WO2010113789A1 - Selex法用のプライマーの設計方法、プライマーの製造方法、アプタマーの製造方法、プライマーの設計装置、プライマー設計用コンピュータプログラムおよび記録媒体 - Google Patents
Selex法用のプライマーの設計方法、プライマーの製造方法、アプタマーの製造方法、プライマーの設計装置、プライマー設計用コンピュータプログラムおよび記録媒体 Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
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- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
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- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
Definitions
- the present invention relates to a primer design method for SELEX method, a primer production method, an aptamer production method, a primer design apparatus, a primer design computer program, and a recording medium.
- Aptamers are nucleic acid ligands that specifically bind to target substances. Aptamers were first reported in 1990 by GOLD et al. And acquired by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method (Patent Document 1, Non-Patent Document 1).
- SELEX Systematic Evolution of Ligands by Exponential Enrichment
- a target substance is immobilized on a carrier such as a bead
- a nucleic acid library such as DNA or RNA is added thereto
- a nucleic acid that binds to the target substance is recovered, the recovered nucleic acid is amplified, and the amplified nucleic acid is amplified.
- a series of steps of adding the protein to the target substance is repeated about 10 times, the nucleic acid having high specificity and binding power to the target substance is concentrated, and its base sequence is determined to obtain an aptamer. is there.
- Aptamers are expected to be applied to pharmaceuticals and sensors. For example, pegaptanib sodium injection, a therapeutic agent for angiogenic age-related macular degeneration (AMD), has been developed and put to practical use as an aptamer drug. .
- the SELEX method it is necessary to design primers for nucleic acid amplification. It is necessary to design primers even in gene amplification methods represented by PCR, and many softwares have been developed to support primer design. However, there is no software that supports the design of primers used in the SELEX method. Also, unlike the gene amplification method primer design that can be designed assuming the sequence of the gene, the primer design in the SELEX method has no information that can be assumed, while calculating the secondary structure, Tm value, etc. from zero It is necessary to repeat trial and error by hand. Moreover, the designed primer does not always work as desired. As described above, the primer design of the SELEX method has a problem that it takes labor, time and cost, is uncertain, and is inefficient.
- the present invention provides a primer design method capable of efficiently designing a primer for the SELEX method, a primer production method using the primer, an aptamer production method, a primer design device, a primer design computer program, and a recording medium
- the purpose is to do.
- the primer design method for the SELEX method of the present invention is: A candidate primer sequence generating step for generating a candidate primer sequence; A candidate primer sequence selection step of evaluating the candidate primer sequence and selecting a candidate primer sequence based on a preset criterion; A random pool generating step for generating a random pool including a plurality of nucleic acid sequences including a random sequence and a candidate primer sequence based on the selected candidate primer sequence; A random pool selection step of predicting the structure of each nucleic acid sequence of the random pool, and evaluating the predicted structure and selecting a random pool based on a preset criterion; A primer sequence determination step for determining that a candidate primer sequence used for the selected random pool is adopted as a primer sequence, and performing each of the steps using a computer to obtain a primer for the SELEX method. It is characterized by designing.
- the method for producing a primer for the SELEX method of the present invention includes a primer design step, A synthesis step of synthesizing a primer based on the primer sequence designed in the design step, The primer designing step is performed by the primer designing method of the present invention.
- the aptamer production method of the present invention is characterized in that an aptamer is produced by the SELEX method using the primer produced by the primer production method of the present invention.
- the primer design apparatus for the SELEX method of the present invention is A data processing device, a storage device, an input device and an output device;
- the data processing device is A candidate primer sequence generating means for generating a candidate primer sequence;
- Primer candidate sequence selection means for evaluating the candidate primer sequence and selecting the candidate primer sequence based on a preset criterion;
- Random pool generating means for generating a random pool including a plurality of nucleic acid sequences including a random sequence and a primer candidate sequence based on the selected primer candidate sequence; Random pool selection means for predicting the structure of each nucleic acid sequence of the random pool, evaluating the predicted structure based on a preset criterion, and selecting a random pool;
- Primer sequence determination means for determining that the candidate primer sequence used in the selected random pool is adopted as a primer sequence.
- the primer design computer program for the SELEX method of the present invention is characterized in that the primer design method of the present invention can be executed on a computer.
- the recording medium of the present invention is characterized by storing a computer program for primer design for the SELEX method of the present invention.
- the present invention it is possible to design a primer suitable for the purpose by simulating with a computer before actually synthesizing the primer. Therefore, it is possible to reduce labor, time and cost, and with excellent reliability and efficiency. Can be designed. Furthermore, according to the present invention, the secondary structure of the nucleic acid in the random pool can be controlled. For example, the structure of the aptamer can be controlled, and the random pool can be designed with reference to the obtained aptamer structure. Therefore, improvement in the acquisition efficiency of the target aptamer can also be expected.
- FIG. 1 is a configuration diagram showing an example of the configuration of the primer designing apparatus of the present invention.
- FIG. 2 is a flowchart showing an example of the primer designing method of the present invention.
- FIG. 3 is a block diagram showing another configuration of the primer designing apparatus of the present invention.
- FIG. 4 is an explanatory diagram showing a state in which the primer designing apparatus of the other example is connected to a server via a communication network.
- FIG. 5 is a class classification diagram based on the graph structure of the secondary structure of the nucleic acid.
- FIG. 6 is a graph showing the distribution of the number of vertices of the secondary structure of only the random area (array) in the random pool according to the embodiment of the present invention.
- FIG. 7 is a graph showing the distribution of the number of vertices of the secondary structure of the entire nucleic acid sequence in the random pool of the example of the present invention.
- FIG. 8 is a graph showing the distribution of the secondary structure class of only the random region (array) in the random pool according to the embodiment of the present invention.
- FIG. 9 is a graph showing the distribution of secondary structure classes of the entire nucleic acid sequence in the random pool of the example of the present invention.
- FIG. 1 shows an example of the configuration of the primer designing apparatus of the present invention.
- the apparatus of this example includes an input device 11, a storage device 12, an output device 13, and a data processing device 14 as main components.
- the input device 11 is for inputting information to the data processing device 14, and examples thereof include a keyboard, a key sheet, a mouse, a terminal such as a USB, a drive such as a CD and a CD-R, and the like.
- the storage device 12 is for storing various information such as information on primer design conditions, information on random pools, information on designed primer sequences, and various programs. For example, flash memory, RAM, ROM, and the like are appropriately stored. Composed in combination.
- the output device 13 is for outputting designed primer sequence information and other information, and examples thereof include a display and a printer.
- the data processing device 14 performs a series of processing for primer design, for example, a central processing unit (CPU).
- the data processing apparatus 14 of this example includes a primer candidate sequence generation unit 141, a primer candidate sequence selection unit 142, a random pool generation unit 143, a random pool selection unit 144, and a primer sequence determination unit 145.
- the primer design apparatus may be constituted by a single computer or a plurality of computers.
- the primer designing apparatus can design the primer as follows, for example, according to the flowchart shown in FIG.
- a candidate primer sequence is generated by a candidate primer sequence generating means.
- the primer sequence is, for example, a combination of A, G, C, T, and U bases. If the sequence is DNA, use T; if it is RNA, use U. It is necessary to generate two types of primer candidate sequences, the 3 ′ end side and the 5 ′ end side.
- the length (number of bases) of the candidate primer sequence is not particularly limited, but is 10 to 100 bases, preferably 15 to 50 bases, and more preferably 20 to 30 bases.
- the candidate primer sequences may be generated, for example, at random or with reference to a database of known primer sequences. In addition, candidate primer sequences may be input from the input device 11.
- the criteria include, for example, the secondary structure formation ability of the primer, Tm value, sodium concentration, GC content, GC content fluctuation width, nucleic acid concentration, bias between G and C in total GC, and between A and T in total AT. There is a bias.
- the secondary structure forming ability of the primer is, for example, Max free energy (a threshold value of free energy allowed as a primer structure).
- primer design software for gene amplification For evaluation of candidate primer sequences, for example, software for designing primers for gene amplification can be used. As such software, a commercially available product may be used, or software on a site published on the Internet may be used. In addition, many of these primer design software for gene amplification can also generate primer sequences. When such software is used, the generation (S1) and selection (S2) of candidate primer sequences are performed. It is also possible to carry out at the same time.
- the random pool is a nucleic acid sequence mixture (library) including a plurality of nucleic acid sequences including random sequences and candidate primer sequences.
- the random sequence is a sequence serving as an aptamer candidate, for example, a sequence in which bases of A, G, C, and U or T are randomly linked.
- the length (number of bases) of the random sequence is, for example, 20 to 120 bases, preferably 30 to 80 bases, and more preferably 30 to 40 bases.
- the types of the nucleic acid sequences contained in the random pool are, for example, 4 20 to 4 120 types (about 10 12 to 10 72 types), preferably 4 30 to 4 60 types (about 10 18 to 10 36 types). ) It is.
- Each nucleic acid sequence included in the random pool may further include a fixed sequence with respect to the random sequence.
- the fixed sequence include a sequence in which G or C bases are linked.
- a GGGG sequence may be added to the terminal side of the forward (Fw) primer candidate sequence
- a CCCC sequence may be added to the tip of the reverse (Rv) primer candidate sequence.
- Random pool selection S4
- the structure of each nucleic acid sequence is predicted, and the predicted structure is evaluated based on a preset criterion to select a random pool.
- known software for predicting the structure of DNA and RNA can be used.
- the criterion a variety of secondary structures can be used.
- classification of secondary structure classification by graph structure can be used (GEVERTZ J. et.al. RNA 2005; 11; 853-863).
- FIG. 5 shows the classification of the secondary structure by the graph structure. As shown in the figure, the class classification represents a stem as a side ( ⁇ ) and a loop as a point (•). In the figure, a graph structure having seven or less points is shown. If the standard is not satisfied (No), the process returns to the random pool generation step again. If the standard is satisfied (Yes), the process proceeds to the next step.
- the candidate primer sequence used for the selected random pool is adopted as the primer sequence, and the sequence is determined as the primer sequence.
- the determined primer sequence information is output from an output device such as a display (S6).
- the determined primer sequence information may be stored in a storage device.
- FIG. 2 The configuration of the apparatus of this example is shown in FIG. In FIG. 3, the same parts as those in FIG.
- the main configuration of the apparatus of this example is the same as that of the apparatus of the first embodiment except that the apparatus includes a communication interface and can be connected to a communication network such as the Internet.
- the apparatus 1 of this example can be connected to a server 3 installed outside the apparatus via a communication network 2 such as the Internet.
- the apparatus of this example is a program in a site of a server outside the apparatus, which includes at least one of primer candidate sequence generation means, primer candidate sequence selection means, random pool generation means, random pool selection means and primer sequence determination means.
- the data processing apparatus of the apparatus according to the present embodiment may not include each of the means to be executed by the program of the server outside the apparatus.
- This embodiment is a method for producing a primer based on the primer sequence designed by the primer designing method of the present invention.
- the primer design method is as described above.
- a known method can be used as a primer synthesis method.
- Specific examples of the synthesis method include a method of chemically synthesizing from a terminal base using dNTP or the like as a material with a DNA synthesizer or an RNA synthesizer.
- the synthesizer a commercially available product may be used.
- This embodiment is an example of a method for producing an aptamer by the SELEX method using the primer produced by the primer production method of the present invention.
- the method for producing the primer is as described above. In the following, the SELEX method will be described.
- the aptamer is a nucleic acid molecule that can specifically bind to a specific target substance.
- single-stranded nucleic acid such as single-stranded RNA and single-stranded DNA
- double-stranded RNA and two-stranded RNA examples include double-stranded nucleic acids such as double-stranded DNA.
- the nucleic acid aptamer may have, for example, a secondary structure by self-annealing, and examples of the secondary structure include a stem-loop structure.
- the nucleic acid aptamer may be, for example, a naturally derived nucleic acid sequence or a synthesized nucleic acid sequence.
- the synthesis method is not limited at all, and examples thereof include a method of chemically synthesizing from a terminal base using dNTP or the like as a material with a DNA synthesizer or an RNA synthesizer.
- Examples of the nucleic acid aptamer include DNA and RNA, and may include a peptide nucleic acid such as PNA.
- the nucleic acid aptamer may include natural nucleic acids (non-artificial nucleic acids) such as A, C, G, T and U, 2′-fluorouracil, 2′-aminouracil, 2′-O-methyluracil, An artificial nucleic acid such as 2-thiouracil may be contained.
- natural nucleic acids non-artificial nucleic acids
- 2-thiouracil an artificial nucleic acid such as 2-thiouracil may be contained.
- the production of the nucleic acid aptamer by the SELEX method is not particularly limited, but can be performed, for example, as follows. First, a nucleic acid pool containing a plurality of nucleic acids is prepared, and the nucleic acid library and the target substance are bound (associated) to form a complex of the nucleic acid pool and the target substance. Then, by collecting only the nucleic acid probe involved in the formation of the complex from the complex, a nucleic acid aptamer that can specifically bind to the target substance can be prepared.
- a method for preparing a nucleic acid aptamer that can specifically bind to the target substance using the SELEX method will be described in detail, but the present invention is not limited thereto.
- the nucleic acid pool is, for example, a library (mixture) of nucleic acids having a random sequence.
- the nucleic acid in the library include polynucleotides such as RNA and DNA.
- the random region (sequence) is, for example, a region (sequence) in which A, G, C, and U or T bases are randomly linked, and its length is, for example, 20 to 120 mer.
- the nucleic acid pool preferably contains, for example, 4 20 to 4 120 types (about 10 12 to 10 72 types) of nucleic acids, and more preferably 4 30 to 4 60 types (about 10 18 to 10 36 types). is there.
- the polynucleotide contained in the nucleic acid pool has, for example, a primer sequence used in nucleic acid amplification described later, a polymerase recognition sequence recognized by a polymerase, and the like at at least one of the 5 'end and the 3' end of the random sequence.
- the primer sequence is a sequence designed by the aforementioned primer design method.
- the polymerase recognition region can be appropriately determined according to, for example, the type of polymerase used in nucleic acid amplification described later in aptamer preparation.
- the polymerase recognition sequence is preferably, for example, a DNA-dependent RNA polymerase recognition sequence (hereinafter also referred to as “RNA polymerase recognition sequence”), and specific examples include a T7 RNA polymerase recognition sequence. And the T7 promoter.
- RNA polymerase recognition sequence for example, from the 5 ′ end side, the RNA polymerase recognition sequence and the primer sequence (hereinafter also referred to as “5 ′ end side primer sequence”) are linked in this order, The random sequence was linked to the 3 ′ end of the 5 ′ end primer sequence, and the primer sequence (hereinafter also referred to as “3 ′ end primer sequence”) was linked to the 3 ′ end of the random sequence.
- the 5 ′ terminal primer sequence in the RNA is, for example, a sequence complementary to the 3 ′ end of a DNA antisense strand synthesized using the RNA as a template, that is, bound to the 3 ′ end of the antisense strand.
- the sequence is preferably the same as possible primers.
- the RNA pool may further include, for example, a nucleic acid having a region (sequence) that assists in binding to a target substance.
- the polynucleotides contained in the nucleic acid pool may have different random sequences, or may have a random sequence in which some sequences are common. In the polynucleotide, each sequence may be directly adjacent (linked) or indirectly adjacent (linked) via an intervening sequence, for example.
- the method for preparing the nucleic acid pool is not particularly limited, and a known method can be adopted.
- the nucleic acid pool is an RNA pool
- an initial pool containing DNA can be used and the DNA can be prepared as a template.
- a DNA strand that serves as a template for RNA in a nucleic acid pool is also referred to as an antisense strand
- a DNA strand having a sequence in which U of the RNA is replaced with T is also referred to as a sense strand.
- Examples of the initial pool containing the DNA include, for example, DNA (antisense strand) obtained by substituting T in the complementary strand of the random region (sequence) in the RNA pool with T, and U in the random region (sequence) as T It is preferable that any one of DNAs (sense strands) having a sequence substituted with is included.
- transcription reaction is performed using DNA-dependent RNA polymerase using the obtained DNA amplification product as a template. Nucleic acid pools can be prepared.
- an initial pool containing DNA in which U in the random region is replaced with T is prepared, and using this as a template, a primer containing the RNA polymerase recognition sequence and a sequence complementary to the 5′-end primer sequence
- a nucleic acid pool of RNA can also be prepared by annealing and nucleic acid amplification.
- the binding form between the nucleic acid pool and the target substance is not particularly limited, and examples thereof include intermolecular forces such as hydrogen bonding.
- Examples of the binding treatment between the nucleic acid pool and the target substance include a method of incubating the both in a solvent for a certain period.
- the solvent is not particularly limited, but is preferably one that retains the binding between the two, and examples thereof include various buffer solutions.
- the complex of the nucleic acid pool and the target substance is recovered.
- a nucleic acid pool that does not participate in the complex formation hereinafter referred to as “unreacted nucleic acid pool”. included. Therefore, for example, it is preferable to separate the complex and the unreacted nucleic acid pool from the reaction solution.
- the separation method is not particularly limited, and examples thereof include a method using a difference in adsorptivity between the target substance and the nucleic acid pool and a method using a difference in molecular weight between the complex and the nucleic acid pool.
- Examples of the method using the former difference in adsorptivity include the following methods. That is, first, a carrier having adsorptivity to the target substance is brought into contact with the reaction solution containing the complex. At this time, the unreacted nucleic acid pool is not adsorbed on the carrier, while the complex of the target substance and the nucleic acid pool is adsorbed on the carrier. Thereby, the unreacted nucleic acid pool and the complex can be separated. Then, after removing the unreacted nucleic acid pool, the complex adsorbed on the carrier may be recovered. Moreover, before recovering the complex from the carrier, for example, it is preferable to wash the carrier in order to sufficiently remove the unreacted nucleic acid pool.
- the carrier having adsorptivity to the target substance is not particularly limited, and can be appropriately selected according to, for example, the type of target substance.
- examples of the adsorptive carrier include a nitrocellulose membrane.
- a method using a carrier can be exemplified.
- the carrier include a carrier having a pore of a size that allows the nucleic acid pool to pass through and the complex cannot pass through.
- the separation may be electrical separation using, for example, an agarose gel or a polyacrylamide gel.
- a method of using a target substance immobilized on a carrier at the time of forming the complex can be mentioned. That is, the target substance is immobilized on a carrier in advance, the carrier and the nucleic acid pool are brought into contact with each other, and a complex of the immobilized target substance and the nucleic acid pool is formed. Then, after removing the unreacted nucleic acid pool not bound to the immobilized target substance, the complex of the target substance and the nucleic acid pool may be dissociated from the carrier.
- the method for immobilizing the target substance on the carrier is not limited at all, and a known method can be adopted.
- a tag is bound to the target substance, and the target substance bound to the tag is brought into contact with a carrier having a binding group with the tag.
- the tag include His-tag
- the binding group include metal ions such as nickel ions (Ni 2+ ) and cobalt ions (Co 2+ ).
- the carrier include Ni-agarose and Ni-Sepharose based on the metal ions.
- the nucleic acid pool involved in the complex formation is recovered from the recovered complex.
- the nucleic acid pool involved in the complex formation can be recovered, for example, by releasing the binding between the target substance and the nucleic acid pool for the complex.
- nucleic acid amplification is performed on the collected nucleic acid pool involved in the complex formation.
- the nucleic acid amplification method is not particularly limited, and can be performed by a known method, for example, depending on the type of the nucleic acid pool.
- the nucleic acid pool is an RNA pool
- cDNA is prepared by a reverse transcription reaction using an RNA-dependent DNA polymerase
- the nucleic acid is amplified by PCR using the cDNA as a template, and the obtained amplification product is used as a template.
- RNA can be transcribed using a DNA-dependent RNA polymerase.
- the RNA pool involved in the complex formation described above can be amplified.
- RNA pool has, for example, the RNA polymerase recognition sequence, the 5′-end primer sequence, the random sequence, and the 3′-end primer sequence as described above
- an amplification method using these sequences can be used. can give.
- a polynucleotide containing a sequence complementary to the 3 'terminal primer sequence of the RNA pool is preferably used as a primer.
- a polynucleotide containing the 5′-end primer sequence and a polynucleotide containing a complementary strand of the 3′-end primer sequence are used.
- the former polynucleotide preferably further has the RNA polymerase recognition sequence on the 5 'end side and the 5' end side primer sequence on the 3 'end side.
- the DNA amplification product is used as a template, and 5 'terminal primer sequence and 3' terminal primer sequence contained in the DNA are used.
- Nucleic acid amplification such as PCR is performed using a sex DNA polymerase.
- the former polynucleotide preferably further has the RNA polymerase recognition sequence on the 5 'end side and the 5' end side primer sequence on the 3 'end side. Then, using the obtained amplification product as a template, using the RNA polymerase recognition sequence contained in the amplification product, and performing the transcription reaction in vitro using the DNA-dependent RNA polymerase, the complex Nucleic acid amplification of RNA pools involved in formation can be performed.
- the DNA of the antisense strand has the RNA polymerase recognition sequence on the 3 ′ end side, so the DNA-dependent RNA polymerase binds to this region, and the antisense strand is used as a template.
- the RNA can be synthesized.
- the RNA-dependent DNA polymerase used in the reverse transcription reaction is not particularly limited, and for example, avian myeloblastosis virus-derived reverse transcriptase (AMV Reverse Transscriptase) can be used.
- AMV Reverse Transscriptase avian myeloblastosis virus-derived reverse transcriptase
- the nucleic acid amplification method is not particularly limited, and for example, a polymerase chain reaction (PCR) method or various isothermal amplification methods can be employed. Also, the conditions are not particularly limited.
- PCR polymerase chain reaction
- nucleic acid pool that has formed a complex with the target substance is recovered, and in the same manner as described above, the formation of the complex using the target substance, the recovery of the complex, and the nucleic acid pool involved in the complex formation
- the nucleic acid aptamer having the binding property to the target substance can be finally obtained by repeatedly performing the separation of the above and the amplification of the separated nucleic acid pool.
- the target substance is not particularly limited, and examples thereof include proteins such as enzymes, antibodies, and structural proteins, nucleic acids, lipids, and organic polymers.
- FwTm limit 75.0 (Tm value including t7 sequence of Fw primer is about 75)
- RvTm limit 65.0 (Rm primer Tm value is around 65)
- Ct concentration 1.0 (Nucleic acid concentration during PCR is 1.0 ⁇ mol / L ( ⁇ M))
- Na + concentration 50.0 (Na + concentration during PCR is 50.0 mmol / L (mM))
- GC% 0.6 (GC% of Fw and Rv primers excluding t7 sequence is about 60% each)
- GC% range 0.1 (The above GC% fluctuation width is allowed to ⁇ 10%.
- (Generate random pool) 10000 sets of candidate primer sequences generated as described above are prepared for each of a set that is likely to form a secondary structure and a set that is difficult to form, and a random sequence of 40 bases of 40 for each primer set. was added to generate a random pool.
- a primer set in which GGGG was added to the Fw-side end of the generated primer set and CCCC was added to the tip of the Rv side was separately generated, and a random sequence was added in the same manner as described above to generate a random pool.
- RNAfold of ViennaPackage for example, literature Hofacker, IL, Nucl. Acids Res. 2003 31: 3429-3431, http://www.tbi.univie.ac.at/RNA/
- RNAfold execution the -noLP option was added, and a stem formed by only one base pair was not allowed.
- the primer candidate sequence among the obtained secondary structures was replaced with a loop.
- str represents a random pool of primer sets that are likely to form secondary structures
- nstr represents a random pool of primer sets that are difficult to form secondary structures
- str_GC represents a secondary structure.
- a random pool in which GGGG + CCCC is added to an easy primer set is shown
- nstr_GC is a random pool in which GGGG + CCCC is added to a primer set that is difficult to form a secondary structure.
- FIG. 6 shows the distribution of the number of vertices of the secondary structure with only a random arrangement.
- the horizontal axis is the number of vertices
- the vertical axis is the number of random arrays.
- FIG. 7 shows the distribution of the number of vertices of the secondary structure of the entire nucleic acid sequence.
- the horizontal axis is the number of vertices
- the vertical axis is the number of random arrays.
- each graph showed the same distribution, and there was no difference in diversity depending on the primer set.
- Fig. 8 shows the class distribution of the secondary structure with only random sequences.
- the horizontal axis represents class
- the vertical axis represents the number of random arrays.
- the numbers of the classes correspond to the numbers of the classes shown in FIG.
- the random pool using a primer set that easily forms a secondary structure concentrates secondary structures around classes 3 to 5, whereas secondary pools concentrate.
- secondary structures appeared in classes 6 and 7 as well as in classes 3 to 5.
- FIG. 9 shows the class distribution of the secondary structure of the entire nucleic acid sequence.
- the horizontal axis represents the class
- the vertical axis represents the number of random arrays. Note that the numbers of the classes correspond to the numbers of the classes shown in FIG. As shown in the figure, there was no difference between the primer sets, but when GGGG and CCCC were added, the probability that a linear secondary structure appeared was high.
- the present invention it is possible to efficiently design primers used in the SELEX method, and it can be expected that the aptamer acquisition efficiency will be improved. Therefore, the present invention can be applied to a wide range of fields such as the field of drugs and sensors using aptamers.
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Abstract
Description
プライマー候補配列を生成するプライマー候補配列生成工程と、
予め設定した基準に基づき、前記プライマー候補配列を評価してプライマー候補配列を選択するプライマー候補配列選択工程と、
前記選択されたプライマー候補配列に基づき、ランダム配列およびプライマー候補配列を含む核酸配列を複数含むランダムプールを生成するランダムプール生成工程と、
前記ランダムプールの各核酸配列の構造を予測し、予め設定された基準に基づき、前記予測構造を評価してランダムプールを選択するランダムプール選択工程と、
前記選択されたランダムプールに使用されたプライマー候補配列を、プライマー配列として採用することを決定するプライマー配列決定工程とを含み、前記各工程を、コンピュータを用いて実施してSELEX法用のプライマーを設計することを特徴とする。
プライマー設計工程と、
前記設計工程で設計されたプライマーの配列に基づきプライマーを合成する合成工程とを有し、
前記プライマー設計工程が、前記本発明のプライマー設計方法により実施されることを特徴とする。
データ処理装置、記憶装置、入力装置および出力装置を備え、
前記データ処理装置が、
プライマー候補配列を生成するプライマー候補配列生成手段と、
予め設定した基準に基づき、前記プライマー候補配列を評価してプライマー候補配列を選択するプライマー候補配列選択手段と、
前記選択されたプライマー候補配列に基づき、ランダム配列およびプライマー候補配列を含む核酸配列を複数含むランダムプールを生成するランダムプール生成手段と、
前記ランダムプールの各核酸配列の構造を予測し、予め設定された基準に基づき、前記予測構造を評価してランダムプールを選択するランダムプール選択手段と、
前記選択されたランダムプールに使用されたプライマー候補配列を、プライマー配列として採用することを決定するプライマー配列決定手段とを含むことを特徴とする。
図1に、本発明のプライマー設計装置の一例の構成を示す。図示のように、本例の装置は、入力装置11、記憶装置12、出力装置13およびデータ処理装置14を主要構成要素とする。入力装置11は、データ処理装置14に情報を入力するためのものであり、例えば、キーボード、キーシート、マウス、USB等の端子、CDやCD-R等のドライブ等があげられる。記憶装置12は、プライマー設計条件に関する情報、ランダムプールに関する情報、設計されたプライマー配列に関する情報等の各種情報および各種プログラムを記憶するためのものであり、例えば、フラッシュメモリ、RAMおよびROM等を適宜組み合わせて構成される。出力装置13は、設計されたプライマー配列情報およびその他の情報を出力するためのものであり、例えば、ディスプレーおよびプリンターがあげられる。データ処理装置14は、プライマー設計のための一連の処理を行うものであり、例えば、中央演算処理装置(CPU)があげられる。本例のデータ処理装置14は、プライマー候補配列生成手段141、プライマー候補配列選択手段142、ランダムプール生成手段143、ランダムプール選択手段144およびプライマー配列決定手段145を備える。本発明において、プライマー設計装置は、一台のコンピュータで構成してもよいし、複数のコンピュータにより構成してもよい。
まず、プライマー候補配列生成手段により、プライマー候補配列を生成する。プライマー配列は、例えば、A,G,C,T,Uの塩基を組み合わせたものである。配列がDNAの場合は、Tを使用し、RNAの場合は、Uを使用する。プライマー候補配列は、3’末端側および5’末端側の二種類を生成する必要がある。プライマー候補配列の長さ(塩基数)は、特に制限されないが、10~100塩基数であり、好ましくは、15~50塩基数であり、より好ましくは、20~30塩基数である。プライマー候補配列は、例えば、ランダムに配列を生成してもよいし、既知のプライマー配列のデータベースを参照して生成してもよい。また、入力装置11からプライマー候補配列を入力してもよい。
つぎに、生成されたプライマー候補配列を、予め設定された基準に従い評価し、前記基準を満たすプライマー候補配列を選択する。この場合、前記基準を満たすプライマー候補配列でない場合(No)は、再度、プライマー候補配列を生成し、前記基準を満たすプライマー候補配列の場合(Yes)は、次のステップに進む。前記基準は、例えば、プライマーの二次構造形成能、Tm値、ナトリウム濃度、GC含量、GC含量のぶれ幅、核酸濃度、総GCにおけるGおよびC間の偏り、総ATにおけるAおよびT間の偏り等があげられる。前記プライマーの二次構造形成能は、例えば、Max free energy(プライマーの構造として許容する自由エネルギーの閾値)があげられる。プライマー候補配列の評価は、例えば、遺伝子増幅のプライマーを設計するためのソフトウエアを使用することができる。このようなソフトウエアは、市販品を用いてもよいし、インターネットで公開されているサイトにあるものを使用してもよい。また、これらの遺伝子増幅のプライマー設計ソフトウエアは、プライマー配列を生成することもできるものも多く、このようなソフトウエアを使用した場合は、プライマー候補配列の生成(S1)および選択(S2)を同時に実施することも可能である。
前記ランダムプールは、ランダム配列およびプライマー候補配列を含む核酸配列を、複数含む核酸配列混合物(ライブラリー)である。前記ランダム配列は、アプタマー候補となる配列であり、例えば、A,G,C,およびUまたはTの塩基をランダムに連結した配列である。前記ランダム配列の長さ(塩基数)は、例えば、20~120塩基数であり、好ましくは、30~80塩基数であり、より好ましくは、30~40塩基数である。前記ランダムプールに含まれる前記核酸配列の種類は、例えば、420~4120種類(約1012~1072種類)であり、好ましくは、430~460種類(約1018~1036種類)
である。
次に、生成したランダムプールについて、各核酸配列の構造を予測し、予め設定された基準に基づき、前記予測構造を評価してランダムプールを選択する。前記構造の予測には、例えば、DNAおよびRNA等の構造を予測する既知のソフトウエアを使用することができる。前記基準としては、二次構造の多様性を使用することができる。二次構造の多様性としては、グラフ構造によるクラス分類を使用することができる(GEVERTZ J. et.al. RNA 2005;11;853-863)。二次構造の前記グラフ構造によるクラス分類を、図5に示す。同図に示すように、前記クラス分類は、ステムを辺(-)として表し、ループを点(・)として表すものである。同図では、点の数が7以下のグラフ構造を示すものである。そして、前記基準を満たさない場合(No)は、再度、ランダムプールの生成ステップに戻り、前記基準を満たす場合(Yes)は、次のステップに進む。
次に、前記選択されたランダムプールに使用されたプライマー候補配列を、プライマー配列に採用し、前記配列をプライマー配列として決定する。決定されたプライマー配列情報は、ディスプレー等の出力装置から出力される(S6)。その他、決定されたプライマー配列情報は、記憶装置に記憶されてもよい。
次に、通信インターフェース(通信装置)を備えたプライマー設計装置の例について説明する。本例の装置の構成を図3に示す。図3において、図1と同一部分には同一符号を付している。図示のように、本例の装置は、通信インターフェースを備えてインターネット等の通信回線網に接続可能な他は、前記実施形態1の装置と主要構成は同様である。図4に示すように、本例の装置1は、インターネット等の通信回線網2を介して、装置外に設置されたサーバー3に接続可能である。したがって、例えば、プライマー候補配列の生成、プライマー候補配列の評価、ランダムプールの生成、およびランダムプールの各核酸配列の構造予測等の各種の工程を、装置外のサーバーにあるサイトのプログラムにより、実施可能である。したがって、本例の装置は、プライマー候補配列生成手段、プライマー候補配列選択手段、ランダムプール生成手段、ランダムプール選択手段およびプライマー配列決定手段の少なくとも一つを、装置外のサーバーのサイトにあるプログラムで実行することが可能であり、その場合は、本例の装置のデータ処理装置は、装置外のサーバーのプログラムで実行する各前記手段については含まなくてもよい。
本実施形態は、前記本発明のプライマー設計方法で設計されたプライマー配列に基づき、プライマーを製造する方法である。プライマーの設計方法は、前述のとおりである。また、プライマーの合成方法は、既知の方法を使用できる。前記合成方法としては、具体的には、例えば、DNA合成装置やRNA合成装置により、dNTP等を材料として、末端塩基から化学合成する方法等があげられる。前記合成装置としては、市販品を使用してもよい。
本実施形態は、前記本発明のプライマー製造方法で製造したプライマーを用い、SELEX法によりアプタマーを製造する方法の一例である。プライマーの製造方法は、前述のとおりである。下記において、SELEX法について、説明する。
本発明において、前記アプタマーとは、特定の標的物質に特異的に結合可能な核酸分子であり、例えば、一本鎖RNAおよび一本鎖DNA等の一本鎖核酸や、二本鎖RNAおよび二本鎖DNA等の二本鎖核酸があげられる。前記核酸アプタマーが、後者の二本鎖核酸の場合、例えば、使用に先立って、変性等により一本鎖にすることが好ましい。前記核酸アプタマーは、例えば、自己アニーリングによる二次構造を有してもよく、前記二次構造としては、例えば、ステムループ構造があげられる。
プライマー候補配列の生成および選択は、例えば、Oligo Calculator(http://www.genosys.jp/whatsnew/active/active_manual.html、シグマジェノシス社製)等を使用して行った。主要な評価基準として、二次構造の形成能を採用した。二次構造を形成し易いプライマーセットと形成し難いプライマーセットの差異として、Max free energy(プライマーの構造として許容するエネルギーの閾値)のパラメーターを用い、形成し易いものは、-10kcal/molに設定し、形成し難いものは-1kcal/molに設定した。その他の共通のパラメーターは、前記ソフトウエアのデフォルト値に従って以下の通りにした。
FwTm limit=75.0(Fwプライマーのt7配列込みのTm値が75程度)
RvTm limit=65.0(RvプライマーのTm値が65程度)
Ct concentration=1.0(PCR時の核酸濃度が1.0μmol/L(μM))
Na+ concentration=50.0(PCR時のNa+濃度が50.0mmol/L(mM))
GC%=0.6(t7配列を除くFwとRvのプライマーのGC%がそれぞれ60%程度)
GC% range=0.1(上記GC%のぶれ幅を±10%まで許容。50%~70%)
Minimum Na%=0.4(t7配列を除くFwとRvに含まれる総GCにおけるGおよびC間の偏り、ならびに、総ATにおけるAおよびT間の偏りを、それぞれ、G:C=4:6~6:4、A:T=4:6~6:4まで許容)
前述のようにして生成したプライマー候補配列を、二次構造を形成し易いセットと形成し難いセットのそれぞれに10000セットずつ用意し、それぞれのプライマーセットに対して100本の塩基数40のランダム配列を付加し、ランダムプールを生成した。また、生成したプライマーセットのFw側の末端にGGGGを、Rv側の先端にCCCCを付加したプライマーセットを別途生成し、前述と同様にしてランダム配列を付加し、ランダムプールを生成した。
二次構造予測には、ViennaPackageのRNAfold(例えば、文献Hofacker, I. L., Nucl. Acids Res. 2003 31:3429-3431、http://www.tbi.univie.ac.at/RNA/)を用いた。RNAfoldの実行には-noLPオプションを付加し、一つの塩基対のみで形成されるステムを許容しないこととした。また、ランダム配列のみの二次構造を予測するために、RNAfoldの実行により二次構造を得た後、かつ後述の多様性の評価の前に、得られた二次構造のうち、プライマー候補配列で形成されるステムについてはループに置換した。
二次構造の多様性の評価は、例えば、論文"GEVERTZ J. et.al. RNA 2005;11:853-863"に基づいて行った。この論文では、二次構造予測結果を入力として、二次構造の構造クラスを判定することが提案されている。二次構造の構造クラスについては、前記論文で採用されているグラフ構造によるクラス分類を採用した。
図6および図7のグラフに、ランダムプールの二次構造の頂点の数の分布を示す。前記両図において、strは、二次構造を形成しやすいプライマーセットのランダムプールを示し、nstrは、二次構造を形成し難いプライマーセットのランダムプールを示し、str_GCは、二次構造を形成しやすいプライマーセットにGGGG+CCCCを付加したランダムプールを示し、nstr_GCは、二次構造を形成し難いプライマーセットにGGGG+CCCCを付加したランダムプールを示し、これらは、下記の図8および図9においても同じである。
前述のように、GGGGおよびCCCCを付加した場合に、直線型の二次構造が出現する確率が高くなった。このため、取得されるランダムプールの核酸配列の小型化を目的とする場合は、GGGGおよびCCCCを付加したプライマー配列を採用すればよく、逆に、取得されるランダムプールの核酸配列の小型化を目的とせず、二次構造の多様化を目的とする場合は、GGGGおよびCCCCを付加しないプライマー配列を採用すればよい。
前述のように、str_GC、nstr_GC、strおよびnstrの順序で二次構造の多様性が低くなっているので、二次構造の多様性を目的とする場合は、二次構造を形成し難く、かつGGGG+CCCCを付加しないプライマー配列を採用すればよい。これとは逆に、アプタマー取得後の後加工を容易にするためには、二次構造の多様性は低い方がよいので、この目的のためには、二次構造を形成し易く、かつGGGG+CCCCが付加されたプライマー配列を採用すればよい。
2 通信回線網
3 サーバー
11 入力装置
12 記憶装置
13 出力装置
14 データ処理装置
15 通信インターフェース
141 プライマー候補配列生成手段
142 プライマー候補配列選択手段
143 ランダムプール生成手段
144 ランダムプール選択手段
145 プライマー配列決定手段
Claims (14)
- プライマー候補配列を生成するプライマー候補配列生成工程と、
予め設定した基準に基づき、前記プライマー候補配列を評価してプライマー候補配列を選択するプライマー候補配列選択工程と、
前記選択されたプライマー候補配列に基づき、ランダム配列およびプライマー候補配列を含む核酸配列を複数含むランダムプールを生成するランダムプール生成工程と、
前記ランダムプールの各核酸配列の構造を予測し、予め設定された基準に基づき、前記予測構造を評価してランダムプールを選択するランダムプール選択工程と、
前記選択されたランダムプールに使用されたプライマー候補配列を、プライマー配列として採用することを決定するプライマー配列決定工程とを含み、前記各工程を、コンピュータを用いて実施してSELEX法用のプライマーを設計することを特徴とするプライマーの設計方法。 - 前記プライマー候補配列選択工程の前記基準が、プライマーの二次構造形成能に関する基準であることを特徴とする請求の範囲1記載のプライマー設計方法。
- 前記ランダムプール選択工程の前記基準が、前記核酸配列の前記ランダム配列が取り得る二次構造の多様性に関する基準であることを特徴とする請求の範囲1記載のプライマー設計方法。
- 前記核酸配列が、さらに、ランダム配列に対する固定配列を含むことを特徴とする請求の範囲1記載のプライマー設計方法。
- プライマー設計工程と、
前記設計工程で設計されたプライマーの配列に基づきプライマーを合成する合成工程とを有し、
前記プライマー設計工程が、請求の範囲1記載のプライマー設計方法により実施されることを特徴とするSELEX法用のプライマーの製造方法。 - 請求の範囲5記載の製造方法により製造されたプライマーを用いたSELEX法によりアプタマーを製造することを特徴とするアプタマーの製造方法。
- データ処理装置、記憶装置、入力装置および出力装置を備え、
前記データ処理装置が、
プライマー候補配列を生成するプライマー候補配列生成手段と、
予め設定した基準に基づき、前記プライマー候補配列を評価してプライマー候補配列を選択するプライマー候補配列選択手段と、
前記選択されたプライマー候補配列に基づき、ランダム配列およびプライマー候補配列を含む核酸配列を複数含むランダムプールを生成するランダムプール生成手段と、
前記ランダムプールの各核酸配列の構造を予測し、予め設定された基準に基づき、前記予測構造を評価してランダムプールを選択するランダムプール選択手段と、
前記選択されたランダムプールに使用されたプライマー候補配列を、プライマー配列として採用することを決定するプライマー配列決定手段とを含むことを特徴とするSELEX法用のプライマー設計装置。 - 前記プライマー候補配列選択工程の前記基準が、プライマーの二次構造形成能に関する基準であることを特徴とする請求の範囲7記載のプライマー設計装置。
- 前記ランダムプール選択工程の前記基準が、前記核酸配列の前記ランダム配列が取り得る二次構造の多様性に関する基準であることを特徴とする請求の範囲7記載のプライマー設計装置。
- 前記核酸配列が、さらに、ランダム配列に対する固定配列を含むことを特徴とする請求の範囲7記載のプライマー設計装置。
- さらに、通信装置を備え、前記通信装置により、装置外の通信回線網を介してサーバーに接続可能であり、前記サーバー内のプログラムにより、前記プライマー候補配列生成手段、前記プライマー候補配列選択手段、前記ランダムプール生成手段、前記ランダムプール選択手段および前記プライマー配列決定手段からなる群の少なくとも一つの手段を実行可能であることを特徴とする請求の範囲7記載のプライマー設計装置。
- 前記データ処理装置が、前記サーバー内のプログラムにより実行される手段を含まないことを特徴とする請求の範囲11記載のプライマー設計装置。
- 請求の範囲1記載のプライマー設計方法をコンピュータ上で実行可能なことを特徴とするSELEX法用のプライマー設計用コンピュータプログラム。
- 請求の範囲13記載のSELEX法用のプライマー設計用コンピュータプログラムが格納されていることを特徴とする記録媒体。
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US13/259,784 US20120083418A1 (en) | 2009-04-01 | 2010-03-26 | Method for designing primers for selex method, method for producing the primers, method for producing aptamers, system for designing the primers, and computer program and recording medium for designing the primers |
AU2010231741A AU2010231741B2 (en) | 2009-04-01 | 2010-03-26 | Method for designing primer for selex method, method for producing primer, method for producing aptamer, device for designing primer, and computer program and recording medium for designing primer |
EP10758561.4A EP2415867A4 (en) | 2009-04-01 | 2010-03-26 | METHOD FOR DESIGNING A PRIMER FOR A SELEX PROCESS, METHOD FOR PRODUCING THE PRIMER, METHOD FOR PRODUCING AN ADAPTER, DEVICE FOR DESIGNING THE PRIMER AND COMPUTER PROGRAM, AND RECORDING MEDIUM FOR DESIGNING THE PRIMER |
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JP2009089607A JP5561755B2 (ja) | 2009-04-01 | 2009-04-01 | Selex法用のプライマーの設計方法、プライマーの製造方法、アプタマーの製造方法、プライマーの設計装置、プライマー設計用コンピュータプログラムおよび記録媒体 |
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WO2013134341A1 (en) | 2012-03-07 | 2013-09-12 | Dow Agrosciences Llc | Primer designing pipeline for targeted sequencing |
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US11066704B2 (en) | 2016-06-10 | 2021-07-20 | Seegene, Inc. | Methods for preparing tagging oligonucleotides |
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- 2010-03-26 EP EP10758561.4A patent/EP2415867A4/en not_active Withdrawn
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WO2013134341A1 (en) | 2012-03-07 | 2013-09-12 | Dow Agrosciences Llc | Primer designing pipeline for targeted sequencing |
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US20120083418A1 (en) | 2012-04-05 |
JP2010239873A (ja) | 2010-10-28 |
AU2010231741A1 (en) | 2011-11-17 |
JP5561755B2 (ja) | 2014-07-30 |
AU2010231741B2 (en) | 2014-04-10 |
EP2415867A1 (en) | 2012-02-08 |
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