WO2009113564A1

WO2009113564A1 - Luciferase-binding aptamer

Info

Publication number: WO2009113564A1
Application number: PCT/JP2009/054617
Authority: WO
Inventors: 一典池袋; 広司早出; 直樹梶山; 恵子黒澤
Original assignee: 国立大学法人東京農工大学
Priority date: 2008-03-11
Filing date: 2009-03-11
Publication date: 2009-09-17
Also published as: JP2009240306A

Abstract

Disclosed is means for simply, promptly and sensitively detecting a disease marker without performing B/F separation. A novel aptamer molecule which has a specific nucleotide sequence and binds to luciferase, particularly, a novel aptamer molecule which binds to luciferase and also suppresses the enzymatic activity of luciferase is provided. For example, if an aptamer capable of suppressing luciferase activity is combined with another aptamer recognizing a desired target substance such as a disease marker, a detection system with very high sensitivity using luciferase luminescence can be constructed.

Description

Luciferase binding aptamer

The present invention relates to an aptamer that binds to luciferase.

Detecting proteins that serve as disease markers is extremely important for early diagnosis and treatment of various diseases. Currently, the most commonly used technique for detecting such disease markers is ELISA (Enzyme Linked Immunosorbent Assay) using antibodies. The ELISA method is a method in which a trace amount of a target substance contained in a sample is quantitatively detected using an enzyme-labeled antibody or antigen and utilizing an antigen-antibody reaction. The ELISA method (1) can detect the target substance with a high sensitivity of 1 attomole (= 1 mol of 10 to the power of 18) and has excellent quantitative properties. (2) Utilizes antigen-antibody reaction. It can be measured in the crude extraction stage for detection, and sample preparation does not require complicated steps such as purification and pretreatment required by other inspection methods. (3) Large amount of sample in a short time There are advantages such as being able to measure.

However, ELISA has the following problems. First, since the antibody does not emit a signal when bound to the target, two types of antibodies that bind to different portions of the target molecule are required. It is difficult to produce two types of antibodies that bind to different parts of a single target molecule, and antibodies are time-consuming and laborious to produce, and are expensive. Secondly, since a signal is transmitted by modifying a molecule such as an enzyme in one antibody, a complicated B / F separation operation that removes the antibody that has not bound to the target molecule is essential. is there.

For early treatment of diseases, early diagnosis is essential, and for this purpose, a simple and rapid detection system capable of measuring collected blood on the spot is required. However, since ELISA requires a separation operation, it takes time and cannot be said to be a rapid system.

On the other hand, aptamers that are oligonucleotides that specifically bind to arbitrary molecules are known. Since aptamers can be chemically synthesized using a commercially available nucleic acid synthesizer, they are much cheaper than specific antibodies and can be easily modified. Therefore, aptamers are expected to be applied as sensing elements. Aptamers that specifically bind to a desired target molecule can be produced by a method called SELEX® (SystematicSystemEvolution of Ligands by EXponential Enrichment) (Non-patent Document 1). In this method, a target molecule is immobilized on a carrier, a nucleic acid library consisting of nucleic acids having a large number of random base sequences is added to the target molecule, nucleic acids that bind to the target molecule are recovered, and this is amplified by PCR. Again, the target molecule is added to the immobilized carrier. By repeating this step about 10 times, aptamers having high binding power to the target molecule are concentrated, the base sequence thereof is determined, and the aptamer that recognizes the target molecule is obtained. The nucleic acid library can be easily prepared by binding nucleotides at random using an automatic nucleic acid synthesizer. Thus, an aptamer that specifically binds to an arbitrary target substance can be produced by a method that actively uses chance by using a nucleic acid library having a random base sequence.

As a disease marker detection technique that does not require a B / F separation operation, the inventors of the present application link a recognition aptamer that recognizes a target molecule and an enzyme-controlled aptamer that binds to an enzyme and changes the activity of the enzyme. Thus, AES (AptamerictaEnzyme Subunit), which is a sensing technique using aptamers as enzyme subunits, has been constructed (Patent Documents 1 and 2). The detection principle is that when the molecule to be measured is present, the molecule binds to the recognition aptamer, which causes a change in the structure of the enzyme-controlled aptamer linked to it, resulting in the activity of the enzyme contained in AES. Therefore, the target molecule is detected by measuring the change in its activity. The advantage of this detection method is that, unlike the detection by ELISA, the binding of the target molecule is detected directly as a signal of the enzyme activity, so that rapid and simple detection without requiring B / F separation is possible. It is done. In addition, once an aptamer that inhibits enzyme activity is acquired, an aptamer that binds to the target molecule to be detected can be arbitrarily selected to detect various target molecules. Furthermore, aptamers are easier to make and less expensive than antibodies.

However, AES described in Patent Documents 1 and 2 uses a thrombin aptamer as an enzyme-controlled aptamer, and detects a target molecule by a change in fibrin clotting time. Such a detection method has a drawback in that it takes a relatively long time for measurement and thus lacks rapidity.

As described above, in order to provide an inexpensive and quick detection technique, it is a problem to facilitate measurement of enzyme activity in AES. That is, it has been a challenge to create an aptamer that controls an enzyme whose activity can be easily measured.

However, since the aptamer creation method is a method that actively uses chance as described above, whether or not an aptamer having a high binding ability to a target substance can be obtained by actually conducting an enormous experiment. I don't know without it. In particular, when constructing AES, as an enzyme-controlled aptamer, an aptamer having a binding ability with an enzyme used as a sensing element and having a high ability to inhibit or increase the activity of the bound enzyme must be created. Therefore, it is not easy to obtain an aptamer that can be used as an AES enzyme-controlled aptamer.

International Publication WO2005 / 049826 International Publication WO2007 / 032359

Therefore, an object of the present invention is to provide a means capable of detecting a desired measurement object such as a disease marker easily, quickly and with high sensitivity without performing a B / F separation operation.

The inventors of the present application paid attention to luciferase as an enzyme applied to AES, and as a result of earnest research, obtained a new aptamer having binding ability to luciferase. Furthermore, an aptamer that can desirably inhibit the activity of luciferase was found out of the aptamers, and the present invention was completed.

That is, the present invention provides an aptamer consisting of any of the following polynucleotides and having the ability to bind to luciferase:
(a) a polynucleotide having the base sequence represented by any of SEQ ID NOs: 4 to 24,
(b) a polynucleotide in which one or more than ten bases are substituted, deleted and / or inserted in the polynucleotide of (a),
(c) A polynucleotide comprising the polynucleotide of (a) or (b).

In addition, the present invention has the ability to bind to luciferase, including a step of crossing and / or shuffling a plurality of aptamers, and a step of selecting an aptamer having the ability to bind to luciferase among the obtained aptamers. Provide a method for producing aptamers. Furthermore, this invention provides the manufacturing method of a luciferase binding aptamer including manufacturing the aptamer produced by this method.

The present invention provides for the first time a luciferase-binding aptamer that can also be used as an AES enzyme-controlled aptamer. By combining an aptamer that can control luciferase activity with another aptamer that recognizes a desired target substance such as a disease marker, a very sensitive detection system using luminescence of luciferase can be constructed. The enzymatic activity of luciferase can be easily measured by measuring the luminescence generated by the reaction with the substrate using a luminescence detector such as a commercially available luminometer, so that rapid detection is possible. Since aptamers can be chemically synthesized using a DNA synthesizer, they require less labor and are less expensive than antibodies and can reduce the price of the measurement kit itself.

It is a figure which shows the result of having investigated the binding ability to the luciferase by aptamer blotting about 24 types of ssDNA considered that the secondary structure is stabilized among ssDNA acquired by the screening. It is a figure which shows the result of having investigated the luciferase inhibitory effect by changing the density | concentration of aptamer Lap 1-6. FIG. 2 is a secondary structure diagram of an aptamer Lap 1-6 having high ability to inhibit luciferase activity. FIG. 3 is a secondary structure diagram of aptamer Lap 1-20 having high ability to inhibit luciferase activity.

Luciferase is a general term for enzymes that catalyze bioluminescence, such as fireflies, and is generally used as a reporter gene for gene expression assays and the like. The luciferin / luciferase reaction can be roughly divided into two stages (T. Nakatsu, S Ichiyama, J Hiratake, A Saldanha, N Kobashi, K Sakata, H Kato: Structural basis for the spectral difference in luciferase bioluminescence. Nature. 2006 Mar 16; 440 (7082): 372-6) In the first step, the luminescent substrate luciferin reacts with ATP in the presence of Mg ²⁺ to produce a luciferyl AMP intermediate in luciferase. In the subsequent second stage, the luciferyl AMP intermediate reacts with oxygen molecules and decomposes into excited oxyluciferin and CO ₂ . When excited oxyluciferin bound to luciferase returns to the base low state, it emits light at 562 nm. Therefore, by measuring the amount of luminescence using a known luminescence detector such as a luminometer, the activity of luciferase can be measured using the amount of luminescence as an index. In a detection system using luciferase as a reporter, in order to utilize this reaction, it is usually necessary to add luciferin as a substrate, ATP, and Mg. In addition, since the enzyme activity is measured using the amount of luminescence as an index, a luminescence detector such as a luminometer is required. Note that assay systems themselves that use luciferase are already well known, and various reagents containing luciferase substrates that can be preferably used in such assay systems are commercially available.

The aptamer of the present invention has the ability to bind to luciferase and consists of any of the following polynucleotides (consists essentially of).
(a) A polynucleotide having the base sequence shown in any one of SEQ ID NOs: 4 to 24.
(b) In the polynucleotide of (a), 1 to a dozen (maximum 19), preferably 1 or several (maximum 9), more preferably 1 or 2 bases are substituted and missing. Lost and / or inserted polynucleotide.
(c) A polynucleotide comprising the polynucleotide of (a) or (b).
Note that “having a base sequence” means that bases are arranged in the sequence in the polynucleotide. For example, a polynucleotide “having the base sequence shown in SEQ ID NO: 4” means that the polynucleotide Means that the base is composed of 66 bases arranged in the sequence shown in SEQ ID NO: 4.

The polynucleotide may be DNA or RNA, or may be an artificial nucleic acid such as PNA, but DNA is preferred from the viewpoint of stability.

The nucleotide sequences shown in SEQ ID NOs: 4 to 24 are the initial live sequences of SEQ ID NO: 1 in the binding ability evaluation by the aptamer blotting method among the ssDNA obtained by screening the ssDNA library (SEQ ID NO: 1) containing a 30-mer random region. This is a base sequence of ssDNA that has a higher binding ability to luciferase than rally (see Examples below). The three-dimensional structure formed by these aptamers under predetermined conditions (folding conditions) can be easily determined by a conventional method using a computer. Various programs for predicting the three-dimensional structure of nucleic acids are known. For example, m-fold (trade name, Nucleic® Acids® Res. 31 (13), which is a well-known nucleic acid structure prediction program using the closest base pair method. 3406-15, (2003), The Bioinformatics Center at Rensselaer and Wadsworth can be downloaded), but is not limited thereto. FIG. 3 and FIG. 4 show secondary structure prediction diagrams of aptamers having the base sequences shown in SEQ ID NOs: 5 and 9 by m-fold (trade name).

The “folding condition” is a condition in which a part of complementary regions of one molecule of aptamer forms a stem part consisting of a double strand by base pairing within the molecule. It is also a usage condition. Usually, it is in an aqueous buffer solution having a predetermined salt concentration at room temperature and optionally containing a surfactant. For example, in addition to TBS (10 mM Tris / HCl, pH 7.0, 100 mM NaCl) and TBST (TBS containing 0.05% v / v Tween 20) employed in the following examples, an aqueous solution containing 10 mM MOPS and 1 mM CaCl ₂ , A buffer solution such as an aqueous solution containing 20 mM Tris-HCl and 150 mM NaCl can be used. After heat denaturation by heating to about 95 ° C. in these buffers, the solution gradually reaches room temperature (if the amount is about 100 μL). By cooling (over about 30 minutes), the aptamer molecule can be folded. In this specification, the term “folding” refers to the formation of a stem portion by pairing complementary bases within a molecule of one aptamer, as well as the split aptamer described later. In particular, it includes the formation of a desired three-dimensional structure by pairing complementary bases within and / or between a plurality of polynucleotide molecules constituting a split aptamer.

The binding ability of the aptamer of the present invention may be capable of binding to other proteins other than luciferase as long as it has the ability to bind to luciferase, but has high specificity and affinity for luciferase, and other proteins It is preferable that there is no binding, or if any, a relatively small amount of binding is possible (hereinafter, such binding ability may be expressed as “specifically bind”). The specificity and affinity of the luciferase-binding aptamer can be evaluated by, for example, the aptamer blotting method described in the following examples. Specifically, for example, luciferase and any other protein (competitive protein) are immobilized on a support such as a nitrocellulose membrane by a conventional method, and the protein-immobilized support and aptamer such as TBS are immobilized. By reacting in an appropriate buffer and examining how many aptamer molecules are bound to each of luciferase and the competitor protein, the specificity and affinity of the aptamer for luciferase can be evaluated. The amount of aptamer molecule bound can be determined by, for example, aptamer molecules that have been labeled with biotin or FITC in advance, reacted with a protein-immobilized support, and then subjected to immunoassay using an antibody against the labeling substance in a conventional manner. The amount of binding can be examined.

Aptamers generally have the same aptamer activity (binding ability to a target molecule and, when the target molecule is an enzyme, an enzyme, as long as the positional relationship and size of the stem and loop portions are equal in the three-dimensional structure. Control ability). For example, even when a small number of bases are deleted from the terminal, the original aptamer activity can be maintained. The base forming the stem part may have a base sequence in which the positions of the bases to be paired with each other are replaced with each other, or the base pair to be paired may be replaced with, for example, an at-t pair to a g-c pair. As for the base forming the loop part, other base sequences may be adopted as long as a loop of the same size is formed at that position. In addition, if the region is not important for aptamer binding ability, even if a small number of bases are inserted, the same aptamer activity as that of the original aptamer can usually be maintained. Therefore, in the polynucleotide (a), 1 to 10 or more (up to 19, preferably 1 or several (up to 9), more preferably 1 or 2) bases are as exemplified above. As long as the aptamer composed of the polynucleotide substituted, deleted and / or inserted (polynucleotide (b) above) also has the same aptamer activity as the aptamer having the original base sequence, the scope of the present invention Is included.

As the luciferase-binding aptamer comprising the polynucleotide of (b), one to ten or more bases (preferably one or several (up to 9), more preferably, one or both ends of the polynucleotide of (a) Is preferably an aptamer comprising a deleted polynucleotide. In general, the activity of the original aptamer is often maintained even if a small number of bases at one or both ends of the aptamer are deleted.

Further, the inventors of the present application randomize a partial region of an aptamer that binds to a certain substance, and perform screening using the binding property to the substance as an index, thereby increasing the binding ability to the substance compared to the original aptamer. (Non-Patent Documents 2 to 4). In addition, when the substance to which the aptamer binds is a substance having an activity such as an enzyme, an aptamer that binds to the substance and inhibits the function of the substance can also be produced by this method. Therefore, by randomizing the sequence of a part of the aptamer of (a), preferably 6 to a dozen (maximum 19), and screening with the ability to bind to or inhibit the substance as an indicator It is possible to produce an aptamer that is superior in binding ability or inhibition ability than the original aptamer. In particular, as specifically described in Non-Patent Documents 2 to 4, the double-stranded part of the aptamer is changed to a double-stranded part composed of another base sequence (that is, the double-stranded part is maintained). The base sequence can be changed without changing the three-dimensional structure of the aptamer, so that the binding activity of the aptamer can be maintained. In addition, there is a high probability that an aptamer having higher binding ability or inhibition ability will be obtained. Such a polynucleotide is also included in the “polynucleotide with one or more than ten bases substituted, deleted and / or inserted” defined in (b) of the present invention.

The polynucleotide (c) is a polynucleotide containing the polynucleotide (a) or (b). This includes not only those containing the polynucleotide (a) or (b) as a continuous partial region, but also those containing a fragment obtained by dividing the polynucleotide (a) or (b) at any site as a partial region. Nucleotides are also included. When a fragmented fragment is included as a partial region, all fragments may be included as a partial region in one molecule of polynucleotide, or may be included separately in a plurality of polynucleotide molecules. Good. Even if an arbitrary base sequence is added to one end or both ends of the aptamer, the aptamer region can have the same aptamer activity as long as the positional relationship and size of the stem portion and the loop portion are equal in the aptamer region. Furthermore, as described in Patent Document 2, the aptamer is divided in a loop region that does not participate in the binding to the target molecule, and two complementary polynucleotide sequences are linked to the divided sites, respectively. Even if it is prepared, it is known that when it is used by being folded, aptamer activity similar to that of an aptamer consisting of one original molecule of polynucleotide can be exhibited. Therefore, the luciferase-binding aptamer of the present invention can be similarly divided into a loop region and used as a bimolecular aptamer. As described above, the three-dimensional structure of a polynucleotide having a certain base sequence can be easily known by a conventional method using a known program, so which region becomes a loop region in a certain base sequence. Can know easily. Accordingly, aptamers composed of the polynucleotide (c) are also included in the scope of the present invention, as are aptamers composed of the polynucleotides (a) and (b).

The polynucleotide (c) includes a polynucleotide having an arbitrary sequence added to one or both ends of the polynucleotide (a) or (b), and the polynucleotide (a) or (b) within the loop region. Two molecules of polynucleotide each containing a fragment fragmented in two is preferred. Among them, a polynucleotide in which an arbitrary sequence is added to one end or both ends of the polynucleotide (a) or (b) is more preferable. However, when the aptamer of the present invention is used as an enzyme-controlled aptamer of AES, two molecules of polynucleotide each containing the latter fragment are more preferable (described later). The size of the sequence added to the polynucleotide (a) or (b) or a fragment thereof is not particularly limited. However, if the total length is too long, it takes time and cost for aptamer synthesis. Accordingly, the size of the additional sequence is generally 40 mer or less, preferably 10 mer or less, more preferably about 1 to 2 mer in total. The total length of the aptamer molecule is preferably about 100 mer or less. When an aptamer composed of a plurality of molecules of polynucleotide is prepared by dividing a luciferase-binding aptamer, the size of each molecule is preferably about 100 mer or less. However, this is not the case when the additional sequence is another aptamer sequence, and the size is determined according to the chain length of the aptamer to be added. For example, when constructing AES using the luciferase-binding aptamer of the present invention, the size of the aptamer molecule can be 100 mer or more depending on the chain length of the aptamer that recognizes the measurement target by AES.

For example, as a technique for increasing the binding ability of aptamers, a method of linking a plurality of aptamers to the same target molecule is known (Non-patent Document 5). Also in the luciferase-binding aptamer of the present invention, by linking two or three or more luciferase-binding aptamers (hereinafter sometimes referred to as “monomers”) comprising the polynucleotide of (a) or (b) above, The ability to bind to luciferase can be increased, and preferably the ability to control luciferase (described later) can also be enhanced. Such a linked aptamer is also included in the scope of the present invention as an aptamer composed of the polynucleotide (c). The size of the linked aptamer is determined according to the number of monomers to be linked, and can be 100 mer or larger.

Since luciferase is a monomeric protein, when preparing a linked aptamer, it is preferable to link monomers that bind to different sites on the luciferase protein. The structure may be such that the monomers are directly linked, or may be linked via a linker consisting of, for example, only adenine or only thymine (preferably only thymine). The chain length of the linker is not particularly limited, but is usually about 1 mer to 30 mer, particularly about 5 mer to 15 mer.

In the present invention, “Xnt” (X is a number) represents the Xth base from the 5 ′ end in the sequence. “Mer” indicates the number of nucleotides.

Preferably, the luciferase-binding aptamer of the present invention further has an ability to change the enzyme activity of the bound luciferase. Here, “change the enzyme activity” means that the enzyme activity of the luciferase to which the aptamer is bound is increased or decreased compared to the luciferase to which the aptamer is not bound, and is not particularly limited. Means that the enzyme activity decreases. Such aptamers having the ability to control luciferase activity (hereinafter sometimes referred to as “luciferase-controlled aptamers”) can be preferably employed as enzyme-controlled aptamers in known AES. Whether or not the aptamer has the ability to control luciferase activity is determined by, for example, mixing the aptamer with luciferase, luciferin as a substrate, and ATP and Mg necessary for the luciferase reaction, using a commercially available luminescence detector. It can be evaluated by measuring the amount of luminescence and examining how much the amount of luminescence has changed compared to the amount of luminescence when no aptamer is added.

As described in the Examples below, the luciferase-binding aptamer obtained by screening a random ssDNA library has the ability to change luciferase activity. In particular, aptamers having the base sequences shown in SEQ ID NOs: 5 and 9, respectively, have a high ability to inhibit luciferase activity, and are particularly preferable as the luciferase-controlled aptamer of the present invention.

FIGS. 3 and 4 show secondary structure prediction diagrams based on m-fold (trade name) of aptamers having the base sequences shown in SEQ ID NOs: 5 and 9, respectively. In the aptamers of SEQ ID NOS: 5 and 9 (Lap 高い 1-6 and Lap９1-20) with particularly high luciferase inhibitory ability, stem loop structures of the same size exist in the region of 10nt to 20nt. Exactly the same structure does not exist in other aptamers obtained in the following examples. Therefore, it is considered that the stem-loop structure having a 5-mer loop present at 10 nt to 20 nt is important for the high luciferase activity inhibiting ability of the aptamers of SEQ ID NOs: 5 and 9. Therefore, when the aptamers of SEQ ID NOs: 5 and 9 are divided and used, it is preferable to divide at a loop site other than the loop part (13nt to 17nt loop part) existing in this region. For example, in the case of the aptamer Lap 1-6 of SEQ ID NO: 5, loops also exist in the region of 32 nt to 34 nt and the region of 49 nt to 53 nt, so that it is preferable to divide at any site in these regions. Further, in the case of the aptamer Lap 1-20 of SEQ ID NO: 9, since loops also exist in the 23nt to 26nt region and the 35nt to 39nt region, it is preferable to break at any site within these regions.

A method for constructing AES using a split aptamer is known as described in Patent Document 2. More specifically, for example, the luciferase-regulated aptamer of the present invention is divided into two at a loop site that is not important for aptamer activity, and an aptamer (recognition aptamer) for a desired measurement object at one fragmented site. Concatenate arrays. A complementary sequence of about half or less (or about 3 to 20 mer) of the total length of the recognition aptamer sequence is linked to the fragmentation site of the other fragment. It may be directly linked to the split site, or may be linked via a linker of about 1 mer to 10 mer consisting of only adenine or thymine, for example. A so-called split AES as described in Patent Document 2 is prepared by mixing and folding two molecules of a polynucleotide having such a sequence, mixing this with luciferase, and binding the luciferase to a luciferase-regulated aptamer site. Can be built. As the recognition aptamer, any aptamer that can specifically bind to a desired measurement object can be used. Examples of known aptamers include adenosine aptamers and IgE aptamers described in Patent Document 1 and Patent Document 2, for example.

The method for measuring (including detection, quantification, and semi-quantification) of a measurement target in a sample using AES employing the luciferase-controlled aptamer of the present invention can be performed as follows, for example. That is, AES that is folded and bound with luciferase is brought into contact with a specimen, luciferin serving as a luciferase substrate, and ATP and Mg necessary for the luciferase reaction are mixed, and the amount of luminescence is measured with a known luminescence detector. This amount of luminescence is compared with the amount of luminescence (control) when the substrate is reacted with AES alone that is not brought into contact with the specimen, and the amount of change in the amount of luminescence is examined. For example, since the aptamers of SEQ ID NOs: 5 and 9 both have the effect of inhibiting luciferase, AES employing the aptamer as an enzyme control site reduces the luciferase inhibitory action of the aptamer due to the presence of the measurement object. Apparently, the luciferase activity is increased. In this case, it is possible to measure the measurement object in the specimen by examining how much the light emission amount has increased from the light emission amount of the control. If a calibration curve is created using a solution having a known concentration of the measurement object, the measurement object can be quantified. In addition, when the luciferase-controlled aptamer has the ability to increase luciferase activity, naturally, in AES employing this as an enzyme-controlled aptamer, the activity of luciferase apparently decreases due to the presence of the measurement object.

In addition, as described in Non-Patent Documents 2 to 4, by crossing or shuffling a plurality of aptamers capable of binding to a certain substance, it is possible to inhibit aptamers with higher binding ability or binding substances. It is known that aptamers that exhibit such functions can be created. Here, “intersection” and “shuffle” are to connect partial regions of different aptamer molecules, and are shuffled when the number of partial regions to be connected is large (although there is no particular rule, usually 3 or more). The aptamer having a higher ability to bind to luciferase than the original aptamer by selecting the aptamer having the ability to bind to luciferase among the obtained aptamers, the step of crossing or shuffling the aptamers of the present invention described above. Can be created. In this method, an aptamer having the ability to inhibit luciferase can also be produced by selecting an aptamer that can inhibit the activity of luciferase. Once such an aptamer is obtained, such a desired aptamer can be produced by analyzing the base sequence and producing it by chemical synthesis using a DNA synthesizer or the like.

The aptamer of the present invention can be easily prepared by a conventional method using a commercially available nucleic acid synthesizer. The AES polynucleotide adopting the aptamer of the present invention also adopts an aptamer whose sequence is specified as the recognition aptamer for the measurement target, and thus can be easily obtained by a conventional method using a commercially available nucleic acid synthesizer. Can be prepared.

The preferred use of the aptamer of the present invention is the use as a sensing element of a biosensor, specifically, the use for AES as described in Patent Documents 1 and 2. However, the use of the aptamer of the present invention is not limited to this, and it can also be used for measurement of luciferase by a method known per se using the binding ability to luciferase.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Screening for luciferase-binding aptamers [oligonucleotides used in screening]
Random library (modified with FITC at the 5 'end):
5'-FITC-ATAGTCTATCCATCAATT- (N30) -AGATAGCAAGTGTATTCA-3 '(SEQ ID NO: 1)
Forward primer (modified at the 5 'end with FITC):
5'-FITC-ATAGTCTATCCATCAATT-3 '(SEQ ID NO: 2)
Reverse primer (5 'end modified with Biotin):
5'-Biotin-TGAATACACTTGCTATCT-3 '(SEQ ID NO: 3)
(All the above libraries and primers were commissioned by Invitrogen)

[Reagent used]
Glucose dehydrogenase (PQQGDH), Hybond ™ -ECL ™ Nitrocellulose membrane (Amersham Bioscience), frozen pool serum L-Consera I EX for quality control (Nissui Pharmaceutical Co., Ltd.), 2-amino-2-hydroxyethyl 1,3-propanediol, NaCl, Tween21 (all Kanto Chemical Co., Ltd.), EDTA (Dojin Chemical Co., Ltd.), anti-FITC antibody HRP (Dako Cytomation), Immobilon (trademark) Western Chemiluminescent HRP Substrate (MILLIPORE ), Urea, chloroform (all Kanto Chemical Co., Ltd.), phenol (Wako Pure Chemical Industries, Ltd.), HCl, sodium acetate, isopropanol, ethanol, NaOH (all Kanto Chemical Co., Ltd.), glycogen (Roche) , 20 bp ladder (Takara), AmpliTaq Gold with Gene Amp (Applied Biosystems), Agarose 21 (Nippon Gene Co., Ltd.), Glycogen (Roche)

[Devices used]
Fluorescence scanner Typhoon 8600, quantitative software Image Quant (Amaersham Pharmacia Biotech), centrifuge, spectrophotometer UV-1200, UV-1600 (Shimadzu Corporation), high-speed shaker (EYELA), thermal cycler PC-700, PC-801-05 (Astech), ABI Prizm 3100 Genetic Analyzer (Applied Biosystem), Thermal cycler PC-700, PC-801-05 (Astech), Sequence Software (Genetics), Hybond-ECL Nitro Cellulose membrane (Amersham Biosciences), automatic fluorescence depolarizer (JASCO Corporation), BIAcore X (BIACORE), sensor chip SA (BIACORE), sensor chip CM5 (BIACORE), centrifuge, spectrophotometer UV-1200 , UV-1600 (Shimadzu Corporation), Thermal cycler PC-700, PC-801-05 (Astech), Fluorescence scanner Typhoon8600, Quantification software Image Quant (Amaersham Pharmacia Biotech)

[Method and results]
Aptamer screening was performed twice in total using the method described below (Screening 1, Screening 2). As a random library, the following 66-mer ssDNA (SEQ ID NO: 1) in which a primer binding region was added to both ends of a 30-mer random sequence was used. TBS (10 mM Tris / HCl, pH 7.0, 100 mM NaCl) and TBST (TBS containing 0.05% v / v Tween 20) with a surfactant added to TBS were used as the binding buffer for screening.

(a) Selection of DNA that binds to luciferase and evaluation of DNA library binding ability 22.5 pmol of luciferase and PQQGDH are dropped on each side of the nitrocellulose membrane (dropping to form a spot with a diameter of 2 mm) and air-dried To fix. The membrane was blocked by immersing the membrane immobilizing the protein in serum prepared to 10% by TBST and incubating at room temperature for 1 hour. Thereafter, the plate was washed with TBST for 10 minutes, followed by washing for 5 minutes twice.

A DNA library modified with FITC at the 5 ′ end was prepared to 1 nmol / 100 μL with TBS buffer, heated at 95 ° C. for 3 minutes, and then slowly cooled to 25 ° C. over 30 minutes to be folded. This DNA library was prepared to a final concentration of 90 nM and incubated with the protein-immobilized membrane at room temperature for 1 hour. Then, repeat the operation of immersing the membrane in Binding buffer (TBST, 5 ml volume per 1 cm ^{2 of} immobilized membrane) and gently washing it manually twice, and then wash with Binding buffer (TBST) for 10 minutes while stirring. Washing was performed twice for 5 minutes.

(b) Extraction of DNA bound to luciferase In the nitrocellulose membrane after incubation with the library and washing, the portion where luciferase was blotted was cut out, 222 μl of 7 M urea and 666 μl of phenol chloroform were added and stirred for 3 minutes Later, it was left at room temperature for 30 minutes. To this, 111 μl of MilliQ was added, stirred for 3 minutes, and centrifuged (12000 rpm, 10-20 ° C.), and the resulting supernatant was transferred to a new eppen. Chloroform in the same amount as the supernatant was added here, stirred for 3 minutes, then centrifuged (12000 rpm, 10-20 ° C), the supernatant was transferred to a new eppen, and the ssDNA molecules contained therein were precipitated by ethanol precipitation. It was collected. The obtained pellet was dissolved in 30 μl of TE buffer (10 mM Tris, 1 mM EDTA).

(c) Amplification of the extracted DNA Thirty PCR reactions (100 μl) containing 20 mol dNTP, 0.1 nmol primer (SEQ ID NOs: 2 and 3), 2.5 U Taq DNA polymerase, and 60 fmol template DNA were prepared. After heating at 95 ° C for 30 minutes with a thermal cycler, one cycle was 95 ° C for 1 minute, 56 ° C for 1 minute, 72 ° C for 1 minute, and 40 cycles were repeated. Each PCR product was subjected to electrophoresis in TAE buffer using a 3.5% agarose 21 gel, and it was confirmed that the DNA obtained in (b) used as a template was amplified. For confirmation, a 20 bp ladder was used as a marker for electrophoresis.

(d) Single-stranded amplification of amplified DNA 75 μl of avidin-immobilized agarose was washed twice with a 5-fold amount of Column buffer (30 mM HEPES, 500 mM NaCl, 5 mM EDTA, pH 7.0). To the PCR product, 1/10 volume of x50 TE Buffer and 1/5 volume of 5 M NaCl were added, and this solution was added to the washed avidin-immobilized agarose and incubated for 30 minutes. After incubation, the supernatant was removed, and the agarose was washed twice with 5 times the amount of Column buffer. Then, 1.5 times the amount of 0.15 M NaOH of agarose was added and stirred for 10 minutes. After collecting the supernatant, the same amount of 0.15 M NaOH was again added to the agarose and stirred for 10 minutes to elute ssDNA and collect the supernatant. The supernatant containing ssDNA was neutralized with 2M HCl, and ssDNA was recovered by ethanol precipitation. The obtained pellet was dissolved in 30 μl of TE buffer, and the absorbance at 260 nm was measured using a spectrophotometer to calculate the DNA concentration. All operations were performed at room temperature. The DNA obtained here was used as the library for the next round.

The above operations (a) to (d) were set as one round, and a total of four rounds were performed for one screening. This screening operation was repeated twice (screening 1, screening 2). In each round, in order to confirm the amount of DNA bound to the protein, the nitrocellulose membrane after incubation and washing with the library in the step (a) and an anti-FITC antibody modified with HRP (horseradish peroxidase) (1000 times) Diluted) was incubated in 1000 μl of Binding buffer (TBST) for 1 hour at room temperature. After the same washing as after incubation with the library, ECLEPlus Western blotting detection reagents was added to the membrane, and the binding of the ssDNA molecule to the protein was observed by chemiluminescence. As a result, in each round, a stronger signal was detected from the portion where luciferase was immobilized than the portion where PQQGDH was immobilized. Moreover, the tendency for the signal from the luciferase immobilization site to become stronger as the round progressed was observed.

2. Base sequence analysis of ssDNA obtained by screening [Reagents used]
Marmaid kit (Funakoshi), Agarose 21 (Nippon Gene), EDTA (Dojin Chemical), pGEM T-Vector System (Promega), Ampicillin (Sigma), IPTG (Takara), X-gal, Agar for medium [INAAGAR (BA-10)] (Ina Food Industry Co., Ltd.), Bactotrypsin, East Extract (DIFCO), NaCl, Tris, Ethanol (all Kanto Chemical Co., Ltd.), Formamide, RNase (Nippon Gene) ), DNA sequence kit, M13 primer (Applied Biosystem), DH5α competent cell

[Method and results]
(a) TA cloning Ten PCR solutions having the same composition as in 1- (c) were prepared, and after PCR amplification, ethanol precipitation was performed (primers were used without modification). The precipitate was dissolved, electrophoresed in TAE buffer using 3% agarose X gel, and about 66 bp DNA was extracted from the gel using MERmaid (registered trademark) kit to obtain 10 μl of DNA extract. The protocol attached to the kit was followed for DNA extraction from the gel. 3 μl of DNA extract, 5 μl Rapid Ligation buffer, 1 μl of pGEM T-Vector (50 ng) and 1 μl of T4 DNA ligase were added, and ligation was performed at 16 ° C. for 1 hour. JM109 competent cells were transformed with the ligation reaction solution by heat shock method, seeded on LB agar medium containing ampicillin (Amp) and X-gal (both final concentration 100 μg / ml) and cultured at 37 ° C overnight .

(b) Sequence White colonies were selected from the TA-cloned plate, incubated overnight in LB medium, and then the plasmid was extracted by the alkali-SDS method. The extracted plasmid was digested with restriction enzymes and then electrophoresed in the same manner as above to confirm that the target fragment was inserted.

For the plasmid in which the insert fragment was confirmed, the base sequence of the insert fragment (that is, the DNA obtained by screening) was determined by the dideoxy method using a kit. In screening 1, 15 nucleotide sequences were obtained, and there was no overlap among them. In screening 2, 22 nucleotide sequences were obtained, of which two types of sequences were duplicated two by two. There was no nucleotide sequence overlap between Screens 1 and 2. That is, 35 types of base sequences were obtained by screening 1 and 2.

3. Evaluation of binding ability of each ssDNA to luciferase by aptamer blotting method and specificity using PQQGDH [Reagent used]
Glucose dehydrogenase, human thrombin (Sigma Aldrich), Hybond ™ -ECL ™ Nitrocellulose membrane (Amersham Bioscience), 2-amino-2-hydroxyethyl-1,3-propanediol, NaCl, Tween21 (all Kanto Chemical) Co., Ltd.), EDTA (Dojindo), anti-FITC antibody HRP (Dako Cytomation), NeutrAvidin Horseradish Peroxidase Conjugated (PIERCE), Immobilon (trademark) Western Chemiluminescent HRP Substrate (MILLIPORE), PQQGDH, NaCl, Tris, Tween 21, urea, chloroform, HCl, sodium acetate, isopropanol, ethanol, NaOH (all Kanto Chemical Co., Ltd.), EDTA (Dojin Chemical Co., Ltd.), Ponceau S (Merck), glycogen (Roche), phenol (Wako Jun) Yakuhin Kogyo Co., Ltd.), 20 bp ladder (Takara), AmpliTaq Gold with Gene Amp (Applied Biosystems), Agarose 21 (Nippon Gene Co., Ltd.), ImmunoPure Immobilized Avidin (PIERCE)

[Devices used]
Fluorescence scanner Typhoon8600, quantitative software Image Quant (Amaersham Pharmacia Biotech), Mild Mixer SI-36 (TAITEC), PLUS SHAKER EP-1 (TAITEC)

The secondary structure of the 66mer base sequence including the primer sequence was predicted using the secondary structure prediction program m-fold (trade name) for the 35 types of base sequences obtained as a result of the sequence analysis. As a result, 9 to 1 base sequences and 15 to 15 base sequences that were considered to have a stable secondary structure from the ΔG value and the Tm value were obtained. A total of 24 of these were evaluated for binding ability to luciferase by the aptamer blotting method. For aptamer blotting, a 66mer oligonucleotide whose 5 ′ end was modified with biotin or FITC was synthesized and used. Moreover, PQQGDH was used as a competitive protein as in the screening.

The results of aptamer blotting are shown in FIG. The binding ability of all 24 types of evaluated polynucleotides to luciferase was confirmed. Since a difference in spot intensity was observed between the arrays, it is considered that there is a difference in binding ability. 21 out of 24 (Table 1) were confirmed to have higher binding ability than the initial library. These 21 nucleotide sequences are shown in SEQ ID NOs: 4 to 24.

4). Examination of aptamer's ability to control luciferase activity Among the ssDNAs obtained in Screens 1 and 2, those that have higher binding ability to luciferase than the initial library (Lap 2-14, 1-20, 1-6) and luciferase binding Those having low ability (Lap 2-26, 2-3) were selected, and the ability to control luciferase activity was examined. The base sequences of Lap 2-26 and Lap 2-3 are shown in SEQ ID NOs: 25 and 26, respectively.

Luciferase (final concentration: 0.9 μM), each ssDNA (final concentration: 9 μM), Tris (10 μmM), NaCl (150 μmM), KCl (5 μmM) were mixed to make a total volume of 18 μL, and allowed to stand at room temperature for 10 minutes. 75 μL of Picker Gene luminescent substrate (Toyo Benet) was added to 5 μL of this sample, allowed to stand for 1 minute, shaken for 10 seconds, and luminescence was measured for 1 second using a luminometer. The luciferase activity control ability of each ssDNA was evaluated by relative evaluation of the luminescence amount of each ssDNA sample with the control luminescence amount being 100%. The results are shown in Table 2 below. The “Δ spot intensity” in Table 2 is the result of digitizing the aptamer blotting spot shown in FIG. 1 using a fluorescence scanner Typhoon 8600 and quantitative software Image-Quant® (Amaersham-Pharmacia Biotech).

As shown in Table 2, large inhibition of luciferase activity was observed with Lap 1-20 and Lap 1-6, which have high binding ability to luciferase.

For Lap 1-6, which had the highest ability to inhibit luciferase activity, the amount of luminescence was measured and evaluated in the same manner as described above by changing the ssDNA concentration to 0, 0.9, 9.0, and 45 μM. However, it inhibited luciferase activity by 40% (FIG. 2).

FIG. 3 and FIG. 4 show the secondary structure diagrams predicted by m-fold (trade name) for Lap 1-6 and Lap 1-20, which have a high ability to inhibit luciferase activity. In Lap 1-6 and Lap 1-20, which were confirmed to have a high ability to inhibit luciferase, the same stem-loop structure was present in the 10 nt to 20 nt region. This stem-loop structure did not exist in any of the other aptamers obtained by the above screening, including other aptamers whose activity control ability was examined above (data not shown). Therefore, it is considered that this stem-loop structure existing at 10 nt to 20 nt is important for the high luciferase activity inhibiting ability of Lap 1-6 and Lap 1-20.

Claims

An aptamer consisting of any of the following polynucleotides and having the ability to bind to luciferase.
(a) A polynucleotide having the base sequence shown in any one of SEQ ID NOs: 4 to 24.
(b) A polynucleotide in which 1 to 10 or more bases are substituted, deleted and / or inserted in the polynucleotide of (a).
(c) A polynucleotide comprising the polynucleotide of (a) or (b).
The aptamer according to claim 1, wherein the polynucleotide (b) is a polynucleotide in which one or several bases in the polynucleotide (a) are substituted, deleted and / or inserted.
The aptamer according to claim 2, wherein the polynucleotide (b) is a polynucleotide in which one or two bases in the polynucleotide (a) are substituted, deleted and / or inserted.
The aptamer according to claim 1, comprising a polynucleotide having the base sequence shown in any one of SEQ ID NOs: 4 to 24 or a polynucleotide containing the polynucleotide.
The aptamer according to claim 4, wherein the polynucleotide has a size of 100 mer or less.
6. The aptamer according to claim 5, comprising a polynucleotide having a base sequence represented by any of SEQ ID NOs: 4 to 24.
The aptamer according to any one of claims 1 to 6, further having an ability to change the enzyme activity of the bound luciferase.
The aptamer according to claim 7, which comprises a polynucleotide having the base sequence represented by SEQ ID NO: 5 or 9, or a polynucleotide containing the polynucleotide.
The aptamer according to claim 8, wherein the size of the polynucleotide is 100 mer or less.
10. The aptamer according to claim 9, comprising a polynucleotide having a base sequence represented by SEQ ID NO: 5 or 9.
A method for producing an aptamer having the ability to bind to luciferase, comprising the steps of crossing or shuffling a plurality of aptamers according to claim 1 and selecting an aptamer having the ability to bind to luciferase among the obtained aptamers. .
The method according to claim 11, further comprising selecting an aptamer having an ability to inhibit luciferase activity.
A method for producing a luciferase-binding aptamer, comprising producing an aptamer produced by the method according to claim 11 or 12.