Smart Turn-on Magnetic Resonance Contrast Agents Based on Aptamer

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Smart Turn-on Magnetic Resonance Contrast Agents Based on Aptamer Powered By Docstoc
					DOI: 10.1002/cbic.200700323                                                         field is the development of novel MRI contrast agents, particu-
                                                                                    larly smart agents that can produce a contrast in response to
Smart “Turn-on” Magnetic                                                            small molecules or biomolecular markers in cells or human
                                                                                    bodies. Such smart MRI contrast agents could dramatically
Resonance Contrast Agents Based                                                     change the way we study cellular function and could help to
                                                                                    achieve early diagnosis for diseases. Toward this goal, organic
on Aptamer-Functionalized                                                           receptors and proteins have been coupled to MRI contrast
Superparamagnetic Iron Oxide                                                        agents, such as gadolinium and superparamagnetic iron oxide
                                                                                    nanoparticles. These contrast agents result in a change of MRI
Nanoparticles                                                                       contrast in the presence of molecules to which these organic
                                                                                    receptors or proteins bind.[34–38] To develop a general strategy
Mehmet Veysel Yigit,[b, d] Debapriya Mazumdar,[a, d]                                         for obtaining smart MRI agents for any molecule of choice, we
Hee-Kyung Kim,[a] Jung Heon Lee,[c, d] Boris Odintsov,[d]                                    report a method for the production of smart contrast agents
and Yi Lu*[a, b, c, d]                                                                       by combining aptamer technology with superparamagnetic
                                                                                             iron oxide nanoparticles—specifically a method based on ade-
Aptamers are single-stranded DNA or RNA molecules that can                                   nosine DNA aptamer-functionalized superparamagnetic iron
bind a variety of chemical and biological molecules with high                                oxide nanoparticles—and show that MRI contrast can be dra-
affinity and selectivity.[1–4] They are isolated from a large                                matically enhanced in the presence of adenosine in human
         random pool of DNA or RNA molecules by using a combinato-                           serum. In addition, this MRI enhancement was highly selective
         rial-biology technique called systematic evolution of ligands by                    for adenosine as the presence of all other nucleobases did not
         exponential enrichment (SELEX).[1, 2] They are often comparable                     result in enhancement effect.
to antibodies in their selective and sensitive binding to a                                     We chose superparamagnetic iron oxide nanoparticles as
broad range of molecules.[5–8] The major advantage of these                                  the contrast agent to be functionalized by aptamers since they
molecules over antibodies lies in the relative ease with which                               are efficient at dephasing the spins of neighboring water pro-
they can be selected for any target analyte and their stability                              tons; this leads to a change in the spin–spin relaxation time
against biodegradation and denaturation. Due to these proper-                                (T2).[39, 40] Weissleder and co-workers have shown that oligonu-
ties aptamers are good candidates for making chemical and                                    cleotide-functionalized, cross-linked dextran-coated superpara-
biological sensors in many fields, such as medical diagnostics                               magnetic iron oxide nanoparticles (CLIOs) can form clusters
and environmental monitoring. Therefore, aptamers have been                                  when linked with a complementary sequence; each nanoparti-
converted into fluorescent,[9–22] colorimetric,[23–29] and electro-                          cle can on average be functionalized with three oligonucleo-
         chemical sensors.[30–33] While aptamer sensors have been wideACHTUNGREly            tides.[41–43] The magnetic properties of dispersed nanoparticles
explored in vitro, their application in vivo, particularly in hu-                   are significantly different from those in clusters, and can be
ACHTUNGREmans, remains a significant challenge because of the difficulty            ACHTUNGREdetected by MRI. As compared to disperse nanoparticles, the
         in light penetration through the skin and signal interference                       clusters are more effective in decreasing the T2 relaxation
         from cellular components.                                                           time, and therefore give rise to a darker T2-weighted MR
            Magnetic resonance imaging (MRI) is a powerful method for                        image.
         noninvasive three-dimensional imaging of cells and human                               To expand the method to aptamer-based sensing of mole-
         bodies. One active area of research in this rapidly advancing                       cules other than nucleic acids, we combined CLIOs with adeno-
                                                                                             sine aptamer (Scheme 1). The adenosine sensor consisted of
[a] D. Mazumdar,+ H.-K. Kim, Prof. Y. Lu                                                     CLIO aggregates that were prepared by using three compo-
    Department of Chemistry                                                                  nents: CLIO functionalized with either 3’ or 5’ thiol-modified
    University of Illinois at Urbana–Champaign                                      DNA (3’Adap–CLIO and 5’Adap–CLIO, respectively) and a linker
    600 S. Mathews Avenue, Urbana, IL 61801 (USA)
    Fax: (+ 1) 217-333-2685
                                                                                    DNA (linker–Adap) that could hybridize to both 3’- and
    E-mail: yi-lu@uiuc.edu                                                          5’Adap–CLIOs; this led to the formation of clusters. One seg-
[b] M. V. Yigit,+ Prof. Y. Lu                                                       ment of the linker was the sequence for the adenosine apta-
    Center for Biophysics and Computational Biology                                 mer (Scheme 1, in green). Seven bases of this aptamer were in-
    University of Illinois at Urbana–Champaign
                                                                                    volved in hybridization with 5’Adap–CLIO. In the presence of
    607 S. Mathews Avenue, Urbana, IL 61801 (USA)
                                                                                    adenosine the aptamer undergoes structure switching in order
[c] J. H. Lee, Prof. Y. Lu
    Department of Materials Science and Engineering                                 to form the adenosine binding pocket;[17, 29, 44, 45] this results in
    University of Illinois at Urbana–Champaign                                               disruption of base-pairing interactions with 5’Adap–CLIO. The
    1304 W. Green Street, Urbana, IL 61801 (USA)                                    five remaining base pairs between linker–Adap and 5’Adap–
[d] M. V. Yigit,+ D. Mazumdar,+ J. H. Lee, Dr. B. Odintsov, Prof. Y. Lu                      CLIO are not enough to hold the clusters together at room
    Beckman Institute for Advanced Science and Technology
                                                                                             temperature; this leads to the disassembly of the clusters. The
    University of Illinois at Urbana–Champaign
    405 N. Mathews Avenue, Urbana, IL 61801 (USA)                                            dispersed nanoparticles result in a higher T2 as compared to
[+] These authors contributed equally to this work.                                          the clusters; thus the adenosine-induced disassembly can be
    Supporting information for this article is available on the WWW under                    monitored as an increase in T2 values and enhancement in
    http://www.chembiochem.org or from the author.                                           brightness of T2-weighted MR images.


ChemBioChem 2007, 8, 1675 – 1678                     2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim                                                1675
Scheme 1. Schematic representation of detection of adenosine by using MRI. The numerals 1 and 2 represent the two kinds of nanoparticles used for detec-
tion—3’Adap–CLIO and 5’Adap–CLIO, respectively. The DNA sequences are shown at the bottom. The two nucleobases highlighted in grey in the adenosine
aptamer segment of linker–Adap are the points of mutation (T!A, G!C) in the mutated linker sequence.



  CLIO was synthesized according to literature procedures and                 plated CLIO structures, lower concentrations of CLIO (~ 20 mg
functionalized with N-succinimidyl 3-(2-pyridyldithio)-propio-                Fe mLÀ1) were used to avoid precipitation.[43] Measurements
nate (SPDP), which could be readily coupled to thiol-modified                 taken by using dynamic light scattering (DLS) showed the
DNA (3’Adap or 5’Adap).[41, 46] To demonstrate sequence specific              average diameter of dispersed CLIO to be (50 Æ 6) nm, while
aptamer-templated assembly, equimolar mixtures of 3’Adap–                     linker–Adap induced hybridization yielded CLIO clusters that
CLIO and 5’Adap–CLIO (100 mg Fe mLÀ1 each) were incubated                     were (187 Æ 21) nm in diameter (see the Supporting Informa-
with linker–Adap; this led to the formation of big clusters of                tion).
nanoparticles that precipitated (Figure 1 C). The same effect                    In order to measure T2, CLIO–DNA conjugates at the lower
was not observed when a noncomplementary sequence was                         concentration of ~ 20 mg Fe mLÀ1 each, were mixed with
used (Figure 1 B). To measure the diameter of the DNA-tem-                    linker–Adap so that the clusters could not precipitate. The pre-
                                                                              pared sensor was then dispensed into the wells of a microplate
                                                                              with progressively increasing amounts of adenosine, and a T2-
                                                                              weighted MR image was obtained. An increase in adenosine
                                                                              concentration led to an increase in the brightness of the
                                                                              image (Figure 2 A). This was attributed to the increase in T2
                                                                              due to the disassembly of CLIO clusters into smaller particles.
                                                                              It is worth noting that a change in contrast was detected even
                                                                              at 10 mm adenosine.
                                                                                 To ensure that the observed effect was solely due to specific
Figure 1. Effect of incubating a mixture of 3’Adap–CLIO and 5’Adap–CLIO
A) without linker, B) with a noncomplementary DNA linker, C) with linker–     binding of adenosine by the aptamer rather than other non-
Adap.                                                                         specific effects, a mutated DNA linker with variation in only


1676          www.chembiochem.org                    2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim               ChemBioChem 2007, 8, 1675 – 1678
                                                                                                                  ameter of the CLIO clusters can
                                                                                                                  be determined by using DLS,
                                                                                                                  the T2 value can be correlated
                                                                                                                  to the number of particles per
                                                                                                                  cluster.[47] In the lower concen-
                                                                                                                  tration range (0–1 mm) T2 was
                                                                                                                  linearly dependent on the con-
                                                                                                                  centration of adenosine, and
                                                                                                                  saturation was reached at
                                                                                                                  2.5 mm. To demonstrate utility
                                                                                                                  and stability of the current
                                                                                                                  system in vivo, the sensor was
Figure 2. T2-weighted MR image of samples in a 96-well microplate. Effect of increasing adenosine concentration
in CLIO–DNA conjugates assembled either with A) linker–Adap, or B) mutated linker. C) Sensor with 5 mm adeno-     prepared in the presence of
sine (A), cytidine (C), uridine (U), and guanosine (G).                                                           human serum (10 %). The activi-
                                                                                                                  ty of the sensor was found to
                                                                                                                  be retained in the presence of
two bases was used as control (Scheme 1, legend). This mutat-                    serum. Upon addition of adenosine the contrast within the
ed linker has been shown not to bind adenosine.[17, 29] As was                   concentration gradient increased (Figure 4).
expected, no change in brightness was observed
with increasing adenosine concentration (Figure 2 B).
In a separate control aimed at investigating the
ACHTUNGREselectivity of the system, the sensor was incubated
         with 5 mm of cytidine, uridine, or guanosine. As
         seen in Figure 2 C, a significant change in contrast
         was not observed in any of these three cases.             Figure 4. T2-weighted MR image of adenosine-induced disassembly of sensor in human
            Quantitative analysis was performed by measuring       serum (10 %).
         the T2 relaxation times of samples with different ad-
         enosine concentrations. A clear increase in T2 values
         from 36 to 63 ms was observed as the adenosine concentra-                 In conclusion, we have demonstrated a general method for
         tion was raised from 0 to 2.5 mm (Figure 3), and the initial T2         aptamer-based biosensing by using MRI. The adenosine apta-
value of dispersed nanoparticles was reached.[41] Since the di-                  mer-based design serves as a model system for similar smart
                                                                                            and functional CLIO contrast agents. Since aptamers
                                                                                            specific for a variety molecular markers of biological
                                                                                            functions and diseases can be obtained through
                                                                                            SELEX, this method can be applied for early molecu-
                                                                                            lar diagnosis for a number of targets.


                                                                                          Experimental Section
                                                                                          Materials: All DNA samples were purchased from Inte-
                                                                                          grated DNA Technologies Inc. (Coralville, IA, USA). Linker
                                                                                          DNA sequences were purified by HPLC whereas thiol-
                                                                                          modified DNA molecules were purified by the standard
                                                                                          desalting method. Adenosine, cytidine, uridine, and
                                                                                          guanosine were purchased from Aldrich (St. Louis, MO,
                                                                                          USA). Cross-linked dextran-coated superparamagnetic
                                                                                          iron oxide nanoparticles (CLIOs; 500 mg Fe mLÀ1) were
                                                                                          synthesized and coupled to N-succinimidyl 3-(2-pyridyl-
                                                                                          dithio)-propionate (SPDP) according to literature proce-
                                                                                          dure and purified by using a PD-10 column.[46] Thiol-
                                                                                          modified oligonucleotides, 3’Adap (5’-TCACAGATGAGT-
                                                                                          A12-SH-3’) and 5’Adap (5’-SH-CCCAGGTTCTCT-3’) were
                                                                                          activated by being incubated with tris(2-carboxyethyl)
                                                                                          phosphine hydrochloride (TCEP; 8 equiv). Excess TCEP
                                                                                          was removed by desalting with a SepPak C-18 cartridge.
                                                                                          TCEP-activated thiol-modified DNA (50 mm final concen-
Figure 3. Disassembly of sensor clusters with increasing concentration of adenosine, as   tration) was mixed with CLIO–SPDP (400 mg Fe mLÀ1) in
detected by change in T2 relaxation times. The lower panel shows the T2-weighted MR       phosphate buffer (100 mm) at pH 8.0, overnight. Excess
image of the samples.                                                                     DNA was removed by using a magnetic separation


ChemBioChem 2007, 8, 1675 – 1678             2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim             www.chembiochem.org                1677
column (Miltenyi Biotec, Auburn, CA, USA) from CLIO–DNA conju-                    [10] S. D. Jhaveri, R. Kirby, R. Conrad, E. J. Maglott, M. Bowser, R. T. Kennedy,
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                                                                                       123, 4928.
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1678             www.chembiochem.org                   2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim                   ChemBioChem 2007, 8, 1675 – 1678