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Fluorescence anisotropy-based structure-switching aptamer assay using a peptide nucleic acid (PNA) probe
Methods xxx (2015) xxx–xxx
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Fluorescence anisotropy-based structure-switching aptamer assay using a peptide nucleic acid (PNA) probe Emma Goux, Quentin Lespinasse, Valérie Guieu ⇑, Sandrine Perrier, Corinne Ravelet, Emmanuelle Fiore, Eric Peyrin ⇑ Département de Pharmacochimie Moléculaire, Université Grenoble Alpes, UMR 5063 CNRS, ICMG FR 2607, Campus universitaire, Saint-Martin d’Hères, France
a r t i c l e
i n f o
Article history: Received 22 July 2015 Received in revised form 4 September 2015 Accepted 18 September 2015 Available online xxxx Keywords: Peptide nucleic acid Aptamer-based assay Fluorescence anisotropy Structure-switching
a b s t r a c t This study describes for the first time the feasibility of using peptide nucleic acids (PNAs) as an alternative to the DNA probes in structure-switching aptamer fluorescence polarisation assays. The effects of experimental parameters such as the length of the PNA strand, the nature of dye and the buffer conditions on the assay performances are first explored using two different methodologies based on the competition between the PNA/aptamer hydribridisation and the target/aptamer complexation. D-ATP can be detected from 1 to 25 lM in a linear range and a detection limit (LOD) of 3 lM can be reached. For this target, this lowers by a factor >5 the LOD reported with conventional DNA-based fluorescent structure switching aptamer-based assays and by a factor 3 the LOD observed with non-competitive fluorescent sensing platform indicating the usefulness of the PNA-based approach. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Due to the flexibility offered by nucleic acids (stability, ease of chemical modifications and labelling), the development of sensitive and selective nucleic acids probes for bioanalytical sensing has become a very active research field in recent years. Notably, nucleic acid aptamers are DNA or RNA sequences which are selected by an in vitro combinatorial procedure called SELEX (for Systematic Evolution of Ligands by Exponential enrichment), in presence of a specific target. The SELEX process enables the isolation of aptamers, which bind targets including small organic molecules (nucleotides, drugs, toxins, hormones, amino acids . . .), peptides and proteins [1–3]. During the last years, nucleic acid aptamers have become a very promising and exciting field of research in both diagnostic and therapeutic areas as exemplified by the very abundant literature data as well as the creation of several companies devoted to the aptamer selection. As an alternative class of molecular recognition elements, aptamers display several attractive features. They are produced in vitro in a short time (a few weeks vs several months for antibodies) and are characterised by a smaller size (5–15 kDa). They usually possess high affinity (with dissociation constants ranging from the pico- to the micromolar range) and high selectivity for their target.
⇑ Corresponding authors. E-mail addresses: [email protected] ujf-grenoble.fr (E. Peyrin).
(V.
Guieu),
eric.peyrin@
One of the most interesting aspects of adapting aptamers to act as biosensors or molecular probes is their structural flexibility, which means that binding of a target usually results in structural alteration or rearrangement of the aptamer [4–6]. This phenomenon can be monitored by fluorescence anisotropy (FA) that constitutes one of the most versatile and popular fluorescent techniques used for the routine analysis of molecules in a variety of areas. As far as quantitative detection of small target molecules is concerned, FA-based aptamer approaches focus mainly on two different strategies known as direct and indirect structure-switching strategies. Despite its simplicity and sensitivity, the direct, noncompetitive strategy requires either a large aptamer conformational/structural change, the rational engineering of the aptameric structure, the precise identification of responsive locations within the sequence or the involvement of signalling/amplifying element to generate a significant fluorescence anisotropy response [7–13]. In the structure switching format, firstly reported by Li et al. for ATP detection, the capability of aptamers to strongly bind a nucleic acid complementary sequence (CS) thanks to specific buffer conditions is exploited [14]. A competition occurs between the complementary nucleic acid strand that associates with the aptamer to form a duplex [14] and the target recognition phenomenon that enables strand release. If the CS is fluorescently labelled, the structure-switching process can be followed by a variation in the global fluorescence anisotropy signal of the solution. Indeed, considering a complementary sequence shorter than the aptameric sequence, the FA signal of the duplex is expected to be higher than that of the free complementary strand [15–18]. This strategy offers
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several advantages over the direct one: the aptamer does not have to be labelled neither engineered to obtain a significant signal and the response does not depend on specific structural features of the aptamer. Thus, this method is applicable to any aptamer for which the sequence is known. However, as any competitive technique, it suffers from lower sensitivity: the more stable the duplex the lower the apparent binding affinity of the target for the aptamer. Considering two nucleic acid strands high salt conditions of the working buffered solutions improve the formation of a duplex. Nevertheless, high salt conditions (mono and divalent cations) are generally also necessary to favour the interaction between the aptamer and its target. This contradiction causes deterioration of the assay performances [16,19]. In addition, a relatively long DNA CS probe (at least 10–12-nt length) is generally required to disrupt the secondary structure of aptamer (quadruplex, hairpin) and to form the duplex species at room temperature. Thus, in specific FA-based assays, the change in size upon aptamer binding is limited so that small signal variation is typically attained. The substitution of the complementary nucleic acid strand by a peptide nucleic acid (PNA) sequence could be of a great interest. Indeed, PNAs are DNA analogues in which the entire sugarphosphate backbone has been replaced by a polyamide backbone [20,21]. Thanks to the charge neutrality and the high rigidity of its backbone, PNA exhibits high binding affinity to DNA within a wide range of hybridization conditions and has demonstrated to be a smart supplement of DNA in various applications [22–26]. In particular, no salt is needed to achieve the PNA–DNA duplex formation even for short (<10 nt length) PNA CS strands [27,28] and PNA containing aptasensors are expected to exhibit a higher thermal and biological stability. Thus, the optimal conditions for the aptamer–target complexation could be potentially applied without favouring the duplex formation and avoiding a decrease in the assay sensitivity. Furthermore, a greater FA signal is expected as the result of the lower size of the PNA probe. In this study, we examined for the first time the feasibility of using PNA as an alternative to the DNA probe in structureswitching aptamer FA assays. The anti-Adenosine triphosphate (ATP) DNA aptamer (Apt-A) was used as model functional nucleic acid [29,30]. Indeed ATP is a small molecule of a paramount interest in cell biology and biochemistry as it is important for regulating cellular metabolism [31]. The intracellular ATP level is an indicator of the cell viability as well as many diseases such as diabetes [32], ischemia [33], epilepsy [33], Parkinson’s disease [34], and some malignant tumors [35]. Therefore, the determination of ATP is essential for diagnosis and biochemical studies and numerous ATP aptasensors have been recently developed as they take advantage of the high binding affinity as well as structural switching properties of aptamers in combination with amplification strategies [8,36,37]. In the present work, the transduction of the duplex formation as well as analyte-aptamer binding into an analytical signal was achieved by end-labelling the PNA with a fluorophore and monitoring the steady-state fluorescence anisotropy variation of the solution. The effects of experimental parameters such as the length of the PNA strand, the nature of dye and the buffer conditions on the assay performances were first explored using the CS displacement methodology. The optimal conditions were reported and then applied to the another methodology in which the CS PNA strand is added to the aptamer–target complex already formed. This methodology was expected to improve the assay sensitivity. 2. Experimental section 2.1. Chemicals and apparatus D-Adenosine triphosphate (D-ATP), bovine serum albumin (BSA) and tris (hydroxymethyl)aminomethane were obtained from
Sigma Aldrich (Saint-Quentin Fallavier, France) NaCl and MgCl2 were obtained from Chem-Plus Laboratories (Bruyère de poully, France) and Roth (Lauterbourg, France) respectively. L-Adenosine triphosphate (L-ATP) was synthesized, purified and characterised as previously described by He et al. [38] Water was obtained from a Purite Still Plus water purification system (Thame, U.K.) fitted with a reverse osmosis cartridge. The oligonucleotide complementary strands listed in Table 1 were synthesized and HPLC-purified by Eurogentec (Angers, France). The identity of the oligonucleotides was confirmed by MALDI-TOF mass spectrometry. Fluorescence anisotropy was measured on a Tecan Infinite F500 microplate reader (Männedorf, Switzerland) using black, 96-well Greiner Bio-One microplates (ref: 675086). Excitation was set at 585 ± 20 nm and emission was collected with 635 ± 30 nm bandpass filters for Texas Red dye and excitation was set at 485 ± 20 nm and emission was collected with 535 ± 25 nm bandpass for fluorescein dye. 2.2. Sample preparation Unless otherwise stated, the binding buffer (BB) consisted of 10 mM Tris, pH 8.5, 10 mM MgCl2 and 10 mM NaCl and filtered before use through 0.22 lm membrane filter. The aptamer stock solution was prepared in filtered (0.22 lm) pure water at 104 M and stored at 20 °C. All working vials and plates were coated with BSA (10 mg/mL) during 15 min and rinsed with BB. The PNA stock solution was prepared daily at 105 M in filtered (0.22 lm) pure water. 2.3. Methodologies In the CS displacement methodology, the working PNA (250 nM) and PNA–aptamer (250–500 nM) solutions were prepared in 1.25 more concentrated BB. They were heated at 80 °C during 5 min and then cooled 5 min at room temperature and 30 min at 4 °C. The analyte solutions were prepared in filtered (0.22 lm) pure water. The analyte final concentrations varied from 1 to 500 lM (except for L-ATP). To establish the titration curves, PNA–aptamer (80 lL) and analyte solutions (20 lL) were mixed in individual wells at room temperature, resulting in PNA and aptamer concentration of 200 and 400 nM respectively. Buffer blank wells received 100 lL of BB, and PNA blank wells received PNA working (80 lL) solution and water (20 lL). All experiments were done in triplicate. The microplate was placed into the microplate reader for measurement with no delay. For the improved assay scheme (where the target:Apt complex was formed before addition of the PNA probe), the working PNA (500 nM) and aptamer (1000 nM) solutions were prepared in 1.25 more concentrated BB. They were heated at 80 °C during 5 min and then cooled 5 min at room temperature and 30 min at 4 °C. The analyte solutions were prepared in filtered (0.22 lm) pure water. The analyte final concentrations varied from 1 to 500 lM (except for L-ATP). To establish the titration curves, aptamer (40 lL) and analyte solutions (20 lL) were mixed in individual wells and incubated during 15 min at room temperature. Then PNA
Table 1 Oligonucleotide and PNA complementary strand sequences used in the study. AptaA PNA7-TR PNA6-TR PNA5-TR PNA7-F PNA6-F PNA5-F
50 N N N N N N
– CCT GGG GGA GTA TTG CGG AGG AAG G – 30 ter – TCC TCC G – Lys – TR – C ter ter – TCC TCC – Lys – TR – C ter ter – CCT CC – Lys – TR – C ter ter – TCC TCC G – Lys – F – C ter ter – TCC TCC – Lys – F – C ter ter – CCT CC – Lys – F – C ter
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solution is added in each well, resulting in PNA and aptamer concentration of 200 nM. Buffer blank wells received 100 lL of BB, and PNA blank wells received PNA working solution (40 lL), 1.25 more concentrated BB (40 lL) and water (20 lL). All experiments were done in triplicate. The microplate was placed into the microplate reader for measurement 5 min after PNA addition. 2.4. Fluorescence anisotropy measurement Anisotropy (r) was calculated by the instrument software, as classically reported:
r¼
Ivv GIv h Ivv þ 2GIv h
where Ivv and Ivh are the vertically and horizontally polarised components of the emission after excitation by vertically polarised light. The instrumental correction factor G was determined from standard solutions according to the manufacturer’s instructions. Fluorescence anisotropy change Dr was calculated as follows:
Dr ¼ r r f
ð1Þ
where r is the anisotropy of the duplex (dye-labelled PNA complementary strand with aptamer) and rf is the anisotropy of the free dye labelled PNA complementary strand. Drmax is the maximal fluorescence anisotropy difference, obtained in the absence of target. Relative Dr was obtained as follows:
relative Dr ¼
Dr 100 Dr max
ð2Þ
3. Results and discussion The schematic principle of the indirect aptamer fluorescence anisotropy assay can be seen on Fig. 1. Free PNA strand exhibits low anisotropy that shall increase upon duplex formation. In the presence of the target, structure-switching shall occur that leads to the release of the PNA strand and then a decrease of the fluorescence anisotropy signal. The aptamer chosen for this study is a 25-nt DNA anti-ATP aptamer AptaA (Table 1) that presents high binding affinity for D-adenosine and its phosphate derivatives (Kd 5–10 lM). This choice was motivated by the fact that the binding and structural properties of Apt-A as well as the buffer working conditions have been very well studied and characterised for aptamer–target recognition in a previous work [8].
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(Tris 10 mM, NaCl, 10 mM, MgCl2 10 mM, pH = 7.5). It has to be mentioned here that for better reproducibility, all vials and fluorescence polarisation plates were coated with BSA before use (see Section 2). Indeed the BSA treatment decreased the PNA adsorption on plastic surfaces and this resulted into a better repeatability of the FA measurement. Typically, variability decreased from approximately 21–8% for a solution containing 200 nM of PNA-7TR without and with BSA coating respectively. More results about the experimental influence of BSA coating are described in supporting information: influence of the PNA strand length, influence of the fluorophore, influence on duplex formation. 3.1.1. Influence of the labelling dye PNA probes of three different lengths (5, 6, 7-nt) were designed taking into account the adenosine binding sites as illustrated in Fig. 2. Numerous studies have shown that the fluorophore nature significantly influences the response of the FA-based assays involving labelled species [12,16,39]. Thus, two different fluorophore were investigated as dyes: Texas Red (TR) and Fluorescein (F). Both were attached to the C-terminal extremity of the PNA sequence (Table 1). An increase in the FA response was observed for the three PNA strands tagged with TR when Apt-A was added to the reaction solution. The two shorter PNA sequences led to relatively weak variation (Dr = 0.040 ± 0.006 and Dr = 0.049 ± 0.004 for PNA5-TR and PNA6TR respectively) while PNA7-TR triggered an intense FA increase at nearly PNA saturating concentration (Dr = 0.146 ± 0.004). This signal enhancement can be typically attributed to the expected increase in the molecular weight of PNA upon aptamer binding, theoretically from 2.6 kDa for the free PNA to 10.5 kDa for the duplex. This data reveals successful hybridization of the PNA probe to the aptamer. This is consistent with previous works which have demonstrated that small PNA strands are able to disrupt efficiently secondary structures such as G-quadruplex and hairpin [26]. In contrast, DNA probes in structure-switching assay generally require at least a 10–12-nt sequence long to hybridize aptamer sequence under room temperature conditions. Moreover, as the result of its
3.1. Study of the PNA–aptamer duplex formation In the initial step, the ability of the PNA probe to hybridize to Apt-A was investigated using previously reported buffer conditions
Fig. 1. Principle of the FA competitive assay. Aptamer (in blue), PNA complementary strand labelled with a fluorophore (in red), Adenosine target (black star).
Fig. 2. Conformation of the anti-Adenosine aptamer (black line) in the presence of its target (triangles) and the three considered PNA complementary sequences (PNA7-Dye in blue, PNA6-Dye in red, PNA5-Dye in green).
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shorter size, the fluorescent free PNA strand showed a greater anisotropy signal variation upon aptamer binding as compared to the use of a 12-nt DNA complementary strand with the longer antityrosinamide aptamer (Dr 0.110) [16]. On the other hand, very low increase in the fluorescence anisotropy signal could be detected in all cases upon aptamer addition in the reaction solution using PNA5, PNA6 and PNA7 labelled with fluorescein (Dr = 0.002 ± 0.001 for PNA6-F for instance). However this is consistent with previous works on the hybridization of DNA targets with fluorescein-tagged synthetic nucleic acid analogues. This behaviour is likely related to the fluorophore local mobility contribution, classically referred as the ‘‘propeller effect”, that overwhelms the size-dependent global diffusion part of FA signal [36].
fluorescence anisotropy (see Section 2) was plotted on Fig. 4a. As expected, it was found that 10 mM NaCl and 10 mM MgCl2 (black circles, Fig. 4a) were necessary to detect a significant PNA release while no NaCl (black squares, Fig. 4a) and low MgCl2 concentrations (2.5 mM black diamonds; 5 mM black triangles, Fig. 4a) were not adequate conditions. Indeed magnesium ion is known to favour the aptamer–target complexation [8]. Considering a 1:8 stoichiometry for the PNA:Apt duplex (Dr = 0.145 ± 1), no fluorescence anisotropy change was observed for [D-ATP] = 200 lM (Dr = 0.150 ± 3.2) confirming that no release of the free PNA fluorescent strand could be occurred as the duplex is too competitive to ATP binding. Increasing pH to 8.5 (grey circles, Fig. 4a) was found to improve the performance of our PNA-based aptamerbased assay.
3.1.2. Influence of the ionic strength Fig. 3 shows the formation of the duplex considering PNA5-TR, PNA6-TR and PNA7-TR under direct format optimised buffer (black markers) or PNA7-TR under high salt concentrations conditions (white markers). Increasing the salt concentrations of the buffer (NaCl = 50 mM instead of 10 mM) had no significant effect confirming the expected advantage brought by the use of PNA and no DNA complementary strands. The ratio Apta/PNA7-TR of 2 was chosen for the rest of the investigation as the duplex had to be strong enough to give a significant signal without competing too much with the ATP binding to the aptamer.
3.2.2. ATP sensing D-ATP could be detected from 1 to 25 lM in a linear range (Fig. 4b) and a detection limit (LOD) of 4.5 lM (calculated for a signal to noise ratio >3) could be reached. This was lowered by a factor >5 relative to the LOD reported with conventional DNAbased fluorescent structure switching aptamer-based assays [14] and was even better (by a factor 3) than the LOD observed with non-competitive fluorescent sensing platform [8]. This indicated the usefulness of the PNA-based approach.
3.2. PNA-based structure-switching assay 3.2.1. Influence of the buffer conditions: salt concentrations and pH The feasibility of the PNA-based approach was subsequently evaluated. D-ATP was chosen to highlight the target binding process and four different NaCl and MgCl2 concentrations were evaluated as well as two different pH (7.5 vs 8.5). The D-ATP target was added to the mixture containing the PNA:Apt (1:2) duplex and the solution was incubated for a 30 min period at room temperature. A decrease in the fluorescence anisotropy signal with increasing D-ATP concentrations was observed that reflects the decrease in the molecular size of the probe upon target binding, confirming that the structured state of the target-bound aptamer allows the release of the PNA from the duplex. Relative
3.2.3. Enantiospecificity To investigate the specificity of the present structure switching aptamer based FP assay, we further examined the aptamer binding reaction with L-ATP. Fig. 4a shows the fluorescence anisotropy changes in response to the addition of various concentrations of L-ATP (white circles) under the same experimental conditions used above. No significant changes were observed upon L-enantiomer
addition, indicating the enantiospecificity of the sensing platform [8]. 3.2.4. Improvement of the assay scheme Using the CS displacement configuration in optimised conditions, only 48% of the maximal FA variation was observed in presence of saturating target concentration (Fig. 4(a), grey circles), indicating that the target binding allowed only partial displacement of the PNA from the duplex. Previous works showed that PNA:DNA
Fig. 3. FA variation (Dr = r rf 103) with hybridization considering PNA5-TR (black triangles), PNA6-TR (black circles), PNA7-TR (black squares), PNA 7-TR under high salt concentrations conditions (white squares). [PNA] = 200 nM against increasing aptamer concentrations.
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Fig. 4. (a) Relative FA variation with increasing D-ATP concentrations considering the ‘‘CS displacement” methodology and [PNA7-TR] = 200 nM, [AptaA] = 400 nM with different Tris 10 mM buffer conditions: NaCl 10 mM, MgCl2 10 mM, pH 7.5 (black circles); MgCl2 10 mM, pH 7.5 (black squares); NaCl 10 mM, MgCl2 2.5 mM, pH 7.5 (black diamonds); NaCl 10 mM, MgCl2 5 mM, pH 7.5 (black triangles); NaCl 10 mM, MgCl2 10 mM, pH 8.5 (grey circles); and L-ATP increasing concentrations for NaCl 10 mM, MgCl2 10 mM, pH 8.5 (white circles). (b) Linear range considering [PNA7-TR] = 200 nM, [AptaA] = 400 nM, NaCl 10 mM, MgCl2 10 mM, pH 8.5.
Fig. 5. (a) Relative FA variation with increasing D-ATP concentrations considering the improved competitive design and [PNA7-TR] = 200 nM, Tris 10 mM pH 8.5, NaCl 10 mM, MgCl2 10 mM with different AptaA concentrations: 200 nM (squares), 400 nM (diamonds), 800 nM (circles), 1600 nM (triangles). (b) Linear range related to (a) from 1 to 25 lM of D-ATP. (c) Relative FA variation with increasing D-ATP concentrations considering [PNA7-TR] = 200 nM, Tris 10 mM pH 8.5, NaCl 10 mM, MgCl2 10 mM and the improved competitive design methodology ([AptaA] = 200 nM, circles) or ‘‘CS-displacement” design ([AptaA] = 400 nM, squares). (d) Linear range related to (c) from 1 to 25 lM of D-ATP.
hybrids are characterised by lower dissociation rate as compared with DNA:DNA hybrids [40–42]. This is very likely responsible for the weak signal change obtained upon target addition. To overcome this pitfall, a second strategy was then investigated by forming the target:Apt complex before addition of the PNA probe, as previously
proposed [43]. The aptamer was first incubated with its target to generate a complex and the PNA complementary TR-labelled strand was then added to the complex solution. In the absence of target, immediate formation of the duplex was observed, monitored by the increase of the fluorescence anisotropy signal. With increasing
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concentrations of D-ATP, a lower increase in the fluorescence anisotropy signal was detected as can be seen from the titration curves plotted on Fig. 5. Fig. 5(a) shows the influence of the PNA:Aptamer ratio using the previously optimised buffer (Tris 10 mM, pH 8.5, 10 mM NaCl, 10 mM MgCl2) and considering ratios of 1:1 (squares), 1:2 (diamonds), 1:4 (circles), 1:8 (triangles): the more concentrated the aptamer, the lower the relative fluorescence anisotropy decrease. A PNA:Aptamer ratio of 1:1 was then chosen and compared to the optimised CS-displacement methodology on Fig. 5 (b). As high as about 75% of the maximal FA change was observed (absolute Dr 0.05), showing a much better assay response as compared to the displacement strategy. Furthermore, the slope of the linear part of the calibration curve (0–25 lM) was found to be better for this design (y = 4.4x + 95.58) than for the CS-displacement format (y = 1.34x + 98.69), highlighting a much better sensitivity. Therefore, the detection limit was lowered by a factor of 1.5 (2.99 ± 1.03 lM vs 4.43 ± 0.94 lM). 4. Conclusion In conclusion, a new aptamer assay based the structure switching strategy was developed by using a peptide nucleic acid (PNA) probe. The capability of PNA strands to bind to an aptamer and to form a duplex was studied through fluorescence polarisation, under different experimental conditions (strand length, ionic strength of the solutions, dye). The fluorescence anisotropy variations upon target binding were measured. A change of as high as about 75% of the maximal fluorescence anisotropy signal could be observed and D-ATP could be detected with a detection limit (LOD) of 3 lM, five times better than LOD reported for this target with conventional DNA-based fluorescent structure switching aptamer-based assays, proving the usefulness of such a strategy. The generalisation of this new assay to real samples could be achieved by overstepping the solubility of PNA under realistic conditions with adding a little amount of organic solvent. Acknowledgement We acknowledge the (ANR-11-LABX-0003-01).
ANR
program
Labex
Arcane
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Fluorescence anisotropy-based structure-switching aptamer assay using a peptide nucleic acid (PNA) probe Emma Goux, Quentin Lespinasse, Valérie Guieu ⇑, Sandrine Perrier, Corinne Ravelet, Emmanuelle Fiore, Eric Peyrin ⇑ Département de Pharmacochimie Moléculaire, Université Grenoble Alpes, UMR 5063 CNRS, ICMG FR 2607, Campus universitaire, Saint-Martin d’Hères, France
a r t i c l e
i n f o
Article history: Received 22 July 2015 Received in revised form 4 September 2015 Accepted 18 September 2015 Available online xxxx Keywords: Peptide nucleic acid Aptamer-based assay Fluorescence anisotropy Structure-switching
a b s t r a c t This study describes for the first time the feasibility of using peptide nucleic acids (PNAs) as an alternative to the DNA probes in structure-switching aptamer fluorescence polarisation assays. The effects of experimental parameters such as the length of the PNA strand, the nature of dye and the buffer conditions on the assay performances are first explored using two different methodologies based on the competition between the PNA/aptamer hydribridisation and the target/aptamer complexation. D-ATP can be detected from 1 to 25 lM in a linear range and a detection limit (LOD) of 3 lM can be reached. For this target, this lowers by a factor >5 the LOD reported with conventional DNA-based fluorescent structure switching aptamer-based assays and by a factor 3 the LOD observed with non-competitive fluorescent sensing platform indicating the usefulness of the PNA-based approach. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Due to the flexibility offered by nucleic acids (stability, ease of chemical modifications and labelling), the development of sensitive and selective nucleic acids probes for bioanalytical sensing has become a very active research field in recent years. Notably, nucleic acid aptamers are DNA or RNA sequences which are selected by an in vitro combinatorial procedure called SELEX (for Systematic Evolution of Ligands by Exponential enrichment), in presence of a specific target. The SELEX process enables the isolation of aptamers, which bind targets including small organic molecules (nucleotides, drugs, toxins, hormones, amino acids . . .), peptides and proteins [1–3]. During the last years, nucleic acid aptamers have become a very promising and exciting field of research in both diagnostic and therapeutic areas as exemplified by the very abundant literature data as well as the creation of several companies devoted to the aptamer selection. As an alternative class of molecular recognition elements, aptamers display several attractive features. They are produced in vitro in a short time (a few weeks vs several months for antibodies) and are characterised by a smaller size (5–15 kDa). They usually possess high affinity (with dissociation constants ranging from the pico- to the micromolar range) and high selectivity for their target.
⇑ Corresponding authors. E-mail addresses: [email protected] ujf-grenoble.fr (E. Peyrin).
(V.
Guieu),
eric.peyrin@
One of the most interesting aspects of adapting aptamers to act as biosensors or molecular probes is their structural flexibility, which means that binding of a target usually results in structural alteration or rearrangement of the aptamer [4–6]. This phenomenon can be monitored by fluorescence anisotropy (FA) that constitutes one of the most versatile and popular fluorescent techniques used for the routine analysis of molecules in a variety of areas. As far as quantitative detection of small target molecules is concerned, FA-based aptamer approaches focus mainly on two different strategies known as direct and indirect structure-switching strategies. Despite its simplicity and sensitivity, the direct, noncompetitive strategy requires either a large aptamer conformational/structural change, the rational engineering of the aptameric structure, the precise identification of responsive locations within the sequence or the involvement of signalling/amplifying element to generate a significant fluorescence anisotropy response [7–13]. In the structure switching format, firstly reported by Li et al. for ATP detection, the capability of aptamers to strongly bind a nucleic acid complementary sequence (CS) thanks to specific buffer conditions is exploited [14]. A competition occurs between the complementary nucleic acid strand that associates with the aptamer to form a duplex [14] and the target recognition phenomenon that enables strand release. If the CS is fluorescently labelled, the structure-switching process can be followed by a variation in the global fluorescence anisotropy signal of the solution. Indeed, considering a complementary sequence shorter than the aptameric sequence, the FA signal of the duplex is expected to be higher than that of the free complementary strand [15–18]. This strategy offers
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several advantages over the direct one: the aptamer does not have to be labelled neither engineered to obtain a significant signal and the response does not depend on specific structural features of the aptamer. Thus, this method is applicable to any aptamer for which the sequence is known. However, as any competitive technique, it suffers from lower sensitivity: the more stable the duplex the lower the apparent binding affinity of the target for the aptamer. Considering two nucleic acid strands high salt conditions of the working buffered solutions improve the formation of a duplex. Nevertheless, high salt conditions (mono and divalent cations) are generally also necessary to favour the interaction between the aptamer and its target. This contradiction causes deterioration of the assay performances [16,19]. In addition, a relatively long DNA CS probe (at least 10–12-nt length) is generally required to disrupt the secondary structure of aptamer (quadruplex, hairpin) and to form the duplex species at room temperature. Thus, in specific FA-based assays, the change in size upon aptamer binding is limited so that small signal variation is typically attained. The substitution of the complementary nucleic acid strand by a peptide nucleic acid (PNA) sequence could be of a great interest. Indeed, PNAs are DNA analogues in which the entire sugarphosphate backbone has been replaced by a polyamide backbone [20,21]. Thanks to the charge neutrality and the high rigidity of its backbone, PNA exhibits high binding affinity to DNA within a wide range of hybridization conditions and has demonstrated to be a smart supplement of DNA in various applications [22–26]. In particular, no salt is needed to achieve the PNA–DNA duplex formation even for short (<10 nt length) PNA CS strands [27,28] and PNA containing aptasensors are expected to exhibit a higher thermal and biological stability. Thus, the optimal conditions for the aptamer–target complexation could be potentially applied without favouring the duplex formation and avoiding a decrease in the assay sensitivity. Furthermore, a greater FA signal is expected as the result of the lower size of the PNA probe. In this study, we examined for the first time the feasibility of using PNA as an alternative to the DNA probe in structureswitching aptamer FA assays. The anti-Adenosine triphosphate (ATP) DNA aptamer (Apt-A) was used as model functional nucleic acid [29,30]. Indeed ATP is a small molecule of a paramount interest in cell biology and biochemistry as it is important for regulating cellular metabolism [31]. The intracellular ATP level is an indicator of the cell viability as well as many diseases such as diabetes [32], ischemia [33], epilepsy [33], Parkinson’s disease [34], and some malignant tumors [35]. Therefore, the determination of ATP is essential for diagnosis and biochemical studies and numerous ATP aptasensors have been recently developed as they take advantage of the high binding affinity as well as structural switching properties of aptamers in combination with amplification strategies [8,36,37]. In the present work, the transduction of the duplex formation as well as analyte-aptamer binding into an analytical signal was achieved by end-labelling the PNA with a fluorophore and monitoring the steady-state fluorescence anisotropy variation of the solution. The effects of experimental parameters such as the length of the PNA strand, the nature of dye and the buffer conditions on the assay performances were first explored using the CS displacement methodology. The optimal conditions were reported and then applied to the another methodology in which the CS PNA strand is added to the aptamer–target complex already formed. This methodology was expected to improve the assay sensitivity. 2. Experimental section 2.1. Chemicals and apparatus D-Adenosine triphosphate (D-ATP), bovine serum albumin (BSA) and tris (hydroxymethyl)aminomethane were obtained from
Sigma Aldrich (Saint-Quentin Fallavier, France) NaCl and MgCl2 were obtained from Chem-Plus Laboratories (Bruyère de poully, France) and Roth (Lauterbourg, France) respectively. L-Adenosine triphosphate (L-ATP) was synthesized, purified and characterised as previously described by He et al. [38] Water was obtained from a Purite Still Plus water purification system (Thame, U.K.) fitted with a reverse osmosis cartridge. The oligonucleotide complementary strands listed in Table 1 were synthesized and HPLC-purified by Eurogentec (Angers, France). The identity of the oligonucleotides was confirmed by MALDI-TOF mass spectrometry. Fluorescence anisotropy was measured on a Tecan Infinite F500 microplate reader (Männedorf, Switzerland) using black, 96-well Greiner Bio-One microplates (ref: 675086). Excitation was set at 585 ± 20 nm and emission was collected with 635 ± 30 nm bandpass filters for Texas Red dye and excitation was set at 485 ± 20 nm and emission was collected with 535 ± 25 nm bandpass for fluorescein dye. 2.2. Sample preparation Unless otherwise stated, the binding buffer (BB) consisted of 10 mM Tris, pH 8.5, 10 mM MgCl2 and 10 mM NaCl and filtered before use through 0.22 lm membrane filter. The aptamer stock solution was prepared in filtered (0.22 lm) pure water at 104 M and stored at 20 °C. All working vials and plates were coated with BSA (10 mg/mL) during 15 min and rinsed with BB. The PNA stock solution was prepared daily at 105 M in filtered (0.22 lm) pure water. 2.3. Methodologies In the CS displacement methodology, the working PNA (250 nM) and PNA–aptamer (250–500 nM) solutions were prepared in 1.25 more concentrated BB. They were heated at 80 °C during 5 min and then cooled 5 min at room temperature and 30 min at 4 °C. The analyte solutions were prepared in filtered (0.22 lm) pure water. The analyte final concentrations varied from 1 to 500 lM (except for L-ATP). To establish the titration curves, PNA–aptamer (80 lL) and analyte solutions (20 lL) were mixed in individual wells at room temperature, resulting in PNA and aptamer concentration of 200 and 400 nM respectively. Buffer blank wells received 100 lL of BB, and PNA blank wells received PNA working (80 lL) solution and water (20 lL). All experiments were done in triplicate. The microplate was placed into the microplate reader for measurement with no delay. For the improved assay scheme (where the target:Apt complex was formed before addition of the PNA probe), the working PNA (500 nM) and aptamer (1000 nM) solutions were prepared in 1.25 more concentrated BB. They were heated at 80 °C during 5 min and then cooled 5 min at room temperature and 30 min at 4 °C. The analyte solutions were prepared in filtered (0.22 lm) pure water. The analyte final concentrations varied from 1 to 500 lM (except for L-ATP). To establish the titration curves, aptamer (40 lL) and analyte solutions (20 lL) were mixed in individual wells and incubated during 15 min at room temperature. Then PNA
Table 1 Oligonucleotide and PNA complementary strand sequences used in the study. AptaA PNA7-TR PNA6-TR PNA5-TR PNA7-F PNA6-F PNA5-F
50 N N N N N N
– CCT GGG GGA GTA TTG CGG AGG AAG G – 30 ter – TCC TCC G – Lys – TR – C ter ter – TCC TCC – Lys – TR – C ter ter – CCT CC – Lys – TR – C ter ter – TCC TCC G – Lys – F – C ter ter – TCC TCC – Lys – F – C ter ter – CCT CC – Lys – F – C ter
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solution is added in each well, resulting in PNA and aptamer concentration of 200 nM. Buffer blank wells received 100 lL of BB, and PNA blank wells received PNA working solution (40 lL), 1.25 more concentrated BB (40 lL) and water (20 lL). All experiments were done in triplicate. The microplate was placed into the microplate reader for measurement 5 min after PNA addition. 2.4. Fluorescence anisotropy measurement Anisotropy (r) was calculated by the instrument software, as classically reported:
r¼
Ivv GIv h Ivv þ 2GIv h
where Ivv and Ivh are the vertically and horizontally polarised components of the emission after excitation by vertically polarised light. The instrumental correction factor G was determined from standard solutions according to the manufacturer’s instructions. Fluorescence anisotropy change Dr was calculated as follows:
Dr ¼ r r f
ð1Þ
where r is the anisotropy of the duplex (dye-labelled PNA complementary strand with aptamer) and rf is the anisotropy of the free dye labelled PNA complementary strand. Drmax is the maximal fluorescence anisotropy difference, obtained in the absence of target. Relative Dr was obtained as follows:
relative Dr ¼
Dr 100 Dr max
ð2Þ
3. Results and discussion The schematic principle of the indirect aptamer fluorescence anisotropy assay can be seen on Fig. 1. Free PNA strand exhibits low anisotropy that shall increase upon duplex formation. In the presence of the target, structure-switching shall occur that leads to the release of the PNA strand and then a decrease of the fluorescence anisotropy signal. The aptamer chosen for this study is a 25-nt DNA anti-ATP aptamer AptaA (Table 1) that presents high binding affinity for D-adenosine and its phosphate derivatives (Kd 5–10 lM). This choice was motivated by the fact that the binding and structural properties of Apt-A as well as the buffer working conditions have been very well studied and characterised for aptamer–target recognition in a previous work [8].
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(Tris 10 mM, NaCl, 10 mM, MgCl2 10 mM, pH = 7.5). It has to be mentioned here that for better reproducibility, all vials and fluorescence polarisation plates were coated with BSA before use (see Section 2). Indeed the BSA treatment decreased the PNA adsorption on plastic surfaces and this resulted into a better repeatability of the FA measurement. Typically, variability decreased from approximately 21–8% for a solution containing 200 nM of PNA-7TR without and with BSA coating respectively. More results about the experimental influence of BSA coating are described in supporting information: influence of the PNA strand length, influence of the fluorophore, influence on duplex formation. 3.1.1. Influence of the labelling dye PNA probes of three different lengths (5, 6, 7-nt) were designed taking into account the adenosine binding sites as illustrated in Fig. 2. Numerous studies have shown that the fluorophore nature significantly influences the response of the FA-based assays involving labelled species [12,16,39]. Thus, two different fluorophore were investigated as dyes: Texas Red (TR) and Fluorescein (F). Both were attached to the C-terminal extremity of the PNA sequence (Table 1). An increase in the FA response was observed for the three PNA strands tagged with TR when Apt-A was added to the reaction solution. The two shorter PNA sequences led to relatively weak variation (Dr = 0.040 ± 0.006 and Dr = 0.049 ± 0.004 for PNA5-TR and PNA6TR respectively) while PNA7-TR triggered an intense FA increase at nearly PNA saturating concentration (Dr = 0.146 ± 0.004). This signal enhancement can be typically attributed to the expected increase in the molecular weight of PNA upon aptamer binding, theoretically from 2.6 kDa for the free PNA to 10.5 kDa for the duplex. This data reveals successful hybridization of the PNA probe to the aptamer. This is consistent with previous works which have demonstrated that small PNA strands are able to disrupt efficiently secondary structures such as G-quadruplex and hairpin [26]. In contrast, DNA probes in structure-switching assay generally require at least a 10–12-nt sequence long to hybridize aptamer sequence under room temperature conditions. Moreover, as the result of its
3.1. Study of the PNA–aptamer duplex formation In the initial step, the ability of the PNA probe to hybridize to Apt-A was investigated using previously reported buffer conditions
Fig. 1. Principle of the FA competitive assay. Aptamer (in blue), PNA complementary strand labelled with a fluorophore (in red), Adenosine target (black star).
Fig. 2. Conformation of the anti-Adenosine aptamer (black line) in the presence of its target (triangles) and the three considered PNA complementary sequences (PNA7-Dye in blue, PNA6-Dye in red, PNA5-Dye in green).
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shorter size, the fluorescent free PNA strand showed a greater anisotropy signal variation upon aptamer binding as compared to the use of a 12-nt DNA complementary strand with the longer antityrosinamide aptamer (Dr 0.110) [16]. On the other hand, very low increase in the fluorescence anisotropy signal could be detected in all cases upon aptamer addition in the reaction solution using PNA5, PNA6 and PNA7 labelled with fluorescein (Dr = 0.002 ± 0.001 for PNA6-F for instance). However this is consistent with previous works on the hybridization of DNA targets with fluorescein-tagged synthetic nucleic acid analogues. This behaviour is likely related to the fluorophore local mobility contribution, classically referred as the ‘‘propeller effect”, that overwhelms the size-dependent global diffusion part of FA signal [36].
fluorescence anisotropy (see Section 2) was plotted on Fig. 4a. As expected, it was found that 10 mM NaCl and 10 mM MgCl2 (black circles, Fig. 4a) were necessary to detect a significant PNA release while no NaCl (black squares, Fig. 4a) and low MgCl2 concentrations (2.5 mM black diamonds; 5 mM black triangles, Fig. 4a) were not adequate conditions. Indeed magnesium ion is known to favour the aptamer–target complexation [8]. Considering a 1:8 stoichiometry for the PNA:Apt duplex (Dr = 0.145 ± 1), no fluorescence anisotropy change was observed for [D-ATP] = 200 lM (Dr = 0.150 ± 3.2) confirming that no release of the free PNA fluorescent strand could be occurred as the duplex is too competitive to ATP binding. Increasing pH to 8.5 (grey circles, Fig. 4a) was found to improve the performance of our PNA-based aptamerbased assay.
3.1.2. Influence of the ionic strength Fig. 3 shows the formation of the duplex considering PNA5-TR, PNA6-TR and PNA7-TR under direct format optimised buffer (black markers) or PNA7-TR under high salt concentrations conditions (white markers). Increasing the salt concentrations of the buffer (NaCl = 50 mM instead of 10 mM) had no significant effect confirming the expected advantage brought by the use of PNA and no DNA complementary strands. The ratio Apta/PNA7-TR of 2 was chosen for the rest of the investigation as the duplex had to be strong enough to give a significant signal without competing too much with the ATP binding to the aptamer.
3.2.2. ATP sensing D-ATP could be detected from 1 to 25 lM in a linear range (Fig. 4b) and a detection limit (LOD) of 4.5 lM (calculated for a signal to noise ratio >3) could be reached. This was lowered by a factor >5 relative to the LOD reported with conventional DNAbased fluorescent structure switching aptamer-based assays [14] and was even better (by a factor 3) than the LOD observed with non-competitive fluorescent sensing platform [8]. This indicated the usefulness of the PNA-based approach.
3.2. PNA-based structure-switching assay 3.2.1. Influence of the buffer conditions: salt concentrations and pH The feasibility of the PNA-based approach was subsequently evaluated. D-ATP was chosen to highlight the target binding process and four different NaCl and MgCl2 concentrations were evaluated as well as two different pH (7.5 vs 8.5). The D-ATP target was added to the mixture containing the PNA:Apt (1:2) duplex and the solution was incubated for a 30 min period at room temperature. A decrease in the fluorescence anisotropy signal with increasing D-ATP concentrations was observed that reflects the decrease in the molecular size of the probe upon target binding, confirming that the structured state of the target-bound aptamer allows the release of the PNA from the duplex. Relative
3.2.3. Enantiospecificity To investigate the specificity of the present structure switching aptamer based FP assay, we further examined the aptamer binding reaction with L-ATP. Fig. 4a shows the fluorescence anisotropy changes in response to the addition of various concentrations of L-ATP (white circles) under the same experimental conditions used above. No significant changes were observed upon L-enantiomer
addition, indicating the enantiospecificity of the sensing platform [8]. 3.2.4. Improvement of the assay scheme Using the CS displacement configuration in optimised conditions, only 48% of the maximal FA variation was observed in presence of saturating target concentration (Fig. 4(a), grey circles), indicating that the target binding allowed only partial displacement of the PNA from the duplex. Previous works showed that PNA:DNA
Fig. 3. FA variation (Dr = r rf 103) with hybridization considering PNA5-TR (black triangles), PNA6-TR (black circles), PNA7-TR (black squares), PNA 7-TR under high salt concentrations conditions (white squares). [PNA] = 200 nM against increasing aptamer concentrations.
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Fig. 4. (a) Relative FA variation with increasing D-ATP concentrations considering the ‘‘CS displacement” methodology and [PNA7-TR] = 200 nM, [AptaA] = 400 nM with different Tris 10 mM buffer conditions: NaCl 10 mM, MgCl2 10 mM, pH 7.5 (black circles); MgCl2 10 mM, pH 7.5 (black squares); NaCl 10 mM, MgCl2 2.5 mM, pH 7.5 (black diamonds); NaCl 10 mM, MgCl2 5 mM, pH 7.5 (black triangles); NaCl 10 mM, MgCl2 10 mM, pH 8.5 (grey circles); and L-ATP increasing concentrations for NaCl 10 mM, MgCl2 10 mM, pH 8.5 (white circles). (b) Linear range considering [PNA7-TR] = 200 nM, [AptaA] = 400 nM, NaCl 10 mM, MgCl2 10 mM, pH 8.5.
Fig. 5. (a) Relative FA variation with increasing D-ATP concentrations considering the improved competitive design and [PNA7-TR] = 200 nM, Tris 10 mM pH 8.5, NaCl 10 mM, MgCl2 10 mM with different AptaA concentrations: 200 nM (squares), 400 nM (diamonds), 800 nM (circles), 1600 nM (triangles). (b) Linear range related to (a) from 1 to 25 lM of D-ATP. (c) Relative FA variation with increasing D-ATP concentrations considering [PNA7-TR] = 200 nM, Tris 10 mM pH 8.5, NaCl 10 mM, MgCl2 10 mM and the improved competitive design methodology ([AptaA] = 200 nM, circles) or ‘‘CS-displacement” design ([AptaA] = 400 nM, squares). (d) Linear range related to (c) from 1 to 25 lM of D-ATP.
hybrids are characterised by lower dissociation rate as compared with DNA:DNA hybrids [40–42]. This is very likely responsible for the weak signal change obtained upon target addition. To overcome this pitfall, a second strategy was then investigated by forming the target:Apt complex before addition of the PNA probe, as previously
proposed [43]. The aptamer was first incubated with its target to generate a complex and the PNA complementary TR-labelled strand was then added to the complex solution. In the absence of target, immediate formation of the duplex was observed, monitored by the increase of the fluorescence anisotropy signal. With increasing
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concentrations of D-ATP, a lower increase in the fluorescence anisotropy signal was detected as can be seen from the titration curves plotted on Fig. 5. Fig. 5(a) shows the influence of the PNA:Aptamer ratio using the previously optimised buffer (Tris 10 mM, pH 8.5, 10 mM NaCl, 10 mM MgCl2) and considering ratios of 1:1 (squares), 1:2 (diamonds), 1:4 (circles), 1:8 (triangles): the more concentrated the aptamer, the lower the relative fluorescence anisotropy decrease. A PNA:Aptamer ratio of 1:1 was then chosen and compared to the optimised CS-displacement methodology on Fig. 5 (b). As high as about 75% of the maximal FA change was observed (absolute Dr 0.05), showing a much better assay response as compared to the displacement strategy. Furthermore, the slope of the linear part of the calibration curve (0–25 lM) was found to be better for this design (y = 4.4x + 95.58) than for the CS-displacement format (y = 1.34x + 98.69), highlighting a much better sensitivity. Therefore, the detection limit was lowered by a factor of 1.5 (2.99 ± 1.03 lM vs 4.43 ± 0.94 lM). 4. Conclusion In conclusion, a new aptamer assay based the structure switching strategy was developed by using a peptide nucleic acid (PNA) probe. The capability of PNA strands to bind to an aptamer and to form a duplex was studied through fluorescence polarisation, under different experimental conditions (strand length, ionic strength of the solutions, dye). The fluorescence anisotropy variations upon target binding were measured. A change of as high as about 75% of the maximal fluorescence anisotropy signal could be observed and D-ATP could be detected with a detection limit (LOD) of 3 lM, five times better than LOD reported for this target with conventional DNA-based fluorescent structure switching aptamer-based assays, proving the usefulness of such a strategy. The generalisation of this new assay to real samples could be achieved by overstepping the solubility of PNA under realistic conditions with adding a little amount of organic solvent. Acknowledgement We acknowledge the (ANR-11-LABX-0003-01).
ANR
program
Labex
Arcane
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