Method for detecting proteins by means of aptamers

Abstract

A method is provided for an in vitro detection of a first protein in a sample, including the steps of: a) separating the sample by a native separation method; b) transferring the separated sample onto a membrane; c) contacting the membrane with an aptamer which specifically binds to the first protein; and d) detecting the first protein by detecting the aptamer bound to the first protein.

Claims

1. A method for the in vitro detection of a first protein in a sample, comprising the steps of: a) separating the sample by means of a native separation method; b) transferring the separated sample onto a membrane; c) contacting the membrane with an aptamer which specifically binds to the first protein, wherein the contacting is performed at a temperature of 30 C., and d) detecting the first protein by detecting the aptamer bound to the first protein.

2. The method according to claim 1, wherein the native separation method is an electrophoretic separation.

3. The method according to claim 1, wherein in the native separation method, a sodium dodecyl sulfate (SDS) concentration of 0% to 0.2% SDS is used.

4. The method according to claim 1, wherein the contacting of the membrane with the aptamer in step c) is performed at a temperature of 30 C. to 70 C.

5. The method according to claim 1, wherein the sample is a protein-containing mixture.

6. The method according to claim 1, wherein the sample is a protein-containing mixture obtained from a cell culture suspension, a cell culture supernatant, a tissue sample or a body fluid, or comprises a cell culture suspension, a cell culture supernatant, a tissue sample or a body fluid.

7. The method according to claim 1, wherein the aptamer includes a modification selected from the group of consisting of: a biotin labelling, fluorescent labelling, radioisotope labelling, digoxigenin labelling, peroxidase labelling, alkaline phosphatase labelling, and a labelling using nanoparticles.

8. The method according to claim 1, wherein the detection of the aptamer is direct or indirect.

9. The method according to claim 1, wherein the detection of the aptamer is performed by means of an enzymatic, auto-radiographic or fluorescent detection method.

10. The method according to claim 1, wherein the detection of the aptamer is indirect and the indirect detection of the aptamer uses an aptamer-specific antibody bound to the aptamer.

11. The method according to claim 1, wherein an aptamer-specific antibody is used for the detection of the aptamer and the aptamer-specific antibody includes a modification selected from the group consisting of: a biotin labelling, fluorescent labelling, radioisotope labelling, digoxigenin labelling, peroxidase labelling, alkaline phosphatase labelling, and a labelling using nanoparticles.

12. The method according to claim 1 for the additional detection of a second protein in the sample, comprising additional steps: i. following step b), the membrane is contacted with a binding molecule which specifically binds to the second protein; and ii. the second protein is detected through detection of the binding molecule bound to the second protein.

13. The method according to claim 1 for additional detection of a second protein in the sample, comprising the additional steps: i. following step b), the membrane is contacted with a binding molecule which specifically binds to the second protein; and ii. the second protein is detected through detection of the binding molecule bound to the second protein; wherein the binding molecule includes a modification selected from the group consisting of: a biotin labelling, fluorescent labelling, radioisotope labelling, digoxigenin labelling, peroxidase labelling, alkaline phosphatase labelling, and a labelling using nanoparticles.

14. The method according to claim 1 for additional detection of a second protein in the sample, comprising additional steps: i. following step b), the membrane is contacted with a binding molecule which specifically binds to the second protein; and ii. the second protein is detected through detection of the binding molecule bound to the second protein; wherein the binding molecule is a further aptamer or an antibody.

15. The method according to claim 2, wherein the native separation method is gel electrophoresis.

Description

FIGURES

(1) FIGS. 1A and 1B Protocols for detecting proteins in the aptamer blot (FIG. 1A) or in the combined aptamer and western blots (FIG. 1B).

(2) FIGS. 2A and 2B Detection of -thrombin (FIG. 2A) and streptavidin (FIG. 2B) after separation under native conditions using specific aptamers. For comparison, -thrombin and streptavidin were separated according to known protocols under denaturating conditions. The concentration of the FAM labelled aptamers was 1 g/ml.

(3) FIGS. 3A-3D Specificity increase of the aptamer blot. The influence of the aptamer incubation at 50 C. was compared with an experimental design that was incubated in parallel at room temperature. (FIG. 3A) 1 g and 2 g of human -thrombin was mixed with HeLa extract and analysed by means of an aptamer blot. The influence of 0.6M NaCl or 6M urea was determined in comparison with an aptamer incubation temperature of 50 C. and with the untreated initial solution. (FIG. 3B) Streptavidin was expressed as a fusion protein with the maltose-binding protein (MBP-Str) in E. coli. A cell extract of the expression strain without an MBP-Str-producing plasmid was tested as a control. (FIG. 3C) and (FIG. 3D) 1 g and 2 g thrombin or streptavidin were mixed with HeLa extract, separated under native conditions including 0.1% SDS, and analysed by means of aptamer blot.

(4) FIGS. 4A and 4B Combination of western and aptamer blot. Human -thrombin (FIG. 4A) and streptavidin (FIG. 4B) were mixed with HeLa extract and separated in a native PAGE including 0.1% SDS. After the transfer of proteins onto a membrane and the blocking of the membrane, the incubation with the primary antibody against actin was carried out. The membrane was then treated with the aptamer and the horseradish peroxidase-labelled secondary antibody at room temperature.

(5) FIG. 5 Multiplex detection of thrombin and streptavidin in the aptamer blot. Thrombin was mixed with streptavidin. The protein mixture was separated in a native PAGE with 0.1% SDS. The concentration of the each aptamer amounted to 1 g/ml. Trace 1: 5 g thrombin/streptavidin each; trace 2: 2 g thrombin/streptavidin each; trace 3: 1 g thrombin/streptavidin each; trace 4: 0.5 g thrombin/streptavidin each; trace 5: 0.25 g thrombin/streptavidin each.

(6) FIG. 6 Comparison between native and denaturating gel electrophoresis of thrombin and streptavidin and the native SDS-PAGE. In the native SDS-PAGE, various SDS quantities (0.01%; 0.05%; 0.1%) were analysed in the loading and running buffer. (A) Coomassie-dyed polyacrylamide gels; (B) aptamer blot.

EXAMPLES

(7) The method according to the invention serves the detection of proteins in protein-containing mixtures, such as cell extracts, tissue samples or the like, by means of specific aptamers. The protein detection protocol is shown in FIGS. 1A and 1B.

(8) The aptamers used in the method contain or consist of a nucleic acid sequence of 15 nucleotides having the SEQ ID No. 1, or of 40 nucleotides having the SEQ ID No. 2. The 15-mer: GGT TGG TGT GGT TGG (SEQ ID No. 1) and the 40-mer: ATC TCC GAT TGC CCC ACG ACG CAG TGG TCG GAG TTA CTT T (SEQ ID No. 2).

(9) The proteins can be separated by means of the following methods:

(10) I. isoelectric focusing

(11) II. native polyacrylamide gel electrophoresis (PAGE)

(12) III. native PAGE including 0.1% SDS.

(13) Following this, the transfer onto the PVDF or nitrocellulose membrane takes place. The membranes are blocked by BSA and then incubated with fluorescently labelled aptamers.

(14) To increase the detection specificity, the incubation with the specific aptamer can take place at higher temperatures (40 C., 45 C., 50 C.). The proteins are detected by way of fluorescent excitation at the fluorophore-specific wavelength.

(15) The simultaneous detection of a plurality of target proteins (multiplex analysis) is enabled by: I. The use of a plurality of aptamers conjugated with different fluorophores. In parallel, the blot membranes are incubated with the various aptamers. II. The combination of the aptamer detection and the antibody detection. For example, the incubation of the aptamer can take place at room temperature in parallel to the incubation with the further antibody. If the aptamer is incubated e.g. at 40-60 C., the incubation with the antibody and the further antibody can be performed after the detection of the aptamer.

(16) The three mentioned aspects allow for the fast and specific detection of several proteins in one test design. Thus, the established protocol for the aptamer blot clearly stands out from the test designs described so far.

(17) Detailed Protocols:

(18) The detection method comprises the following steps: step a) separation of the sample; step b) transfer of the separated sample onto a membrane; step c) contacting the membrane with an aptamer; step d) detection of the aptamer.

(19) Provided that the separation in step a) was performed by means of isoelectric focusing (IEF), the developed protocol was carried out with the below materials and conditions as follows:

(20) Step a): For the IEF, a gel having a pH gradient of pH 3-10 was used (BioRad). The samples were mixed with loading buffer (final concentrations: 2 mM lysin, 2 mM arginine, 5% glycerin) and applied onto the gel. An electric voltage was then applied to the gel and the respective running time was set (100V for 60 min; 250V for 60 min; 500V for 45 min). The cathode buffer contained 2 mM lysin and 2 mM arginine, the anode puffer 0.7 mM phosphoric acid.

(21) Step b): A PVDF or nitrocellulose membrane was used as a blot membrane. The transfer onto the blot membrane was carried out at 10V for 60 min. The transfer buffer contained 0.7% acetic acid.

(22) Subsequently, the membrane was blocked for 60 min at room temperature with 5% BSA in the respective selection buffer of the aptamer+0.1% TWEEN (polysorbate).

(23) Step c): The membrane was incubated with the aptamer for 60 min at room temperature or at 40 C., 45 C. or 50 C. For that purpose, 0.5-1.5 g/ml aptamer was used in the respective selection buffer+0.1% TWEEN (polysorbate)+1% BSA.

(24) Step d): The detection was achieved by fluorescent excitation.

(25) Provided that the polyacrylamide gel electrophoresis (PAGE) was applied for the separation in step a), the developed protocol was performed with the below materials and conditions as follows:

(26) Step a): The PAGE can be carried out both under native and under denaturating conditions. A 10% polyacrylamide gel was used for the native PAGE (without SDS). The samples were mixed with loading buffer (31.25 mM TrisCl; pH 6.8+5% glycerin+bromphenol blue) and applied onto the gel. An electric voltage was then applied to the gel and the respective running time was set (100V for 60 min). A Tris/glycin buffer was used as a running buffer.

(27) A small amount of SDS can be added to the native PAGE. Here, a 10% polyacrylamide gel was used. The sample containing 0.1% SDS (sample+31.25 mM TrisCl pH 6.8+5% glycerol+0.1% SDS+bromphenol blue) was applied onto this gel. An electric voltage was then applied to the gel and the respective running time was set (100V for 60 min). A Tris/glycin buffer including 0.1% SDS was used as a running buffer.

(28) A 10% BisTris polyacrylamide gel was used for the denaturating PAGE. The denaturated sample was then applied onto this gel (sample buffer: 315 mM Tris pH 6.8, 10% SDS, 50 glycerin, 0.05% bromphenol blue, 25% -mercaptoethanol). An electric voltage was then applied to the gel and the respective running time was set (200V for 30 min). A MOPS/Tris buffer including 5 mM EDTA, 1 mM sodium bisulfite and 0.1% SDS was used as a running buffer.

(29) Step b): A PVDF or nitrocellulose membrane was used as a blot membrane. The transfer onto the blot membrane was carried out at 140 mA for 70 min. The transfer buffer contained 25 mM Tris, 192 mM glycine and 20% methanol.

(30) Subsequently, the membrane was blocked for 60 min at room temperature with 5% BSA in the respective selection buffer of the aptamer+0.1% TWEEN 20 (polysorbate 20).

(31) Step c): The membrane was incubated with the aptamer for 60 min at room temperature or at 40 C., 45 C. or 50 C. For that purpose, 0.5-1.5 g/ml aptamer was used in the respective selection buffer+0.1% TWEEN 20 (polysorbate 20)+1% BSA.

(32) Step d): The detection was achieved by means of a fluorescent excitation or ECL development.

(33) The developed protocol also allows for the combination of the aptamer blot with the classic western blot. The incubation with the specific aptamer is performed prior to the treatment of the proteins with the primary and secondary antibodies, provided that the aptamer was incubated at higher temperatures (FIG. 1B). Had the membrane been incubated with the aptamer solution at room temperature, the membrane was initially incubated with the primary antibody and then in parallel with the aptamer and the secondary antibody. After that, the aptamer and the antibodies were detected in parallel.

(34) The detection method comprises the following steps provided that the membrane was initially incubated with the aptamer and then with the antibodies: step a) separating the sample; step b) transferring the separated sample onto a membrane; step c) contacting the membrane with an aptamer; step d) detecting the aptamer, step i) contacting the membrane with the primary antibody, step ii) contacting the membrane with the secondary antibody, and detecting the antibody.

(35) The developed protocol was performed with the below materials and conditions:

(36) Step a): A 10% polyacrylamide gel was used. The sample containing 0.1% SDS was applied onto the gel (loading buffer: 31.25 mM TrisCl pH 6.8+5% glycerin+0.1% SDS+bromphenol blue). An electric voltage was then applied to the gel and the respective running time was set (100V for 60 min). A Tris/glycin buffer including 0.1% SDS was used as a running buffer.

(37) Step b): A PVDF or nitrocellulose membrane was used as a blot membrane. The transfer onto the blot membrane was carried out at 140 mA for 70 min. The transfer buffer contained 25 mM Tris, 192 mM glycine and 20% methanol.

(38) Subsequently, the membrane was blocked for 60 min at room temperature with 5% BSA in the respective selection buffer of the aptamer+0.1% TWEEN 20 (polysorbate 20).

(39) Step c): The membrane was incubated with the aptamer for 60 min at temperatures 40 C. For that purpose, 0.5-1.5 g/ml aptamer was used in the respective selection buffer+0.1% TWEEN 20 (polysorbate 20)+1% BSA.

(40) Step d): The detection was achieved by fluorescent excitation.

(41) Step i): Incubation with the primary antibody at room temperature for 1 h or, as appropriate, overnight at 4 C.

(42) Step ii): Incubation with the secondary antibody for 1 h at room temperature. The ECL method was used for detection.

(43) The detection method comprises the following steps, provided that the membrane was initially incubated with the primary antibody and then in parallel with the secondary antibody and the aptamer: step a) separating the sample; step b) transferring the separated sample onto a membrane; step i) contacting the membrane with a primary antibody, step c) contacting the membrane with the aptamer and the secondary antibody; step d) or ii) detecting the antibody and the aptamer.

(44) The developed protocol was performed with the below materials and conditions as follows:

(45) Step a): A 10% polyacrylamide gel was used. The sample containing 0.1% SDS was applied onto the gel (loading buffer: 31.25 mM TrisCl pH 6.8+5% glycerol+0.1% SDS+bromphenol blue). An electric voltage was then applied to the gel and the respective running time was set (100V for 60 min). A Tris/glycin buffer including 0.1% SDS was used as a running buffer.

(46) Step b): A PVDF or nitrocellulose membrane was used as a blot membrane. The transfer onto the blot membrane was carried out at 140 mA for 70 min. The transfer buffer contained 25 mM Tris, 192 mM glycine and 20% methanol.

(47) Subsequently, the membrane was blocked for 60 min at room temperature with 5% BSA in the respective selection buffer of the aptamer+0.1% TWEEN 20 (polysorbate 20).

(48) Step i): The incubation of the membrane with the primary antibody was performed at room temperature for 1 h or, as appropriate, overnight at 4 C.

(49) Step c): The membrane was incubated with the aptamer and the secondary antibody at room temperature for 60 min. For this purpose, 0.5-1.5 g/ml aptamer and a secondary antibody were used in the selection buffer of the aptamer+0.1% TWEEN 20 (polysorbate 20)+1% BSA.

(50) Step d or ii): The detection was achieved by fluorescent excitation or ECL development, respectively.

Example 1: Comparison Between Denaturating and Native PAGE and Isoelectric Focusing as a Separation Method for the Aptamer Blot (FIG. 2)

(51) The interaction between aptamer and protein is both sequence-specific and structure-specific. Accordingly, native conditions should be of advantage to the separation of proteins for aptamer blot applications. A comparison was made between the aptamer-based detection of thrombin and streptavidin following the separation under native and denaturating conditions (FIG. 2).

(52) Literature has so far only reported on the separation of thrombin in the denaturating PAGE. As the aptamer blot works under these denaturating conditions, it can be assumed that the interaction between thrombin and the thrombin-binding aptamer (TBA) is exclusively based on sequence-specific interactions (FIG. 2A). However, the detection of streptavidin in the denaturating PAGE was not possible (FIG. 2B). It could be seen in Coomassie-dyed gels after the denaturating PAGE that the streptavidin was present in its monomeric form and the homotetrameric form of the native protein had been destroyed. Since streptavidin was not any longer detectable in its denaturated form it can be assumed that the interaction between streptavidin and its specific aptamer is based on structural interactions.

(53) Thrombin was also analysed after a native PAGE in the aptamer blot (FIG. 2A). Compared to the denaturating PAGE, only very weak signals were detected, which also had a size of well over 100 kDa. This can supposedly be attributed to the formation of aggregates. The formation of the aggregates resulted in an aggravated inflow into the PAGE gel on the one hand, and in a very low transfer onto the PVDF membrane on the other. Streptavidin was also separated in a native PAGE and then analysed in the aptamer blot (FIG. 2B). Following the transfer of the protein onto PVDF membranes, the detection by means of the streptavidin-binding aptamer (SBA) was possible.

(54) Furthermore, it was tested if isoelectric focusing (IEF) could be used for the aptamer blot. Both thrombin and streptavidin were applied onto IEF gels. Initially, the focusing was analysed using Coomassie dyes (data not shown). Both thrombin and streptavidin have flown into the IEF gels. Whereas thrombin was very well detectable in the aptamer blot (FIG. 2A), no bands could be detected with the SBA (FIG. 2B).

(55) Another native separation method was analysed where the samples prior to the PAGE were denaturated neither by heat nor by reducing agents (FIGS. 2A and 2B, on the right). The loading and running buffer, however, was mixed with 0.1% SDS so that a uniform negative load of the proteins was achieved and the formation of aggregates was avoided. However, a denaturation of the proteins is not observed under these conditions. Clear signals were observed after the analysis of thrombin and streptavidin in the aptamer blot under these conditions. The streptavidin homotetramers were not destroyed by this treatment. Accordingly, this method seems to be most suitable for detecting proteins by the use of aptamer.

Example 2: Detection of Human -Thrombin and Streptavidin in Protein Mixtures (FIG. 3)

(56) Human -thrombin was added to a HeLa cell extract while streptavidin was expressed as a fusion protein by means of the maltose-binding protein (MBP) in E. coli. The -thrombin/HeLa mixture was separated in a denaturating SDS-PAGE, and the streptavidin/E. coli mixture was separated using a native PAGE.

(57) During the detection of -thrombin it became clear that a number of HeLa proteins were detected in addition to -thrombin (FIG. 3A, on the left). To reduce the number of unspecific bindings, several washing steps were performed following the aptamer incubation. On the one hand, the membranes were washed with 0.6M NaCl, and on the other hand with 6M urea. The unspecific bindings were reduced by each washing step, but the specific -thrombin signal became weaker proportionally and the background became darker (FIG. 3A, centre). Accordingly, the two washing steps were not suited to increase the specificity in the aptamer blot.

(58) Had the aptamer incubation, however, been performed at a temperature of 50 C., there was a strongly reduced background having only a slightly weaker -thrombin signal (FIG. 3A, on the right).

(59) After a heterological expression and native PAGE of streptavidin in E. coli, significantly fewer unspecific bands than for -thrombin were observed in the HeLa mixture (FIG. 3B, on the left). In order to increase the specificity of the detection, this membrane was also incubated with the streptavidin aptamer at 50 C. A reduction of unspecific bands was detectable (FIG. 3B, on the right-hand side).

(60) As it had become clear that a separation of human -thrombin and streptavidin in native acrylamide gels and the use of 0.1% SDS yielded very good results in the aptamer blot, it was also analysed whether an incubation at higher temperatures leads to more specific results (FIGS. 3C and 3D). Consequently, thrombin or streptavidin were mixed with Hela extract and separated by means of native PAGE+0.1% SDS. The proteins were then transferred onto PVDF membranes; the membranes were blocked with BSA and incubated with TBA or SBA. While unspecific signals were observed with thrombin during the aptamer incubation at room temperature, the incubation at 50 C. allowed for a much more specific detection of thrombin (FIG. 3C). The incubation with the streptavidin-binding aptamer did neither produce any unspecific signals at room temperature nor at 50 C. (FIG. 3D). SBA seems to be much more specific than TBA.

Example 3: Combination of Aptamer and Western Blot (FIG. 4)

(61) As considerably more antibodies than aptamers have been available for the detection of proteins until now, the compatibility of western and aptamer blot was analysed. To this end, both human -thrombin and streptavidin were mixed with Hela extract and then actin was tested by means of western blot, and thrombin by means of the aptamer blot.

(62) The protein mixtures were separated in a native PAGE with 0.1%. Next, the proteins were transferred onto a PVDF blot membrane, and the membrane was blocked using BSA. The membrane was then incubated with the primary antibody against actin overnight. This was followed by the incubation of the membrane at room temperature with the TBA or SBA and a secondary antibody conjugated with the horseradish peroxidase. Finally, the aptamer and the secondary antibody were detected in parallel.

(63) This test design made it possible to detect both thrombin and streptavidin jointly with actin (FIG. 4). In the tests without thrombin or streptavidin, no signals were detected in the corresponding molecular weight range. The successful combination of western and aptamer blot could thus be demonstrated.

Example 4: Multiplex Detection of Thrombin and Streptavidin (FIG. 5)

(64) The use of fluorescently labelled aptamers enables the parallel detection of a plurality of proteins in one test. The feasibility was shown by way of example for a thrombin-streptavidin mixture (FIG. 5). The TBA was labelled using Cy5 and the SBA using FAM. The protein mixture was separated in the native PAGE including 0.1% SDS. Clear signals were observed both for thrombin and for streptavidin following the transfer of proteins onto PVDF membranes and the joint incubation with both aptamers. The limit of detection for streptavidin was 0.25 g and for thrombin 0.5 g.

Example 5: Comparison Between Native and Denaturating Separation Methods Using the Native SDS-PAGE (FIG. 6)

(65) Comparing the running behaviour of thrombin in polyacrylamide gels after Coomassie staining you find thrombin to form aggregates under native conditions (FIG. 6A). The formation of aggregates results in very weak signals in the aptamer blot (FIG. 6B). The addition of even small quantities of SDS (starting from 0.01% SDS) to the loading and running buffer during the gel electrophoresis prevents the formation of aggregates. The thrombin detection thus becomes much more sensitive, and it can be assumed that the native structure of thrombin is preserved.

(66) Streptavidin is present as a native, functional protein in its homotetrameric form. This homotetrameric structure is also very well detectable in Coomassie-dyed polyacrylamide gels. Under native conditions and following the addition of 0.01%, 0.05% or 0.1% SDS to the loading and running buffer, one could see that streptavidin as a native protein had flown into the gels because the protein in reference to a marker was detected at a size of approx. 64 kDa (FIG. 6A). Under denaturating conditions, only the streptavidin monomers were visible in the gel. As a result, the streptavidin detection was not possible in the aptamer blot under denaturating conditions (FIG. 6B). The detection of streptavidin in the aptamer blot, however, was possible in the native PAGE or native SDS-PAGE including 0.1%.