ARTICLE pubs.acs.org/ac
Design of Aptamer-Based Sensing Platform Using Triple-Helix Molecular Switch Jing Zheng,† Jishan Li,† Ying Jiang,† Jianyu Jin,† Kemin Wang,† Ronghua Yang,*,† and Weihong Tan†,‡ †
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China ‡ Center for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center and UF Genetics Institute, University of Florida, Gainesville, Florida, 32611-7200, United States
bS Supporting Information ABSTRACT: For successful assay development of an aptamer-based biosensor, various design principles and strategies, including a highly selective molecular recognition element and a novel signal transduction mechanism, have to be engineered together. Herein, we report a new type of aptamer-based sensing platform which is based on a triplehelix molecular switch (THMS). The THMS consists of a central, target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide serving as a signal transduction probe (STP). The STP is doubly labeled with pyrene at the 50 - and 30 -end, respectively, and initially designed as a hairpin-shaped structure, thus, bringing the two pyrenes into spacer proximity. Bindings of two arm segments of the aptamer with the loop sequence of STP enforce the STP to form an “open” configuration. Formation of aptamer/target complex releases the STP, leading to new signal readout. To demonstrate the feasibility and universality of our design, three aptamers which bind to human R-thrombin (Tmb), adenosine triphosphate (ATP), and L-argininamide (L-Arm), respectively, were selected as models. The universality of the approach is achieved by virtue of altering the aptamer sequence without change of the triple-helix structure.
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uccessful design and development strategies for aptamer-based biosensors, or aptasensors, are of great interests in the fields of chemistry and biotechnology.1,2 Aptamers are single-stranded RNA or DNA oligonucleotides with unique intramolecular conformation. They are isolated from large random-sequence nucleic acid libraries by “in vitro selection”.3,4 Ease of chemical synthesis and generally impressive target selectivity and affinities make aptamers ideal recognition elements for biosensor applications.5,6 To design an aptasensor, the most common strategy normally utilizes conformational alteration of the aptamer during the aptamertarget binding event. Essentially, these designs use the spatial change of the aptamer sequence termini to produce a fluorescent or electrochemical signal.714 As such, for any given aptamer sequence, this approach should provide a simple method for aptasensor, but the unpredictability of such structural alteration may not induce perceptible signal transduction. When the molecular beacon concept is adopted for the detection of nucleic acid,15 this problem has been addressed with the design of hairpin structurebased signaling aptamers.1618At the same time, however, labeling the aptamer with another signaling reporter tends to significantly reduce the aptamer’s affinity and specificity to the target molecules.19,20 Therefore, in addition to these strategies, doublehelix DNA molecular switches, which function by switching structures from DNA/DNA duplex to DNA/target complex, have been successfully developed.2126 In the approach, a signal transduction probe (STP) hybridizes with partial or whole aptamer sequence but r 2011 American Chemical Society
allows dehybridization when the aptamer binds to a target molecule. Although this strategy does offer a new means of signaling an aptamertarget binding event without aptamer labeling, it also suffers from intrinsic limitations. That is, in order to achieve suitable hybridization without hindering the recognition and affinity of an aptamer toward its target, lengthy optimization of the STP is often involved. This optimization requires resynthesis of several different STPs which are generally labeled with fluorescent or electrochemical signal elements. Also, generalizability remains a problem since the STP is aptamer sequence specific and, hence, limited by the aptamer-binding target against which it can be deployed. Herein, we describe a new molecular engineering mechanism for the development of a convenient and universal aptamer-based sensing platform. Human R-thrombin (Tmb), adenosine triphosphate (ATP), and L-argininamide (L-Arm) were selected as targets. Conceptually different from the established molecular beacon-based signaling aptamers1618 or double-helix DNA molecular switches,2226 we designed here a triple-helix molecular switch (THMS) based on WatsonCrick and Hoogsteen base pairings.2732 The THMS consists of a central, target specific aptamer sequence flanked by two arm segments and a dual-labeled oligonucleotide serving as a signal transduction probe. Received: April 18, 2011 Accepted: July 28, 2011 Published: July 28, 2011 6586
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Analytical Chemistry Aptamertarget binding results in the formation of a structured aptamer/target complex which disassembles the THMS and releases the STP. Compared to the known molecular beacon-based signaling aptamers and double-helix DNA molecular switches, the THMS possesses some remarkable features. First, by separating the molecular recognition element and signal reporter, there is no need to make any labeling of the original aptamer, which is required in many of the aptamer-based detections to preserve the affinity and specificity of the original aptamer. Second, formation of a triple-helix structure with two short-armed complementary oligos of the aptamer achieves the same stability as designs with a long complementary oligo yet leaves more aptamer sequence free, thereby keeping the binding affinity and specificity of the aptamer, and even enabling higher sensitivity. Most important, this strategy is generalizable; one STP can detect multiplicate targets by selecting different types of aptamers, and this allows for facile introduction of various targets without increasing the complexity and cost of the STP synthesis. Therefore, we demonstrated that this approach is not only sensitive and selective but also convenient and generalizable.
’ EXPERIMENTAL SECTION Materials and Apparatus. All oligonucleotides were synthesized by TaKaRa Biotechnology Co. Ltd. (Dalian, China). All sequences were dissolved in highly pure water (sterile Minipore water, 18.3 MΩ) as stock solutions; the concentrations of the solution were estimated by UV absorption using published sequencedependent absorption coefficients.33 Human R-thrombin (Tmb), adenosine triphosphate (ATP), and L-argininamide (L-Arm) were purchased from Sigma-Aldrich and prepared using sterile water. All other chemical reagents were of analytical reagent grade and were purchased from Fluka (Switzerland). All work solutions were prepared with the sodium phosphate buffer (0.01 M). UVvis absorption spectra were recorded in 1 cm path length quartz cuvettes on a Hitachi U-3010 UV/vis spectrophotometer (Kyoto, Japan). The steady-state fluorescence emission spectra were obtained on a PTI QM4 Fluorescence System (Photo Technology International, Birmingham, NJ) with an accessory of temperature controller. Fluorescence emission spectra were collected using a bandwidth of 5 nm and 0.2 1 cm2 quartz cuvettes containing 500 μL of solution. pH was measured by model 868 pH meter (Orion). Kinetics and Thermodynamic Studies. To study the kinetics and time dependence of the interactions of THMS and subsequently the targets, the excimer fluorescence intensity of STP at 480 nm was recorded at 20 °C. The excimer fluorescence of 500 μL of STP (100 nM) was monitored for a few minutes. Then, Tmb-Apt or ATP-Apt was added to the probe buffer, and the final concentration was 120 nM; excimer fluorescence was measured at room temperature. After confirming that there was no change of fluorescence with time, Tmb or ATP was added and the final concentration was 100 nM or 0.5 mM, respectively; the level of fluorescence was then recorded with time. The thermal denaturation studies of THMS were carried out using quartz cuvettes with an optical path length of 1 cm on a Hitachi U-3010 UV/vis spectrophotometer (Kyoto, Japan) on a sample containing 1 μM STP and 1.2 μM corresponding TmbApts. The temperature control of the cell holder was achieved with a Neslab RTE-111 circulating water bath. The temperature of the water bath was decreased from 91.5 to 1.5 °C and increased
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back to 91.5 °C at a rate of 0.2 °C/min using the thermoprogramer. The absorbances at 245, 260, 295, and 305 nm were recorded every 5 min, whereas absorbance spectra were recorded in the 220420 nm range with a scan speed of 500 nm/min and a data interval of 1 nm. Temperature-dependent studies of the pyrene excimer fluorescence intensity of the triplex formed by STP and Tmb-Apt3 and duplex formed by STP and Tmb-Apt30 were measured on a PTI QM4 Fluorescence System at every 2.5 centigrade ranging from 5 to 80 °C. Binding Affinity Determination. A sensing strategy of binding affinity determination is adapted from the literature.21 For Tmb/Tmb-Apt3 binding affinity determination, the Tmb-Apt3 was labeled with TAMAR at the 50 -end (Table S1, Supporting Information), and the competitor was labeled with Dabcyl at the 30 -end (Table S1, Supporting Information). Before the Tmb was added, the Tmb-Apt3/competitor duplex was formed by adding a few microliters of competitor to 0.5 mL of 100 nM Tmb-Apt3 with a quartz cell in the sodium phosphate buffer containing 20 mM KCl and 2.5 mM MgCl2 at room temperature34 the final concentration of the competitor was 120 nM. For ATP-Apt/ ATP binding affinity determination, the ATP-Apt was labeled with TAMAR at the middle portion (Table S1, Supporting Information), and the competitor was labeled with Dabcyl at 30 -end (Table S1, Supporting Information). Before the ATP was added, the ATP-Apt/competitor duplex was formed by adding a few microliters of competitor to 0.5 mL of 100 nM ATP-Apt with a quartz cell in the sodium phosphate buffer containing 300 mM NaCl, 2.5 mM MgCl2, and 0.1 mM EDTA25 for 30 min; the final concentration of the competitor was 120 nM. Then, Tmb or ATP was added. For each data point, the fluorescence intensity of the solution was recorded and the titration curves were obtained: λex = 560 nm; λem = 580 nm. The dissociation constant (Kd) was obtained by fitting the dependence of fluorescence intensity of specific binding on the concentrations of the ligand to the equation: Y = BmaxX/(Kd + X) using the SigmaPlot software.35 Fluorescence Measurements. Before the targeting molecules were added, the THMS complex was formed by adding a few microliters of Tmb-Apt or ATP-Apt to 0.5 mL of 100 nM STP with a quartz cell; the addition was limited to 50 μL so that the volume change was insignificant. For Tmb detection, the THMS complex was formed in the sodium phosphate buffer containing 20 mM KCl and 2.5 mM MgCl2, pH 6.2, at room temperature for 30 min. For ATP detection, the THMS complex was formed in the sodium phosphate buffer containing 300 mM NaCl, 2.5 mM MgCl2, and 0.1 mM EDTA, pH 6.2, at 30 °C for 30 min. Following the additions of targeting molecules, the solutions were incubated at room temperature for 30 min and then the fluorescence intensities were recorded on a PTI QM4 Fluorescence System (Photo Technology International, Birmingham, NJ).
’ RESULTS AND DISCUSSION The signaling transduction scheme of our THMS is shown in Scheme 1. The THMS consists of a central, target-specific aptamer sequence (in green) flanked by two arm segments (in purple) and a STP (in blue). Originally, the STP adopts a hairpin structure by intramolecular DNA hybridization. Once the two arm segments of the aptamer’s are bound with the loop sequence of STP by WatsonCrick and Hoogsteen base pairings, a THMS structure is formed and thereafter maintained by a triple helical stem region, within which the STP forms an “open” configuration. 6587
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However, upon the introduction of a specific target, aptamertarget binding results in the formation of a structured aptamer/target complex which disassembles the THMS and releases the STP. The released STP then changes from the “open” configuration to the originally “closed” configuration, leading to new signal readout. To demonstrate the feasibility of the THMS principle, human R-thrombin (Tmb),17 adenosine triphosphate (ATP),36 and Largininamide (L-Arm)37 were used as our models. To fluorescently Scheme 1. Design Scheme of THMS for Signaling AptamerTarget Binding Eventa
“-” and “•” represent Watson-Crick and Hoogsteen base pairings, respectively. The detailed principles are described in the main text.
a
Figure 1. Fluorescence emission spectra of 100 nM STP (a), (a) + 120 nM Tmb-Apt3 (b), and (b) + 250 nM thrombin (c) in the sodium phosphate buffer at 20 °C. λex = 340 nm.
signal the aptamer-binding event, the STP was labeled with two pyrene fluorophores at the 30 - and 50 -ends, respectively (Figure S1, Supporting Information), taking advantage of the capability of pyrene to form an excited-state excimer with relatively high quantum yield and long lifetime.3840 Figure 1 shows the fluorescence emission spectra of the STP at different conditions. As expected, the free STP emits strong pyrene excimer emission. Formation of the triple-helix structure separates the pyrene groups at opposite sites, and only pyrene monomer emission is produced. Upon Tmb binding, the released STP forms a hairpin structure, thus reconstituting the proximity of the pyrene groups, which results in pyrene excimer emission and proves, in turn, the feasibility of the design. Because our THMS structure depends on the stability of the triplex stem formed by STP and the two arm segments of aptamer, in this work, a 20-base oligonucleotide was selected as the STP. This oligonucleotide has a hairpin structure, including a 5-base pair stem and a 10-base loop of d(AG)5 (Table 1). The melting temperature measurement (Tm) indicates that this STP has moderate thermal stability with a Tm of 40 °C (Figure S2, Supporting Information). To form THMS between STP and Tmb-Apt, we appended the Tmb-Apt at the 30 - and 50 -ends, respectively, with two arm segments of d(TC)n of varied lengths (n = 24) (Table 1). The two arm sequences were designed to bind the loop sequences of the STP to form a triplex motif through forming T-A•T and C-G•C+ base triplets.28 Then, we optimized the arm length of the aptamers by real-time monitoring of excimer fluorescence changes of STP upon the additions of Tmb-Apt and subsequent Tmb (Figure 2). For Tmb-Apt1, we found that the intermolecular interaction between the aptamer and STP could not disrupt the hairpin stem of STP, remaining high excimer emission. Increasing the sequence length of the arm segments of the aptamer, THMS was formed, and the excimer fluorescence from the STP was, correspondingly, weakened. Interaction of Tmb with the aptamer sequence restores the fluorescence emission of STP. However, once the triplex-stem length is increased to 8 bases, the THMS can not be disassembled even in the presence of a high concentration of Tmb. When 100 nM Tmb is added to the solutions of THMS1 to THMS4, the excimer fluorescence enhancements, E/E0, of STP are 1.08, 1.86, 4.12, and 1.03, respectively, where E0 and E are the pyrene fluorescence intensities at 480 nm in the absence and the presence of Tmb, respectively. From Figure 2, one can see that the fluorescence response of THMS3 to the introduction of Tmb is prompt, which delivered
Table 1. Oligonucleotide Sequences Used in This Worka entry
a
sequence
STP
50 -Py-GAGGAGAGAGAGAGATCCTC-Py-30
STP0 Tmb-Apt1
50 -Py-GGTTGGCCAACCACACCAACC-Py-30 50 -CTCTCGGTTGGTGTGGTTGGCTCTC-30
Tmb-Apt2
50 -CTCTCTGGTTGGTGTGGTTGGTCTCTC-30
Tmb-Apt3
50 -CTCTCTCGGTTGGTGTGGTTGGCTCTCTC-30
Tmb-Apt30
50 -CTCTCTCGGTTGGTGTGGTTGG-30
Tmb-Apt4
50 -CTCTCTCTGGTTGGTGTGGTTGGTCTCTCTC-30
Tmb-Apt0
50 -GGTTGGTGTGGTTGG-30
ATP-Apt1
50 -CTCTCTACCTGGGGGAGTATTGCGGAGGAAGGTTCTCTC-30
ATP-Apt2 ATP-Apt
50 -CTCTCTCACCTGGGGGAGTATTGCGGAGGAAGGTCTCTCTC-30 50 -CTCTCTCTACCTGGGGGAGTATTGCGGAGGAAGGTCTCTCTC-30
ATP-Apt3
50 -CTCTCTCTCACCTGGGGGAGTATTGCGGAGGAAGGTCTCTCTCTC-30
The boldface type is the aptamer sequence; the underlined sequences indicate the bases that form a triplex; and Py represents the fluorophore pyrene. 6588
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>95% response within several minutes, and the excimer fluorescence enhancement of STP is 4.12. To compare the response behavior with double-helix DNA molecular switches, the corresponding kinetic response of unmodified Tmb-Apt0 (Table 1) with long complementary oligos STP0 (Table 1) is shown in Figure S3. In the presence of 100 nM Tmb in the solution, E/E0 of STP0 is 1.20; apparently, the variance by a factor of 4.12/1.20 = 3.43 was achieved, therefore, demonstrating that the two short-armed segments do not affect the aptamer’s spatial folding in the same manner as a typical Tmb aptamer, keeping, as a result, higher recognition affinity, which is required for optimal aptamer probe design and application. Because the stability of the Hoogsteen base pairing is pH dependent,41,42 we studied the effect of pH on the formation of THMS. When the Tm changes of each THMS as a function of pH (Table 2) was taken into account, the triplex stem of THMS should be formed under acidic conditions and the Hoogsteen base pairing between C-G•C+ triplets could be formed as a result of the protonation of cytosine residues,41,42 enforcing efficient interaction between the STP and the arm segments of the aptamer. When
Figure 2. Real-time fluorescence records of STP (100 nM) upon additions of 120 nM Tmb-Apts (from trace a to trace d: Tmb-Apt 1, Tmb-Apt 2, Tmb-Apt 3, and Tmb-Apt 4) and subsequent 100 nM thrombin. For each measurement, we distinguish three steps: (1) the cell was filled with 500 μL of sodium phosphate buffer containing the STP; (2) Tmb-Apt was introduced into the cell; (3) thrombin was added. The transition between each regime is marked with an arrow. Fluorescence emission was recorded at 480 nm with an excitation wavelength of 340 nm.
Table 2. Melting Temperatures (°C) of THMS 1-THMS 4 Measured Under Different pH pH entry
5.0
6.0
7.0
8.0
THMS 1
46
41
30
26
THMS 2
49
45
32
27
THMS 3
54
50
37
32
THMS 4
58
53
42
40
the pH value was increased, the imino group of cytosines could not realize their protonation; the Hoogsteen base pairing became unstable so that triplex stem could not be efficiently formed. On the other hand, considering the binding properties of the aptamer, the dissociation constant, Kd, for the complex of Tmb/Tmb-Apt3 was also assessed under different pH according to a previous report.21 From Table 3, we found that, in neutral binding buffer, the aptamer had a lower Kd, representing higher affinities. As the environment became weakly acidic (pH 6.07.0), the Kd demonstrated less than a 10% increment, which means the weak acidic condition had little effect on the affinity of the aptamer. Especially, the Kd had been determined to be 358.13 ( 1.04 nM (Figure S4, Supporting Information) for the complex of Tmb/Tmb-Apt3 in the sodium phosphate buffer under pH 6.2, which was comparable with the reported Kd values.21 Therefore, with overall consideration, pH 6.2 was selected in the subsequent experiments. On the basis of the fact that hybridization between two singlestranded DNAs can form a duplex DNA with only WatsonCrick base pairing, a key question that arises now is just whether triplehelix stem is formed between the aptamer and STP in order to disrupt the hairpin stem of STP. Therefore, to clarify if the decreased excimer fluorescence of STP by Tmb-Apt3 is actually the result of the triple-helix formation, a controlled oligonucleotide, Tmb-Apt30 (Table 1), was designed. Tmb-Apt30 had the same aptamer sequences as that of Tmb-Apt3. However, it did not have the complementary stem-forming bases on one end and thus could not form a triple-helix structure with STP. We found the hybrid complex formed by Tmb-Apt30 and the STP displayed strong excimer fluorescence even at a temperature of
