. Yang, J. Am. Chem. Soc., 2009, 131, 11286;
J. P. Schneider, D. J. Pochan, B. Ozbas, K. Rajagopal, L. Pakstis
and J. Kretsinger, J. Am. Chem. Soc., 2002, 124, 15030;
C. M. Micklitsch, P. J. Knerr, M. C. Branco, R. Nagarkar,
D. J. Pochan and J. P. Schneider, Angew. Chem., Int. Ed., 2011,
50, 1577.
11 Y. Gao, Y. Kuang, Z. F. Guo, Z. H. Guo, I. J. Krauss and B. Xu,
J. Am. Chem. Soc., 2009, 131, 13576; H. M. Wang, C. H. Yang,
L. Wang, D. L. Kong, Y. J. Zhang and Z. M. Yang, Chem.
Commun., 2011, 47, 4439.
12 B. Francois, R. J. M. Russell, J. B. Murray, F. Aboul-ela,
B. Masquida, Q. Vicens and E. Westhof, Nucleic Acids Res.,
2005, 33, 5677.
13 Y. Zhang, Y. Kuang, Y. A. Gao and B. Xu, Langmuir, 2011,
27, 529.
14 A. P. Carter, W. M. Clemons, D. E. Brodersen, R. J. Morgan-
Warren, B. T. Wimberly and V. Ramakrishnan, Nature, 2000,
407, 340.
15 D. M. Chipman, V. Grisaro and N. Sharon, J. Biol. Chem., 1967,
242, 4388.
16 M. Misumi, T. Nishimura, T. Komai and N. Tanaka, Biochem.
Biophys. Res. Commun., 1978, 84, 358.
17 Y. Gao, M. J. C. Long, J. F. Shi, L. Hedstrom and B. Xu, Chem.
Commun., 2012, 48, 8404.
In summary, we have designed and synthesized novel molecular
hydrogelators based on kanamycin A and demonstrated a feasible
approach to explore the potential targets of supramolecular
nanofibers/hydrogels. One potential practical application of this
hydrogel of kanamycin would be the enrichment of 16S rRNA
from cell lysates for typing bacteria. However, the selectivity of the
gel towards 16S remains to be improved, possibly by designing
and screening new kanaymicin containing hydrogelators. The
principle illustrated in this work should help identify new targets of
supramolecular nanostructures,17 which may lead to new insights
or phenomena that are inaccessible by an individual molecule.18
Our future works will focus on the modifications of other amino-
functions of kanamycin A and other aminoglycosides.
18 Z. L. Wu, T. Kurokawa, S. Liang, H. Furukawa and J. P. Gong,
J. Am. Chem. Soc., 2010, 132, 10064; P. Casuso, P. Carrasco,
I. Loinaz, H. J. Grande and I. Odriozola, Org. Biomol. Chem.,
2010, 8, 5455; N. Kameta, K. Yoshida, M. Masuda and
T. Shimizu, Chem. Mater., 2009, 21, 5892; G. M. Price,
K. K. Chu, J. G. Truslow, M. D. Tang-Schomer, A. P. Golden,
J. Mertz and J. Tien, J. Am. Chem. Soc., 2008, 130, 6664;
M. D. Tang, A. P. Golden and J. Tien, Adv. Mater., 2004,
16, 1345; J. F. Lovell, A. Roxin, K. K. Ng, Q. C. Qi,
J. D. McMullen, R. S. DaCosta and G. Zheng, Biomacromolecules,
2011, 12, 3115; G. L. Liang, H. J. Ren and J. H. Rao, Nat. Chem.,
2009, 2, 54; S. W. Choi, Y. Zhang and Y. N. Xia, Angew. Chem.,
Int. Ed., 2010, 49, 7904.
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This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 9257–9259 9259