WO2009113564A1 - Aptamère de liaison à la luciférase - Google Patents

Aptamère de liaison à la luciférase Download PDF

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WO2009113564A1
WO2009113564A1 PCT/JP2009/054617 JP2009054617W WO2009113564A1 WO 2009113564 A1 WO2009113564 A1 WO 2009113564A1 JP 2009054617 W JP2009054617 W JP 2009054617W WO 2009113564 A1 WO2009113564 A1 WO 2009113564A1
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aptamer
luciferase
polynucleotide
ability
binding
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PCT/JP2009/054617
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Japanese (ja)
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一典 池袋
広司 早出
直樹 梶山
恵子 黒澤
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国立大学法人東京農工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the present invention relates to an aptamer that binds to luciferase.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • AES AdimerictaEnzyme Subunit
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • the luminescent substrate luciferin reacts with ATP in the presence of Mg 2+ to produce a luciferyl AMP intermediate in luciferase.
  • the luciferyl AMP intermediate reacts with oxygen molecules and decomposes into excited oxyluciferin and CO 2 .
  • 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.
  • 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.
  • 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).
  • having a base sequence means that bases are arranged in the sequence in the polynucleotide.
  • 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.
  • 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.
  • TBS 10 mM Tris / HCl, pH 7.0, 100 mM NaCl
  • TBST TBS containing 0.05% v / v Tween 20
  • an aqueous solution containing 10 mM MOPS and 1 mM CaCl 2 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 ⁇ L). By cooling (over about 30 minutes), the aptamer molecule can be folded.
  • 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.
  • luciferase and any other 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.
  • a support such as a nitrocellulose membrane
  • aptamer such as TBS
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Non-Patent Documents 2 to 4 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.
  • 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.
  • 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.
  • 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.
  • the luciferase-binding aptamer of the present invention can be similarly divided into a loop region and used as a bimolecular aptamer.
  • 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.
  • 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.
  • the aptamer of the present invention when 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.
  • the size of each molecule is preferably about 100 mer or less.
  • the additional sequence is another aptamer sequence, and the size is determined according to the chain length of the aptamer to be added.
  • 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.
  • Non-patent Document 5 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.
  • luciferase is a monomeric protein
  • 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.
  • Xnt (X is a number) represents the Xth base from the 5 ′ end in the sequence.
  • “Mer” indicates the number of nucleotides.
  • the luciferase-binding aptamer of the present invention further has an ability to change the enzyme activity of the bound luciferase.
  • “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.
  • 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.
  • the luciferase-binding aptamer obtained by screening a random ssDNA library has the ability to change luciferase activity.
  • 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.
  • the aptamers of SEQ ID NOS: 5 and 9 Lap ⁇ ⁇ 1-6 and Lap91-20
  • 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.
  • 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.
  • a loop site other than the loop part (13nt to 17nt loop part) existing in this region.
  • 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.
  • 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.
  • 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.
  • 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.
  • the luciferase activity is increased.
  • 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.
  • 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.
  • 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.
  • “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).
  • 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.
  • 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.
  • 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)
  • a DNA library modified with FITC at the 5 ′ end was prepared to 1 nmol / 100 ⁇ L 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.
  • 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 ⁇ l 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 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.
  • 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 ⁇ G 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.
  • Luciferase (final concentration: 0.9 ⁇ M), each ssDNA (final concentration: 9 ⁇ M), Tris (10 ⁇ mM), NaCl (150 ⁇ mM), KCl (5 ⁇ mM) were mixed to make a total volume of 18 ⁇ L, and allowed to stand at room temperature for 10 minutes.
  • 75 ⁇ L of Picker Gene luminescent substrate (Toyo Benet) was added to 5 ⁇ L 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 “ ⁇ 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).
  • 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.
  • 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.

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Abstract

L'invention porte sur un moyen pour détecter de façon simple, rapide et sensible un marqueur de maladie sans effectuer de séparation B/F. L'invention porte sur une nouvelle molécule d'aptamère qui présente une séquence nucléotidique spécifique et se lie à la luciférase, en particulier sur une nouvelle molécule d'aptamère qui se lie à la luciférase et également qui supprime l'activité enzymatique de la luciférase. Par exemple, si un aptamère capable de supprimer l'activité de la luciférase est combiné avec un autre aptamère reconnaissant une substance cible voulue telle qu'un marqueur de maladie, un système de détection avec une sensibilité très élevée utilisant la luminescence de la luciférase peut être construit.
PCT/JP2009/054617 2008-03-11 2009-03-11 Aptamère de liaison à la luciférase WO2009113564A1 (fr)

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JP2008-061730 2008-03-11
JP2008061730 2008-03-11

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Publication number Priority date Publication date Assignee Title
JP2012024026A (ja) * 2010-07-23 2012-02-09 Tdk Corp Ｓｍ菌特異的アプタマー、Ｓｍ菌増殖抑制剤及びＳｍ菌の検出方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012070649A (ja) * 2010-09-28 2012-04-12 Katayanagi Institute Ｄａｐｏｘｙｌ又はその類縁体に結合するＤＮＡアプタマー及びその使用方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005049826A1 (fr) * 2003-11-22 2005-06-02 Ultizyme International Ltd. Methode de detection d'une molecule cible au moyen d'un aptamere
WO2007032359A1 (fr) * 2005-09-12 2007-03-22 National University Corporation Tokyo University Of Agriculture And Technology Procédé de détection d'une molécule cible à l'aide d'un complexe aptamère/sonde
JP2007325546A (ja) * 2006-06-08 2007-12-20 Kikkoman Corp 分割ルシフェラーゼ

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2005049826A1 (fr) * 2003-11-22 2005-06-02 Ultizyme International Ltd. Methode de detection d'une molecule cible au moyen d'un aptamere
WO2007032359A1 (fr) * 2005-09-12 2007-03-22 National University Corporation Tokyo University Of Agriculture And Technology Procédé de détection d'une molécule cible à l'aide d'un complexe aptamère/sonde
JP2007325546A (ja) * 2006-06-08 2007-12-20 Kikkoman Corp 分割ルシフェラーゼ

Non-Patent Citations (1)

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"88th Annual Meeting on Chemical Society of Japan in Spring Koen Yokoshu II, 12 March, 2008", 12 March 2008, article KAZUNORI IKEBUKURO ET AL.: "Luciferase ni Ketsugo suru DNA Aptamer no Tansaku", pages: 818 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012024026A (ja) * 2010-07-23 2012-02-09 Tdk Corp Ｓｍ菌特異的アプタマー、Ｓｍ菌増殖抑制剤及びＳｍ菌の検出方法

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