The present invention relates to optical sensors. More specifically, the present invention is concerned with optical sensors based on hybrid aptamer/conjugated polymer complexes.
Intense research is being carried out worldwide with the goal of developing rapid, simple, specific, and sensitive detection tools for medical diagnostics and biomedical research applications. Fundamentally, most analytical tests and immunoassays rely on molecular recognition and its transduction into a measurable output. Among all the possible molecular recognition elements, artificial nucleic acid ligands (aptamers) have recently attracted a lot of interest due to their capability of binding various metal ions, amino acids, drugs, proteins, as well as other molecules having high affinity and specificity.1-11
Aptamers are usually isolated from combinatorial libraries of synthetic nucleic acids by an iterative process of adsorption, recovery, and amplification coined as SELEX (Systematic Evolution of Ligands by Exponential Procedure). Aptamer-based ligands constitute highly promising candidates for the specific detection of various molecules. Additionally, they can also be used in competition binding assays, such as for example in high-throughput screening assays7, for the identification of new potential drugs capable of displacing the aptamers from their targets.
The above-mentioned approaches, however, require adequate transducing (i.e. reporting) elements in order to generate a physically measurable signal resulting from the recognition event. Binding of an aptamer to a target protein, for example, has been detected by using fluorescence (e.g. molecular beacons12-13) or by using a quartz microbalance14. In most cases, however, these methods either involve a tagging process or sophisticated experimental techniques. Furthermore, it is worth noting that labeling with various functional groups may even compromise the binding properties of the aptamers.
There thus remains a need to develop a rapid, simple, specific and sensitive detection tool capable of transducing the binding of an aptamer to its target into a clear signal.
The present invention seeks to meet these and other needs.
The present invention refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present invention relates to optical sensors based on hybrid aptamer/conjugated polymer complexes. More specifically, the present invention relates to the use of a water-soluble cationic polythiophene derivative as a “polymeric stain” capable of specifically transducing the binding of an aptamer to its target into a clear optical (calorimetric or fluorometric) signal.
The present invention relates to an optical sensor for detecting a target comprising a single-stranded aptamer complementary to the target, and a water-soluble cationic polythiophene derivative of the following formula:
wherein “n” is an integer ranging from 6 to 100.
The present invention also relates to a method for detecting a target comprising the steps of:
In addition, the present invention also relates to a method for detecting a target comprising the steps of:
Furthermore, the present invention also relates to the use of an optical sensor comprising a single-stranded aptamer and a water-soluble cationic polythiophene derivative of the following formula:
wherein “n” is an integer ranging from 6 to 100, for detecting a target, the aptamer being complementary to the target.
In a particular embodiment of the present invention, the target is human α-thrombin.
In a further particular embodiment of the present invention, the target is D-adenosine.
Further scope and applicability will become apparent from the detailed description given hereinafter. It should be understood however, that this detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.
Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration preferred embodiments thereof, and in which:
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description, of preferred embodiments with reference to the accompanying drawings which is exemplary and should not be interpreted as limiting the scope of the present invention.
Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill. Nevertheless, definitions of selected terms are provided for clarity and consistency.
As used herein, the term “optical sensor” is understood as referring to a complex consisting of a single-stranded aptamer and a water-soluble, cationic polythiophene derivative, the aptamer being complementary to a target to be detected, allowing for the detection of the target via optically measurable means. Without being so limited, these means include UV-visible or fluorescence spectra.
As used herein, the term “aptamer” is understood as being a single-stranded oligonucleotide that binds to a specific molecular target, non limiting examples of which are potassium ions, small organic molecules, amino acids, proteins, whole cells and nucleotides.
As used herein, when referring to an aptamer, the term “complementary” is understood as referring to the ability of the aptamer to hybridize with a specific chemical or biochemical target.
As used herein, the expression “enantiomeric resolution” is understood as referring to a discrimination (identification) between two enantiomers.
As used herein, the expression “folded structure” is understood as referring to a non-linear conformational structure.
As used herein, the term “target” is understood as referring to a charged entity, non-limiting examples of which are potassium ions, small organic molecules, amino acids, proteins, whole cells and nucleotides.
In a broad sense, the present invention relates to optical sensors based on hybrid aptamer/conjugated polymer complexes. More specifically, the present invention relates to the use of a water-soluble cationic polythiophene as a “polymeric stain” capable of specifically transducing the binding of an aptamer to its target into a clear optical (calorimetric or fluorometric) signal. The optical sensors do not require any chemical reaction to take place on the probes or with the analytes. Instead, the use of the optical sensors of the present invention is based on conformational changes as well as on electrostatic interactions between a cationic polythiophene derivative (i.e. poly(3-alkoxy-4-methylthiophene), an anionic single-stranded oligonucleotide (aptamer) and a target to be detected.
The present invention also relates to methods for detecting a charged entity using such optical sensors. Non-limiting examples of such charged entities include potassium ions, small organic molecules, amino acids, proteins, whole cells and nucleotides.
The use of the optical sensors of the present invention, for instance, allows to specifically detect as few as 2×10−15 mol of human thrombin or 2×10−14 mol of D-adenosine in only a few minutes and could be easily adapted for use in detecting many other chemical or biochemical targets non-limiting examples of which are ions, small organic molecules, amino acids, proteins, whole cells and nucleotides. Indeed, it is well known that the optical, electrical and electrochemical properties of polythiophenes are essentially constant after a minimum of 6-8 repeating units (P. Bäuerle, The synthesis of Oligothiophenes, Chapter 3 in Handbook of Oligo- and Polythiophenes, D. Fichou Ed., Wiley-VCH, Weinheim, pp. 89-181, 1999). A person skilled in the art would therefore understand that any polymer 1 having from 6 to 100 repeating units would work within an optical sensor as contemplated by the present invention. Furthermore, it is well known that aptamers with high affinity and selectivity have been created against a variety of targets, such as small organic molecules, peptides, proteins, and even cells (M. Famulok, G. Mayer, M Blind, Acc. Chem. Res. 33, 591-599, 2000). It is to be understood that any aptamer complementary to a target to be detected is within the scope of the present invention.
Single-stranded DNA (aptamer) can specifically bind potassium ions, human α-thrombin, or D-adenosine for instance. When binding takes place, the aptamer undergoes a conformational transition from an unfolded to a folded structure. This conformational change of the negatively-charged oligonucleotide can be detected by adding a water-soluble, cationic polythiophene derivative which transduces the new complex formation into an optical (calorimetric or fluorometric) signal without any labeling of the probe or of the target.
In a particular embodiment, an optical sensor according to the present invention comprises a polythiophene derivative, referred to herein as polymer 1, having the following formula:
wherein “n” is an integer ranging from 6 to 100 (see
The cationic, water-soluble, electroactive, and photoactive polymer 1 was prepared according to known literature procedures.15 As is observed for most poly(3-alkoxy-4-methylthiophene)s,16-19 polymer 1 exhibits chromic properties (color changes) which are due to conformational changes of the flexible conjugated backbone. Moreover, polymer 1 is known to display important optical changes when complexed to ss-DNA or ds-DNA15, making it a good candidate for transducing the binding of an aptamer to a given target.
The monovalent potassium cation is known for its folding-inducing properties in several classes of nucleic acids.20,21 As shown in
A red color (λmax=527 nm) was observed in the presence of LiCl (FIGS. 1A,b and B,b); NaCl (FIGS. 1A,c and B,c) or RbCl (FIGS. 1A,e and B,e) and ss-DNA (sequence X1: 5′-GGTTGGTGTGGTTGG-3′ (SEQ, ID NO 1)). This red color shift is associated with a stoichiometric complexation between the unfolded anionic ss-DNA and the cationic polythiophene derivative (
The optical properties (FIG. 1A,d and B,d), however, are different when potassium ions are present. As a result of the formation of a folded structure (quadruplex form) of oligonucleotide X1, stabilized by potassium ions (K+), polymer 1 is able to wrap itself around this structure through electrostatic interactions (
In a particular embodiment of the present invention, human α-thrombin was selected as an example of a target to be detected since X1 ss-DNA sequence (5′-GGTTGGTGTGGTTGG-3′ (SEQ ID NO 1)) is known to be a specific binding sequence (i.e. an aptamer) of this protein. On the other hand, the oligonucleotide ss-DNA (X2: 5′-GGTGGTGGTTGTGGT-3′ (SEQ ID NO 2)) is known to be a non-binding sequence.22 A conformational change occurs in the aptamer X1 when it binds to the thrombin molecule. Both NMR and X-ray diffraction studies have revealed that the aptamer adopts a compact unimolecular quadruplex structure with two G-quartets.23, 24
As shown in
The specificity of the detection was verified by two control experiments carried out under identical conditions. In a first control experiment a non-binding sequence ss-DNA (X2: 5′-GGTGGTGGTTGTGGT-3′ (SEQ ID NO 2)) was used (
The fluorescent properties of conjugated polymers can also be utilized to detect very small quantities of analytes.25-29 The fluorometric detection of the binding of the thrombin aptamer to human α-thrombin is possible because the fluorescence of poly(3-alkoxy-4-methylthiophene) is quenched in the planar, aggregated form.15-19 The yellow, random-coil form of polymer 1 is fluorescent (
The use of a standard spectrofluorimeter provides for a detection limit of 2×10−15 mole (a concentration of 1×10−11 M in 200 μL) of human α-thrombin.
The resolution of small molecule enantiomers is of great importance in many research fields such as biochemistry and drug analysis. Two enantiomers of a same molecule may have distinct physiological behaviors; one enantiomeric form can be pharmaceutically active whereas the other enantiomeric form can be inactive or toxic.
In a further particular embodiment of the present invention, D-adenosine was selected as an example of a target to be detected.
More specifically, the enantiomeric resolution of D-adenosine and L-adenosine was performed using DNA aptamer (5′-ATTATACCTGGGGGAGTATTGCGGAGGAAGGTATAAT-3′ (SEQ ID NO 3)) (31).
In a first step, a framework composed of two stacked G-quartets is assumed by mixing D-adenosine and DNA aptamer (31) (5′-ATTATACCTGGGGGAGTATTGCGGAGGAAGGTATAAT-3′ (SEQ ID NO 3)). The formed complex is more stable at 5° C. The cationic polymer 1 is then added and is assumed to wrap itself around the previously formed complex. The stoichiometry of the adenosine enantiomer/aptamer/polymer 1 complex is 1:1 1.
A series of identical steps was then performed using L-adenosine. Since L-adenosine is not supposed to induce a conformational change in DNA aptamer (31) (5′-ATTATACCTGGGGGAGTATTGCGGAGGAAGGTATAAT-3′ (SEQ ID NO 3)), the cationic polymer 1 should bind to the aptamer and lead to the formation of a duplex.
The cationic polymer 1 is yellow in aqueous solution (λ=397 nm) (
Since fluorescence spectroscopy is more sensitive than UV-visible absorption spectroscopy, the emission properties of polymer 1 can thus be used to detect even smaller quantities of D-adenosine. The yellow aqueous solution of polymer 1 is fluorescent with a maximum of emission at 525 nm (
UV-Visible Measurements
All UV-Visible absorption spectra were taken using a Hewlett-Packard (model 8452A) spectrophotometer.
Fluorescence Measurements
All fluorescence spectra were recorded on a Carry Eclipse (Varian Inc.) spectrofluorimeter. The excitation was performed at 420 nm.
In a quartz cell having an optical path length of 1.0 cm, 4 μL [2.9×10−9 mole (based on negative charges)] of 15-mer X1 were added to 100 μL of an aqueous solution of a given alkali metal cation (10 mM) (chloride salts), followed by the addition of 4 μL [2.9×10−9 mol (based on positive charge)] of a solution of cationic polymer 1. All UV-Visible absorption spectra were recorded at room temperature. The results are illustrated in
In a UV quartz cell having an optical path length of 1.0 cm, 1.9×10−10 mol of human α-thrombin (Haematologic Technologies Inc.) (the initial concentrated solution was diluted with sterilized water to obtain the appropriate concentration) and 2.9×10−9 mol (based on negative charges or 1.9×10−10 mol of 15-mer) of ss-DNA thrombin aptamer X1 were mixed in 100 μL of pure water at 25° C. This was followed by the addition of 2.9×10−9 mol (based on charge repeat unit) of polymer 1, to form a complex (1/1/1).
Two control experiments were carried out using a non-specific sequence X2 and BSA (bovine serum albumin, obtained from Sigma), under identical conditions. The results are illustrated in
In a UV quartz cell having an optical path length of 1.0 cm, 2.9×10−9 mol of D-adenosine and 1.08×10−7 mol of DNA D-adenosine aptamer (based on monomeric negative charges or 2.9×10−9 mol of 37-mers) were mixed in 200 μL of pure water at 5° C. This was followed by the addition of 1.08×10−7 mol (based on charge repeat unit) of polymer 1, to form a complex (1/1/1).
A control experiment, under identical conditions, was carried out using L-adenosine, to which the D-adenosine aptamer is not complementary. The results are illustrated in
In a fluorescence cell having an optical path length of 3.0 mm, 3.8×10−10 mol of human α-thrombin and 5.7×10−9 mole (based on monomeric negative charge or 3.8×10−10 mol of 15-mer) of ss-DNA thrombin aptamer X1 were mixed in 200 μl of pure water, followed by the addition of 5.7×10−9 mol (based on charge repeat unit) of polymer 1. The fluorescence spectrum of all mixtures was recorded at 5° C. For the lower concentration of human α-thrombin, the excitation was performed at, 420 nm, and the fluorescence emission intensity was measured at 584 nm (without recording the entire emission spectrum).
A control experiment was carried out using a non-specific sequence X2 under identical conditions. The results are illustrated in
In a fluorescence cell having an optical path length of 3.0 mm, 2.9×10−9 mol of D-adenosine and 1.08×10−7 mol of DNA D-adenosine aptamer (based on monomeric negative charge or 2.9×10−9 mol of 37-mers) were mixed in 200 μl of pure water, followed by the addition of 1.08×10−7 mol (based on charge repeat unit) of polymer 1. The fluorescence spectrum of all mixtures was recorded at 5° C. The results are illustrated in
A control experiment, under identical conditions, was carried out using L-adenosine, to which the D-adenosine aptamer is not complementary. The results are illustrated in
Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
Number | Date | Country | Kind |
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2430910 | Jun 2003 | CA | national |
This application is a national stage entry of PCT/CA04/00824 filed on Jun. 3, 2004, which claims priority from U.S. provisional application No. 60/474,950, filed on Jun. 3, 2003. This application claims the benefit of the prior-filed foreign application, CA 2430910, filed Jun. 3, 2003.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/CA2004/000824 | 6/3/2004 | WO | 00 | 1/10/2007 |
Publishing Document | Publishing Date | Country | Kind |
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WO2004/106544 | 12/9/2004 | WO | A |
Number | Name | Date | Kind |
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5631146 | Szostak et al. | May 1997 | A |
Number | Date | Country |
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WO 02081735 | Oct 2002 | WO |
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20070196825 A1 | Aug 2007 | US |
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60474950 | Jun 2003 | US |