JPH1052285A - Preparation of antisense nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain an antisense nucleic acid for medicines, etc., by numerically converting the extent of possibility of the complementary double chain formation between a region of base sequence of mRNA and a region other than the base sequence in each region of base sequence of mRNA and preparing a complementary nucleic acid in a region which is low in numerical value. SOLUTION: The extent of possibility forming a substantially complementary double between a region of base sequence of mRNA and other region different from the above region in each region of a base sequence of mRNA is converted to a numerical value based on a fact that the extent of possibility forming the double chain is maximum when a distance between substantially complementary base sequences holds 3-10 bases or the extent of the possibility is converted into a numerical value based on the fact that a binding energy in forming the double chain becomes a numerical value calculated based on the closest base pair model or the extent of the possibility is numerically converted based on above both facts and a nucleic acid having a base sequence substantially complementary to a region low in the numerical value is prepared as an antisense nucleic acid to efficiently provide the objective antisense nucleic acid used for antisense nucleic acid method useful for preparation, etc., of a medicine or a gene recombinant plant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for preparing an antisense nucleic acid.

[0002]

2. Description of the Related Art The antisense nucleic acid method uses a nucleic acid having a base sequence complementary to a target gene (antisense nucleic acid) or a nucleic acid having a base sequence substantially complementary to a target gene. This is a method of inhibiting the expression of a protein by hybridizing to a target gene, and is used for preparation of medicines, transgenic plants, and the like.

[0003] In the antisense nucleic acid method, it is first necessary to prepare a nucleic acid having a base sequence that has an effect of hybridizing to a target gene and inhibiting the expression of the gene (antisense nucleic acid effect). As a method for preparing a nucleic acid having such a base sequence, a site having an antisense nucleic acid effect is experimentally found, a method of synthesizing a nucleic acid at the site, and a method of synthesizing the antisense nucleic acid effect without performing an experiment. There is a method of predicting and synthesizing a certain base sequence. However, in the former, a partial sequence complementary to the gene of interest must be prepared, and whether or not the sequence has an antisense nucleic acid effect must be examined by actually conducting experiments on the gene of interest. Costly and time-consuming.

[0004] On the other hand, the latter is a method for preparing an antisense nucleic acid by predicting a base sequence having an antisense nucleic acid effect by empirically targeting a translation initiation site or a non-translation site upstream thereof, for example. Is proposed (K.
R. Blake et al., Biochemistry 24 6132-6138 1985
Year; E. Uhlmann, A. Peyman, Chemical Reviews 90 5
43-584 1990), whose certainty is low, and antisense nucleic acids selected by these methods do not always effectively inhibit protein expression (for example, RD Ricker,
A. Kaji, FEBS Letters 309, 363-370, 1992).
Further, a method for calculating and predicting an antisense nucleic acid target site based on the secondary structure formation energy of mRNA or its precursor, the hybridization energy of mRNA or its precursor with an antisense nucleic acid, or the difference between these two energies However, those selected by these methods have low certainty and are not always effective (RA Stull et al., Nucleic Acids Resear
ch 20 volume 3501-3508 1992).

Therefore, in the antisense nucleic acid method,
Effective antisense nucleic acids, that is, antisense nucleic acids having a nucleotide sequence that effectively inhibits the expression of the mRNA encoding the protein of interest or its precursor, are predicted with high probability to facilitate the preparation of antisense nucleic acids. Is required.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing an antisense nucleic acid, which can efficiently obtain an antisense nucleic acid without conducting experiments.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies based on the above-mentioned problems, and as a result, have found that a substantially complementary double region between a specific region of mRNA and another different region on the same mRNA. The magnitude of the possibility of forming a strand is quantified for all other regions on the same mRNA, and a nucleic acid having a base sequence substantially complementary to a specific region of the mRNA having a small sum of these values is prepared. As a result, they have found that an antisense nucleic acid can be prepared, and have completed the present invention.

That is, the present invention quantifies the possibility of forming a substantially complementary double strand between each region of the mRNA base sequence and another region different from the region, A method for preparing an antisense nucleic acid, comprising preparing, as an antisense nucleic acid, a nucleic acid having a base sequence that is substantially complementary to a low region.

The above-mentioned possibility is quantified based on the distance between two base sequence regions forming a substantially complementary double strand, and more specifically, as the possibility decreases as the distance increases. You. In addition, the distance is quantified as the maximum when two to three nucleotides, preferably four to six nucleotides, are sandwiched between the two nucleotide sequence regions. Furthermore, based on the binding energy at the time of forming a double chain, it is quantified more specifically that the larger the binding energy, the greater the possibility. As the binding energy, one obtained based on the nearest base pair model or the like is applied. In particular, the possibility is optimally quantified based on the distance between two base sequence regions and the binding energy for forming a substantially complementary double strand. Hereinafter, the present invention will be described in detail.

[0010]

DETAILED DESCRIPTION OF THE INVENTION An mRNA can form a duplex if a particular region is substantially complementary to another different region on the same mRNA. The present invention takes advantage of this property to make a duplex substantially complementary between a specific region on an mRNA and another different region on the same mRNA (hereinafter also referred to as “substantially complementary duplex”). ) Is quantified for all relevant regions other than the specific region on the same mRNA, and the specific region having a high total value is likely to form a substantially complementary duplex. The specific region having a low numerical value is assumed to be a region in which a substantially complementary double strand is unlikely to be formed (a single-stranded region). An antisense nucleic acid is prepared by preparing a nucleic acid having a sequence.

In the present invention, the possibility of forming a substantially complementary double strand (hereinafter, also referred to as “substantially complementary double strand forming ability”) is quantified. mR
It means that the sequence of a specific region on NA pairs with each other (forms a base pair) with the sequence of another region on the same mRNA. For example, the relationship between adenine (A) and uracil (U), cytosine It refers to the relationship between (C) and guanine (G).
In the present invention, “complementary” means that a specific region and another region on the same mRNA are paired in a direction facing each other, that is, for example, the sequence of a certain region in the 5 ′ → 3 ′ direction is the other region. 3 ′ → 5 ′ sequence.

Further, in the present invention, “substantially” means
As long as the bases are paired with each other, the base pair of G and C or A
And U are not limited to base pairs. Therefore,
For example, in addition to base pairs of G and C and base pairs of A and U,
G and U base pairs are also acceptable. Furthermore, a sufficiently long complementary duplex (for example, 10 bases or more) means that a small number of mismatched base pairs can be tolerated.

Hereinafter, specific means for quantifying the ability to form a substantially complementary double strand will be described. First, for all or a part of the base sequence of mRNA (including the precursor), starting from a base at a specific site, a specific direction (a direction predetermined in either 5 ′ → 3 ′ or 3 ′ → 5 ′) 1 to continue
A region consisting of bases or more, preferably 2 to 4 bases or more,
Whether the RNA is substantially complementary to a region sandwiching three or more bases (preferably four or more bases) (ie, separated by four or more bases (preferably five or more bases))
Examine the relevant area above. If it is substantially complementary, further examine the next adjacent bases to see if they are substantially complementary, and if they are substantially complementary,
Further, the next adjacent bases are examined. Hereinafter, a substantially complementary region as long as possible is found as described above, and a region including a specific site is defined as the specific region here. Then, the possibility that the specific region and the corresponding substantially complementary region form a substantially complementary double strand is quantified. At that time, the numerical value is large when the regions substantially complementary to each other are close as long as they are separated by a certain distance or more on the RNA,
A numerical value is set by setting a calculation program so that it becomes smaller when the distance is far. The calculation program is set so that the larger the energy of forming a double strand between substantially complementary regions is, the larger the energy is, and the smaller the energy is, the smaller the energy is. The value thus set and quantified is defined as the value of each base constituting the specific region (and each base of the region forming a substantially complementary double strand with the specific region).

Next, in the region starting from the base at the specific site, a next substantially complementary region different from the above is similarly found under the same conditions as above, and the substantial region between the complementary region and the specific region is determined. The ability to form a complementary double strand is similarly quantified.
Then, the value is added to the value for each base determined above. In this way, for a region starting from a base at a certain specific site, it is checked whether or not all of the corresponding regions on the RNA are substantially complementary, and if they are substantially complementary, substantially complementary as described above The ability to form complementary duplexes is similarly quantified. Then, if there is a numerical value already obtained for the corresponding base, it is added to the numerical value. If there is no such numerical value (that is, if such a numerical value is calculated for the corresponding base for the first time), the numerical value is added. A new numerical value of the corresponding base is used as it is (when a numerical value is also given to each base in a region that forms a substantially complementary double strand with the specific region, similarly, add or set a new numerical value) .

The above operation is performed on the region starting from the bases at all possible sites, and the sum of the numerical values of the ability of each base to form a substantially complementary double strand is determined. At this time, for a certain substantially complementary base pair region, the substantially complementary base pair region included in the inside is not counted twice again. Therefore, for example, as a specific region, a region of 2 or more bases in the 3 ′ direction starting from the 10th (the 5 ′ end is the first) and a region of 2 or more bases in the 5 ′ direction starting from the 30th position When comparing the substantial complementarity of the 3rd direction starting from the 9th position, if the 4 bases in the 5 ′ direction starting from the 31st position are substantially complementary, the 10th position starts. Possibility of a substantially complementary duplex formed by 3 bases in the 3 'direction and 3 bases in the 5' direction starting from position 30, and 2 bases in the 3 'direction starting from position 11 and from position 29 Numerical values for the possibility of a substantially complementary duplex formed by two bases in the starting 5 'direction are not included.

When the sequence of a certain region is substantially complementary to the sequence of another region, the possibility of forming a double strand between the regions is quantified so as to be a significant value. When the sequence of the region and the other region is not substantially complementary, the possibility that the regions form a double strand is calculated as zero (0). In order to calculate such a possibility, a computer program can be assembled and used.

[0017] The type of the mRNA targeted by the antisense nucleic acid prepared by the method of the present invention is not particularly limited. Examples thereof include mRNA of vascular endothelial cell growth factor derived from animals such as human and rat, mRNA of heme oxygenase type I or II derived from animals such as human and rat, and mRNA of luciferase derived from firefly. FIG. 1 shows an outline (schematic diagram) of a method for quantifying the possibility of forming a duplex between a partial sequence of an mRNA and a partial sequence of another region of the mRNA that does not overlap with the sequence. It is shown in FIG.

The length of the fragment for judging whether or not the sequence length of the specific region on the mRNA is complementary is preferably 2 to 4 bases or more. In FIG. 1, the total length of mRNA is 50
The description will be made assuming that the length of the base and the fragment of the specific region are two or more bases. In Figure 1, the most 5 mRNA '2 base end side (1-2 th base; and a ₁₎ the most 3' 2 bases from the distal side (50 to 49 th bases; and b ₁₎ It is determined whether or not are complementary to each other (A ₁ ). If a ₁ and b ₁ and are not complementary, then the a _1, b ₁ from the 5 'side one base shifted by 2 bases (49 to 48 th bases; and b ₂₎ and the complementary It is determined whether or not (FIG. 1, A ₂ ). This is sequentially repeated, whether there is a sequence complementary to the a ₁ to search for all regions of mRNA. However, the minimum required distance (here, the sandwich the three base between the regions) retrieves except (A ₁ to A ₄₄ in FIG. 1). As a result, the a _1, 2 bases shifted 4 bases from the 3 'end of the mRNA; If (the 46-45 th base and b ₅₎ and is complementary (Fig. 1, A _5),
The next bases, ie, the substantial complementarity between the third base and the 44th base are compared, and if they are substantially complementary, the next bases (ie, the fourth base and the 43rd base) are compared.
Compare substantial complementarity th) (in FIG. 1, A ₅ "→"
And "←"). Hereinafter, the procedure is performed as long as the substantially complementary base sequence continues in this manner (however, the minimum number of bases required to form a loop portion between the substantially complementary base sequences is ensured). The magnitude of the possibility that the complementary base sequences form a double strand is quantified, and the value is expressed as a
It is given to each base a base forming a substantially complementary nucleotide sequence starting from the first (and the base of a ₁ and b ₅ to form a substantially complementary double-strand) contained in _1. Further, as described below, the binding energy can be used as a numerical value reflecting the possibility that the complementary base sequences form a double strand.

Here, as for each base, the greater the ability to form a substantially complementary double strand between a specific site and another site substantially complementary to the specific site, the more easily a stable substantially complementary chain can be formed. Can be evaluated. The ability to form a substantially complementary duplex depends on the binding energy (thermodynamic free energy) experimentally determined when forming a substantially complementary duplex, and the binding energy calculated with the nearest base pair model. The ability to form a substantially complementary double strand is also quantified based on these.

For example, quantification of the ability to form a substantially complementary duplex based on the binding energy is a method called “nearest base pair model” (Naoki Sugimoto, Biophysics, Vol. 33, p1-7, (199)
3)) can be obtained as follows. In addition,
The "nearest base pair model" is a model constructed based on the fact that the next base pair that has already produced the most influence on the base pair generation of nucleic acids is the adjacent base pair that has already been generated. A dimer composed of a certain base and its next base,
The binding energy ΔG generated between the substantially complementary sequence and the sequence is shown in Table 1.

[0021]

[Table 1]

The ability to form a substantially complementary duplex based on the binding energy obtained by the sequences in Table 1 and based on the distance between the two strands is represented by the following formula I: (L + 1) / r) ^F · exp (| ΔG | / RT) (I) (wherein | ΔG | is the absolute value of the binding energy, R is the gas constant, T is the absolute temperature, and L is the minimum of the loop portion. Number of bases,
r represents the distance between one strand and the other strand of the duplex (the number of bases between two strands (regions) +1), and F represents an evaluation index for the distance r. ) Can be calculated.

L is 3-10, preferably 4-6. r
Is an integer of 1 or more. F is 0 or a positive number, and can give a value such as 6, 1, 1/3, or 0.1. For example, the specific site is the first, and the first to fourth
Assuming that there is a “GUAU” sequence that is substantially complementary to the 43rd to 46th nucleotide sequence with respect to the 43rd base sequence “AUGC”, the binding energy ΔG when these form a double strand is shown in Table 1. Can be calculated as follows: ΔG = Δ (AU / UA) + Δ (UG / AU) + Δ (GC / UG) = (− 0.8) + (− 0.5) + (− 1.7) = − 3.0

Also, r = 43-4 = 39. Where L =
Assuming that 4, F = 6 and the temperature is 37 ° C. (T = 310.15 K), the ability to form a substantially complementary double strand in this case can be quantified as follows. Substantially complementary duplex forming ability (1-4 and 46-43) = (5/39) ⁶ · exp (3.0 kcal / RT) = (5/39) ⁶ · exp (3000 / 1.9872 × 310.15) = 5.77 × 10 ^{-4 The} obtained value (5.77 × 10 ^-4 ) is calculated from the first to fourth,
And the 43rd to 46th bases.

Hereinafter, as described above, a site where a specific site is complementary (or substantially complementary) to a base sequence of two or more bases starting from the first position is searched for, and a substantially complementary site is searched for those sites. Calculate heavy chain forming ability. For example, assuming that the 39th to 40th arrays are “AU” and the 15th to 17th arrays are “CAU” in addition to the arrays used in the above calculation example, Bases “AU” and 39-
Since the 40th base “AU” is complementary, and the 1st to 3rd bases “AUG” and the 15th to 17th “CAU” are complementary, a substantially complementary double strand in each case. The forming ability was quantified, and the former (the 39th to 40th duplex) was converted to the first and second bases (and the 39th and 40th bases).
The respective values are added to the 1st to 3rd bases (and the 15th to 17th bases) for the latter (duplex with the 15th to 17th bases).

This is sequentially repeated to obtain a specific area (a ₁ )
And the most 3′-side base and other regions (b ₁ , b
₂ ,. . . . )), The probability is calculated until the distance (r) to the 5'-most base reaches a predetermined number of bases (FIG. 1, _A44 ). The minimum value of r can be set arbitrarily,
For example, it is 4 to 11, preferably 5 to 7. In the above example, since r is set to 4, b ₁ , b
₂ ,. . . . Whether sequences is complementary to the sequence of a _1, will be done until the 7-6-th sequence (Figure 1, A _44).

Thus, the entire region of the mRNA (excluding the region where the distance between the region starting from the specific (first in this case) base and the corresponding complementary region is less than r) is 2 A search is made for a region substantially continuous with bases or more that is substantially complementary, and for those regions, the ability to form a substantially complementary double strand with a region starting from a specific base is quantified, and the numerical value is converted to a specific base (and substantially complementary thereto). (A region forming a double-stranded chain) is added to each base or a new numerical value of each base.

Next, the most 5 of mRNA; 2 bases 'end one base sequence shifted from 2 bases (the first 2-3 th base and a _2), most 3 mRNA' from the distal side ( No.
50-49 th base; and b ₁₎ and it is determined whether substantially complementary (Figure _1, B 1). B _{1 in the same} manner as above
Is shifted by one base at a time, and b ₁ , b ₂ , b
₃ ,. . . It is determined whether it is substantially complementary to or not. When there is a substantially complementary portion, the substantial complementarity between the next bases is determined as described above. For example, in B _2,
The substantial complementarity between the base next to a ₂ (the fourth base from the 5 ′ end) and the 47 th base of b ₂ (the fourth base from the 3 ′ end) is determined. If it is substantially complementary, further fifth next base between (a ₂ and b
(46th of No. ₂ ) to determine the substantial complementarity (FIG. 1, B ₂
Arrow)).

Such an operation is sequentially repeated, and A in FIG.
In the substantially complementary region as in ₅ (FIG. 1 in the example _{B 2, C 1, C 3} ), the magnitude of the potential to form a substantially complementary double-stranded quantified for each case, substantially A numerical value is assigned to each base of the specific region constituting the complementary double strand (and the other region forming a substantially complementary double strand with the specific region), and the process is terminated (FIG. 1, Z).

The calculation program for quantifying the possibility of forming a substantially complementary double strand is a program that can form a substantially complementary double strand for both one strand (region) and the other strand (region). A value obtained by quantifying the magnitude of the sex may be set to be assigned to each base, or a value obtained by quantifying the possibility of forming a substantially complementary double strand in only one strand (region) is assigned. It can be set as follows. The example of FIG. 1 is an outline of a method of assigning a numerical value to the magnitude of the possibility of forming a substantially complementary duplex to both strands (regions) forming a duplex. In the case where only one of the strands (regions) is set to be assigned a value obtained by quantifying the possibility of forming a substantially complementary double strand, for example, a ₁ , a ₂ , a
₃ ,. . . Are assigned numerical values of the possibility of forming a substantially complementary double strand only for the region (conversely, only for the regions b ₁ , b ₂ , b ₃ , ...). It is also possible to assign a numerical value indicating the possibility of forming a heavy chain). That is, a ₁ , a ₂ ,
a ₃ ,. . . When assigning the value only for the _{region, b 1, b 2, b} 3,. . . No value is assigned to the region of. When the value is assigned to only one strand (region) of the substantially complementary double strand in this manner, the second to first base sequence is finally added to the 49th to 50th base sequence which is the specific region. Judge whether or not the base sequence forms a substantially complementary double strand, and if so, quantify the magnitude of the possibility, and use that value as the value of the 49th and 50th bases (Or add to the previous value). In addition, the calculation program for evaluating the magnitude of the possibility of forming a substantially complementary double strand is set so as to numerically reduce the possibility of forming a substantially complementary double strand when the value of r is large. I do. For example, in FIG. 2A, m ₁ and m ₂ , and m ₁
And m ₄ are each substantially complementary,
Since the distance r ₁ between m ₁ and m ₂ is shorter than the distance r ₂ between m ₁ and m _4, the substantially complementary duplex forming ability higher in between m ₁ and m _2, or lower Will be calculated.
Incidentally, when the m ₁ and m ₂ to form a substantially complementary to duplex, single-stranded loop can be formed between the m ₁ and m ₂ (loop 1 in Figure 2 (2) ). In this case, m ₁ and m ₂
Single strand (loop 1 in Fig. 2 (2))
Is more or less than the possibility that a single strand (loop 2 in FIG. 2 (3)) connecting m ₁ and m ₄ is formed.

In FIGS. 3 (1) and 3 (2), m ₁ and m ₃ , and m ₂ and m ₄ are substantially complementary to each other to form a double chain (so-called “stem”; stem 4) is formed, it is assumed that the length of the loop 5 which connects the m ₁ and m _3, and the length of the loop 6 for connecting the m ₂ and m ₄ equals. If the value of the binding energy for forming a duplex is between m ₁ and m ₃ is greater than the value between m ₂ and m ₄ , the larger is the substantially complementary duplex. The calculation program is set so that the forming ability is calculated high. Therefore, in FIG. 3, the possibility that a double strand (stem 3) formed between m ₁ and m ₃ is formed is as follows.
It is more likely that a duplex (stem 4) formed between m ₂ and m ₄ will be formed.

Next, the possibility of forming a substantially complementary double strand quantified above is added up for the bases in each region.
According to the example shown in FIG. 1, the total value P1 possibilities 1st base is complementary to the bases of the other regions, P1 = p ₁
, And the sum P2 possibility the second base is complementary to the base in the other region is P2 = p ₁ + p _2, similarly, the third sequence P3 = p ₂ + p ₃ +
For p ₄ and the fourth sequence, P 4 = p ₃ + p ₄ (FIG. 1). Hereinafter, similarly, the total value P5, P
6,. . . , P50 are calculated.

When the obtained sum is low, it means that the possibility of forming a substantially complementary double strand is low, and since the possibility of forming a single strand is high, the base having such a value is an antisense. Preferred as a target site for a sense nucleic acid. In particular, in a region where the low total value is 6 bases or more, preferably 10 bases or more, particularly preferably 15 bases or more, the possibility of forming a substantially complementary double strand is continuously low, and the antisense nucleic acid Suitable as a target site. In this way, an antisense nucleic acid target site can be predicted and an antisense nucleic acid substantially complementary to the site can be designed.

Based on the above design, natural oligodeoxyribonucleotides, phosphorothioate oligodeoxyribonucleotides or natural R
Oligodeoxyribonucleotides or oligoribonucleotides with modified bases, phosphates or sugar moieties of NA or nucleic acids can be synthesized.

As a synthesis method, a liquid phase synthesis method such as a solid phase synthesis method or a triester method performed by an automatic synthesizer based on the amidite method or the thiophosphite method can be used. In addition, the obtained nucleic acid is a reversed-phase or ion-exchange H
An antisense nucleic acid can be prepared by purification using a PLC or a cartridge. For a program that calculates according to the method described above, for example, BASIC
, FORTRAN, or C language, and languages derived or developed therefrom.

FIG. 4 shows an example of a flowchart of the above calculation program. In FIG. 4, first, parameters L, D, F, and NAS and dimensions (the total number of bases) are set (step 1). NAS is the length of the antisense nucleic acid, and is 10 to 30, preferably 15 to 25.

Next, data (base sequence) is read (step 2). After the data reading is completed, the complementarity is determined. The position of each base in the base sequence starting from the base (referred to as i) at a specific site is defined as I (= i, i + 1, i + 2, etc.), and a base sequence to check whether or not the base is substantially complementary to the specific site The position of each base in J (= j, j
-1, j-2, etc.). First, I = i and J = j
Before comparing the substantial complementarity of the regions beginning with
1 and J = j + 1 (where i-1 ≧ 1, j + 1 ≦ N)
Are compared for substantial complementarity (step 3). This is because when checking the complementarity of the region starting from the I = i-th and J = j-th, it is checked whether or not it is a part of the already counted substantial complementary chain region. I = i-1 and J = j
When +1 is substantially complementary, it is not necessary to check the substantial complementarity of the combination starting from I = i and J = j, since the complementarity has already been checked.

If it is determined in step 3 that there is no need to check for substantial complementarity (ie, I = i-1 and J = j + 1)
Are substantially complementary to each other. In step 3, “Ye
In the case of "s"), I = i-1 is compared with J = j (hereinafter, in the case of "Yes", the same is repeated). On the other hand, I =
If i-1 and J = j + 1 are not substantially complementary,
It is checked whether I = i to i + D-1 and J = j to j-D + 1 are substantially complementary (step 4). If it is not substantially complementary, the process returns to step 3 to check the substantial complementarity for I = i-1 and J = j. If it is not substantially complementary, I = i to i + D-1 and J = j The substantial complementarity of j-1 to j-D is represented by I = i to i + D-1 and J = j to j-D + 1
Investigate as you did for. On the other hand, I = i to i +
When D-1 and J = j to j-D + 1 are substantially complementary, the substantial complementarity between I = i + D and J = j-D is checked, and when it is substantially complementary, Is I = i + D + 1
And J = j−D−1 until they are not substantially complementary (step 5). Then, ΔG and r are determined for the thus obtained double strand formed by the substantially complementary regions (step 6). For ΔG, the nearest base pair parameter (see Table 1) is used, and for r, the distance (expressed in bases) between the closest sites in both substantially complementary strand regions.
Is used. Next, using these values, a substantially complementary duplex forming ability is calculated for the double strand formed by the substantially complementary regions obtained here based on the formula I (step 7). Each of the bases (i-i + D-1 th (or i-i + D + D)
+ X-th) and j-th to j-D + 1-th (or j-th)
(-Jx-th) base) (or a new value) (EFB # (I) and EFB # (J)).
Here, x is 0 or a positive integer, and I is I = i to i + D
-1 or i to i + D + x, and J is J = j
~ J-D + 1 or j ~ j-Dx.

Next, a base sequence starting from I = i and J =
It is determined whether or not a substantial complementarity with the base sequence starting from the j-1th position can be compared (step 8). (J-
D)-(i + D-1) is equal to or greater than L + 1, "Y
es ", otherwise" No ".

If "No" in step 8, I = i +
It is determined whether or not 1 may be set (step 9). That is,
It is determined whether the value obtained by adding D to the i-th sequence is equal to or less than the total number of bases. If "Yes" in step 9, I = i +
Step 3 is repeated to compare the complementarity of the first to i + D-th sequences. If "No", I
= The value (EFB # (I)) added for each base with respect to the i-th to (i + NAS-1) -th sequence is further added,
Value for NAS base starting from i-th (AS # (i))
(Step 10). The value of AS # (i) is the sum of the ability to form a substantially complementary double-strand for NAS bases from the ith base to the (i + NAS-1) th base. It can be used as an index indicating effectiveness. As is apparent from the above description, the smaller the value of AS # (i), the more the antisense nucleic acid can be expected to be effective. After the end of step 10, the result is displayed (printout).

[0041]

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

[Example 1] A portion containing a base sequence encoding VEGF (SEQ ID NO: 1) in a plasmid having a base sequence of human-derived vascular endothelial cell growth factor (VEGF121; hereinafter referred to as "VEGF") 635 bases from the 38th position to the 672nd position in the base sequence), the ability of each base to form a substantially complementary double strand was evaluated using a program created by BASIC.

In this embodiment, in the flowchart of FIG. 4, the parameters are L = 4, D = 4, F = 6, and NAS.
= 20 was set. That is, in the mRNA of 635 bases, complementarity between a specific site consisting of 4 bases or more and another site 5 bases or more away from the specific region was examined for the entire base sequence of the RNA.

Of the base sequence represented by SEQ ID NO: 1, 38
Investigate the magnitude of the possibility of forming a substantially complementary double strand with another different region in a specific region consisting of a part of the bases 672 to 672, quantify it and contribute to the numerical value Bases were assigned and the values were summed for each base. The numerical value reflecting the possibility of forming a substantially complementary duplex was set to be larger when the possibility of forming a substantially complementary duplex was larger. Here, as a substantially complementary double strand, GU base pairs were allowed in addition to GC and AU base pairs.

In addition, the ability to form a substantially complementary double strand is determined by the thermodynamic free energy (S.
M. Freier et al. Proc. Natl. Acad. Sci. USA 83 9373-
9377 (1986)).
The parameters obtained by Naoki Sugimoto et al. Were used as parameters of the closest base pair model used here (Table 1).
Further, the interval between the base sequences constituting the substantially complementary double strand to be formed is such that when the closest bases are separated by 5 bases, the ability to form the most substantially complementary double strand is maximized; and The greater the distance, the lower the ability to form a substantially complementary duplex. Then, the specific sites were shifted one base at a time, and the ability of each specific site to form a substantially complementary double strand on each base constituting the specific sites was added up. Then, the values were further summed for 20 bases, and the numerical value was assigned to the base with the lowest number among the 20 bases.

The logarithms of some of the numerical values thus obtained were taken and plotted against the base numbers (数 in FIG. 5). FIG. 5 shows cell-free transcription and
The experimental results obtained with the translation system (indicated by a triangle; WO 96/00286) are also shown. Here, the vertical axis is higher as the ability to form a substantially complementary duplex is larger in the case of the calculation result, and is higher as the expression rate of VEGF is larger in the case of the experimental result. Therefore, if both results are correlated, both plots should show similar patterns.

In FIG. 1, it can be seen that the outlines of both plots match. That is, from base number 400 around 5
Near to 00, the expression of VEGF is remarkably suppressed and the antisense nucleic acid effect is remarkable, but the ability of this region to form a substantially complementary double strand is low. In the experimental results, base numbers 77, 173, 209, 245, 269, 335, 371
At the 533rd and 533rd bases, VEGF expression is high, and in most of them, the ability to form a substantially complementary double strand is high.

The total value of the bases for the ability to form a substantially complementary double strand for each base is NAS (= 20) bases and the logarithmic value (the value on the right vertical axis) is 5.5 or less. 8
As for 2%, the expression rate of VEGF was 10% or less, and it was revealed that both results showed a good correlation. Therefore, by preparing a nucleic acid having a sequence complementary to the sequence of the region having a low logarithmic value (5.5 or less) by a known synthetic means, an antisense nucleic acid exhibiting an effective antisense nucleic acid effect can be easily prepared. Obtainable.

Example 2 In the example shown in Example 1, where the inhibition of VEGF expression in the cell-free transcription / translation system was marked (◆ in FIG. 5), the concentration of antisense nucleic acid and RNase
The concentrations of VEGF were reduced to 1/5 (80 nM) and 1/50 (0.0092 units / μl), respectively.
Was similarly examined (FIG. 6). Here, the antisense nucleic acid number such as A143T shown in FIG. 6 is the same as SEQ ID NO: 1 as shown in the above-mentioned patent (WO96 / 00268).
Means an antisense nucleic acid for a 20-mer starting from the position of the number. For example, A143T is an antisense nucleic acid (natural oligo DNA) for a 20-mer starting from the 143rd position of the nucleotide sequence represented by SEQ ID NO: 1. Similarly, A197
T, A227T, etc. are antisense nucleic acids (natural oligo DNA) for 20-mers starting from positions 197 and 227 of SEQ ID NO: 1, respectively.

As a result, as shown in FIG.
T, A479T, A485T, A491T, A497
VEGF expression was strongly suppressed when T, A503T, and A509T coexisted, particularly when A485T and A491T coexisted, and the antisense nucleic acid effect by these was remarkable. This result is shown in FIG.
(A; lowest value from A491T to A509T), indicating that a site having an antisense nucleic acid effect can be found by the method of the present invention.

When a similar experiment was performed using a plasmid having a base sequence encoding luciferase instead of VEGF, it was found that the antisense nucleic acid shown in FIG. 6 did not inhibit the expression of luciferase. Therefore, it was shown that the antisense nucleic acid shown in FIG. 6 had no effect of inactivating the cell-free transcription / translation system itself. That is, it can be concluded that the expression inhibitory effect shown in FIG. 6 is due to the antisense nucleic acid effect.

[Example 3] The calculation of the nucleotide sequence encoding human VEGF, including the 5 'upstream region and the 3' downstream region, was performed by the method shown in Example 1.
The nucleotide sequence used here is described in the literature (J. Biol. Chem. 266 1
1947-11954 1991 and Science 246
Vol. 1309-1312, 1989), and is composed of 1873 bases, and the nucleotide sequence is shown in SEQ ID NO: 2. In consideration of the correspondence with SEQ ID NO: 1, when the first base of SEQ ID NO: 2 (which corresponds to the transcription start site) is named No. 63, the translation start site is 110
No. 1 (corresponding to No. 1039 in the sequence of SEQ ID NO: 2).
Corresponds to No. 1. As a result of the calculation, in particular, among the seven types that can be expected to have an antisense nucleic acid effect,
Phosphorothioate-type antisense nucleic acids were synthesized by a phosphoramidite method using an automatic synthesizer. Then, its antisense nucleic acid effect was examined using A549 cells derived from human lung cancer and HT1080 derived from human fibroblasts.

[0053]

[Table 2]

That is, A549 cells were used in a 48-well plate, and 5% C in 10% FBS-containing DMEM medium.
The cells were cultured at 37 ° C. in an O ₂ atmosphere. Cell number is about 1-3 × 1
5.25 μg / 200 μ at the point of 0 ⁵ pieces / hole
l (equivalent to about 15 μM) of Tfx-50 (Promega)
OP containing 2.3 μM antisense nucleic acid
5% CO 2 for 2 hours in TI-MEM medium (in the absence of serum)
_The treatment was performed at 37 ° C. in _two atmospheres. Then, the medium was added to 10% F
The medium was replaced with a DMEM medium containing BS, and the cells were treated at 37 ° C. in a 5% CO ₂ atmosphere for 3 hours. The amount of VEGF released into the medium during these three hours was measured by ELISA using an anti-VEGF polyclonal antibody as a primary antibody and a peroxidase-labeled anti-VEGF polyclonal antibody as a secondary antibody (WO96 / 00286).

Further, HT1080 cells were used in a 48-well plate, and 5% C in an EMEM medium containing 10% FBS.
The cells were cultured at 37 ° C. in an O ₂ atmosphere. 4-7 × 10 cells
2.5-3.5 μg / 20 at ⁴ / hole
0 μl (corresponding to about 7 to 10 μM) of Tfx-50 (Pro
(Mega) and 1.6 to 2.3 μM of antisense nucleic acid in an OPTI-MEM medium (in the absence of serum) for 4 hours at 37 ° C. in a 5% CO ₂ atmosphere. Thereafter, the medium was replaced with an EMEM medium containing 10% FBS,
Treated at 37 ° C. for 5 hours in a 5% CO ₂ atmosphere. The amount of VEGF released into the medium during these two hours was determined by using an anti-VEGF polyclonal antibody as a primary antibody, a peroxidase-labeled anti-VEGF polyclonal antibody as a secondary antibody, and
It was measured by the ISA method (WO96 / 00286).

A control phosphorothioate oligo DNA (RA419T-S; base sequence was CTAGACTGTGTG
Compared with TTCTGGAG (SEQ ID NO: 3)), + indicates that VEGF production was significantly suppressed, of which 2
Table 2 shows the case where the number is less than 1 / min, expressed by ++. In addition, "-S" described in the antisense nucleic acid # of Table 2 and "-S" in RA419T-S and the like described above.
Indicates a phosphorothioate type.

That is, the antisense nucleic acids selected from the calculated predictions all significantly inhibit VEGF, and reduce the expression of VEGF to less than half that of the control in about half of the seven species tested. It has been shown.
In each of the cells, the decrease in the number of cells was smaller than in the case where the phosphorothioate-type oligo DNA was not added.
It was less than 30%, confirming that these effects were not due to mere toxicity.

Example 4 A calculation similar to the calculation performed in Example 1 was performed for the luciferase gene. As a gene encoding the luciferase gene, SEQ ID NO: 1
The calculation was performed on the base sequence consisting of 1150 bases shown in FIG. Here, the first base number was 38 and the last base number was 1187. Five species (A65) predicted from calculation results to be particularly suitable as antisense nucleic acids
3T, A960T, A996T, A1405T, and A1446T) and 50 species (A085T to A575T) which were systematically randomly selected by shifting 10 bases at a time.
In the cell-free transcription / translation system experiment performed in Example 1, the inhibition of luciferase expression was examined. Table 3 shows the results.

[0059]

[Table 3]

In Table 3, the numbers of antisense nucleic acids such as A653T have the same meaning as in the above patent (WO96 / 00286) and the above. For example, A653T is an antisense nucleic acid (natural oligo DNA) for a 20-mer starting from SEQ ID NO: 3 at position 653 (where the first base is at position 38 and the last base is at position 1187). means. That is, the nucleotide sequence of A653T is CATT.
ATCAGTGCAATTGTTT (SEQ ID NO: 12). Similarly, A
960T, A990T and the like mean that they are antisense nucleic acids (natural oligo DNA) for the 20-mer starting from the 960th position and the 990th position in the base sequence represented by SEQ ID NO: 11, respectively.

As shown in Table 3, the expression of 5 species, which was predicted from the calculation results to be particularly suitable as an antisense nucleic acid, was significantly suppressed as compared with 50 species randomly selected systematically. . For example, looking at the ratio of those with an expression rate of 10% or less, it is expected that the ratio is 22/50 (44%) when systematically selected at random, whereas it is particularly suitable as an antisense nucleic acid. 4/5 (8
0%). Therefore, it was found that the antisense nucleic acid prepared by the method of the present invention extremely effectively suppressed gene expression.

[0062]

According to the present invention, a target site of an antisense nucleic acid can be predicted with high probability without performing an experiment for a target RNA base sequence, and this method can be used for biochemistry and molecular biology. Useful nucleic acids, particularly antisense nucleic acids useful as therapeutic agents, diagnostic agents, and research reagents for diseases can be easily prepared.

[0063]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 774 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: mRNA Sequence: UUAUGUAUCA UACACAUACG AUUUAGGUGA CACUAUAGAA UACAAGCUUA UGCAUGCGGC 60 CGCAUCUGCUCUGAGCAUGAGGAGCUCUGACGUCUGACU UGCUGCUCUA CCUCCACCAU GCCAAGUGGU CCCAGGCUGC 180 ACCCAUGGCA GAAGGAGGAG GGCAGAAUCA UCACGAAGUG GUGAAGUUCA UGGAUGUCUA 240 UCAGCGCAGC UACUGCCAUC CAAUCGAGAC CCUGGUGGAC AUCUUCCAGG AGUACCCUGA 300 UGAGAUCGAG UACAUCUUCA AGCCAUCCUG UGUGCCCCUG AUGCGAUGCG GGGGCUGCUG 360 CAAUGACGAG GGCCUGGAGU GUGUGCCCAC UGAGGAGUCC AACAUCACCA UGCAGAUUAU 420 GCGGAUCAAA CCUCACCAAG GCCAGCACAU AGGAGAGAUG AGCUUCCUAC AGCACAACAA 480 AUGUGAAUGC AGACCAAAGA AAGAUAGAGC AAGACAAGAA AAAUGUGACA AGCCGAGGCG 540 GUGAGCCGGG CAGGAGGAAG GAGCCUCCCU CAGGGUUUCG GGAACCAGAU CCACUAGUUC 600 UAGAUGCAUG CUCGAGCGGC CGCCAGUGUG AUGGAUAUCU GCAGAAUUCC AGCACACUGG 660 CCGUUACUAG UGGAUCCGAG CUCCCAAAAA AAAAAAAAAA AAAAAAAAAA AAAAACCGAA 720 U UAAUUCGUA AUCAUGGUCA UAGCUGUUUC CUGUGUGAAA UUGUUAUCCG CUCA 774

SEQ ID NO: 2 Sequence length: 1873 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: mRNA sequence: UCGCGGAGGC UUGGGGCAGC CGGGUAGCUC GGAGGUCGUG GCGCUGGGGG CUAGCACCAG 60 CGCUCUGUCG GGAGGCGACGCGGACGAGGACGGCGACGGGGACGG 120 CUCGGUGCUG GAAUUUGAUA UUCAUUGAUC CGGGUUUUAU CCCUCUUCUU UUUUCUUAAA 180 CAUUUUUUUU UAAAACUGUA UUGUUUCUCG UUUUAAUUUA UUUUUGCUUG CCAUUCCCCA 240 CUUGAAUCGG GCCGACGGCU UGGGGAGAUU GCUCUACUUC CCCAAAUCAC UGUGGAUUUU 300 GGAAACCAGC AGAAAGAGGA AAGAGGUAGC AAGAGCUCCA GAGAGAAGUC GAGGAAGAGA 360 GAGACGGGGU CAGAGAGAGC GCGCGGGCGU GCGAGCAGCG AAAGCGACAG GGGCAAAGUG 420 AGUGACCUGC UUUUGGGGGU GACCGCCGGA GCGCGGCGUG AGCCCUCCCC CUUGGGAUCC 480 CGCAGCUGAC CAGUCGCGCU GACGGACAGA CAGACAGACA CCGCCCCCAG CCCCAGCUAC 540 CACCUCCUCC CCGGCCGGCG GCGGACAGUG GACGCGGCGG CGAGCCGCGG GCAGGGGCCG 600 GAGCCCGCGC CCGGAGGCGG GGUGGAGGGG GUCGGGGCUC GCGGCGUCGC ACUGAAACUU 660 UUCGUCCAAC UUCUGGGCUG UUCUCGCUUC GGAGGAGCCG UGGUCC GCGC GGGGGAAGCC 720 GAGCCGAGCG GAGCCGCGAG AAGUGCUAGC UCGGGCCGGG AGGAGCCGCA GCCGGAGGAG 780 GGGGAGGAGG AAGAAGAGAA GGAAGAGGAG AGGGGGCCGC AGUGGCGACU CGGCGCUCGG 840 AAGCCGGGCU CAUGGACGGG UGAGGCGGCG GUGUGCGCAG ACAGUGCUCC AGCCGCGCGC 900 GCUCCCCAGG CCCUGGCCCG GGCCUCGGGC CGGGGAGGAA GAGUAGCUCG CCGAGGCGCC 960 GAGGAGAGCG GGCCGCCCCA CAGCCCGAGC CGGAGAGGGA GCGCGAGCCG CGCCGGCCCC 1020 GGUCGGGCCU CCGAAACCAU GAACUUUCUG CUGUCUUGGG UGCAUUGGAG CCUUGCCUUG 1080 CUGCUCUACC UCCACCAUGC CAAGUGGUCC CAGGCUGCAC CCAUGGCAGA AGGAGGAGGG 1140 CAGAAUCAUC ACGAAGUGGU GAAGUUCAUG GAUGUCUAUC AGCGCAGCUA CUGCCAUCCA 1200 AUCGAGACCC UGGUGGACAU CUUCCAGGAG UACCCUGAUG AGAUCGAGUA CAUCUUCAAG 1260 CCAUCCUGUG UGCCCCUGAU GCGAUGCGGG GGCUGCUGCA AUGACGAGGG CCUGGAGUGU 1320 GUGCCCACUG AGGAGUCCAA CAUCACCAUG CAGAUUAUGC GGAUCAAACC UCACCAAGGC 1380 CAGCACAUAG GAGAGAUGAG CUUCCUACAG CACAACAAAU GUGAAUGCAG ACCAAAGAAA 1440 GAUAGAGCAA GACAAGAAAA AUGUGACAAG CCGAGGCGGU GAGCCGGGCA GGAGGAAGGA 1500 GCCUCCCUCA GGGUUUCGGG AACCAGAUCU CUCACCAGGA AAGACUGAUA CAGAAC GAUC 1560 GAUACAGAAA CCACGCUGCC GCCACCACAC CAUCACCAUC GACAGAACAG UCCUUAAUCC 1620 AGAAACCUGA AAUGAAGGAA GAGGAGACUC UGCGCAGAGC ACUUUGGGUC CGGAGGGCGA 1680 GACUCCGGCG GAAGCAUUCC CGGGCGGGUG ACCCAGCACG GUCCCUCUUG GAAUUGGAUU 1740 CGCCAUUUUA UUUUUCUUGC UGCUAAAUCA CCGAGCCCGG AAGAUUAGAG AGUUUUAUUU 1800 CUGGGAUUCC UGUAGACACA CCCACCCACA UACAUACAUU UAUAUAUAUA UAUAUUAUAU 1860 AUAUAUAAAU UAA 1873

SEQ ID NO: 3 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTAGACTGTG TGTTCTGGAG 20

SEQ ID NO: 4 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACCTCTTTCC TCTTTCTGCT 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Sequence: CTCTCTCTTC CTCGACTTCT 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: ACCCCGTCTC TCTCTTCCTC 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTCCTCTTCC TTCTCTTCTT 20

SEQ ID NO: 8 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTTCTGTATC AGTCTTTCCT G 21

SEQ ID NO: 9 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTCATTTCA GGTTTCTGGA TTAA 24

SEQ ID NO: 10 Sequence length: 20 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TCTTTCTTTG GTCTGCATTC 20

SEQ ID NO: 11 Sequence length: 1150 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: mRNA sequence: GAAUACAAGC UUAUGCAUGC GGCCGCAUCU AGAGGGCCCG GAUCCAAAUG GAAGACGCCA 60 AAAACAUAAA GAAAGGCCCG GCGCCAUUGAGACUGA GCAU 120 AACUGCAUAA GGCUAUGAAG AGAUACGCCC UGGUUCCUGG AACAAUUGCU UUUACAGAUG 180 CACAUAUCGA GGUGAACAUC ACGUACGCGG AAUACUUCGA AAUGUCCGUU CGGUUGGCAG 240 AAGCUAUGAA ACGAUAUGGG CUGAAUACAA AUCACAGAAU CGUCGUAUGC AGUGAAAACU 300 CUCUUCAAUU CUUUAUGCCG GUGUUGGGCG CGUUAUUUAU CGGAGUUGCA GUUGCGCCCG 360 CGAACGACAU UUAUAAUGAA CGUGAAUUGC UCAACAGUAU GAACAUUUCG CAGCCUACCG 420 UAGUGUUUGU UUCCAAAAAG GGGUUGCAAA AAAUUUUGAA CGUGCAAAAA AAAUUACCAA 480 UAAUCCAGAA AAUUAUUAUC AUGGAUUCUA AAACGGAUUA CCAGGGAUUU CAGUCGAUGU 540 ACACGUUCGU CACAUCUCAU CUACCUCCCG GUUUUAAUGA AUACGAUUUU GUACCAGAGU 600 CCUUUGAUCG UGACAAAACA AUUGCACUGA UAAUGAAUUC CUCUGGAUCU ACUGGGUUAC 660 CUAAGGGUGU GGCCCUUCCG CAUAGAUGUGUGAGAG CUCGCAU GCCAGAGAUC 720 CUAUUUUUGG CAAUCAAAUC AUUCCGGAUA CUGCGAUUUU AAGUGUUGUU CCAUUCCAUC 780 ACGGUUUUGG AAUGUUUACU ACACUCGGAU AUUUGAUAUG UGGAUUUCGA GUCGUCUUAA 840 UGUAUAGAUU UGAAGAAGAG CUGUUUUUAC GAUCCCUUCA GGAUUACAAA AUUCAAAGUG 900 CGUUGCUAGU ACCAACCCUA UUUUCAUUCU UCGCCAAAAG CACUCUGAUU GACAAAUACG 960 AUUUAUCUAA UUUACACGAA AUUGCUUCUG GGGGCGCACC UCUUUCGAAA GAAGUCGGGG 1020 AAGCGGUUGC AAAACGCUUC CAUCUUCCAG GGAUACGACA AGGAUAUGGG CUCACUGAGA 1080 CUACAUCAGC UAUUCUGAUU ACACCCGAGG GGGAUGAUAA ACCGGGCGCG GUCGGUAAAG 1140 UUGUUCCAUU 1150

SEQ ID NO: 12 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CATTATCAGT GCAATTGTTT 20

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of a method for calculating the possibility of forming an mRNA duplex.

FIG. 2 is a diagram showing an outline of a method for calculating the possibility of forming an mRNA duplex.

FIG. 3 is a diagram showing an outline of a method for calculating the possibility of forming an mRNA duplex.

FIG. 4 shows a flowchart of a method for calculating the possibility of forming an mRNA duplex.

FIG. 5 is a diagram showing a calculation result of the ability to form a substantially complementary double strand for VEGF mRNA and an expression result of VEGF by an experiment.

FIG. 6 is a diagram showing the results of examining VEGF expression in the presence of an antisense nucleic acid by a cell-free transcription / translation system experiment.

[Explanation of symbols]

1: loop, 2: loop, 3: stem, 4: stem,
5: loop, 6: loop

Claims (7)

[Claims] 1. For each region of the mRNA base sequence,
The possibility of forming a substantially complementary duplex with another region different from the region is quantified, and a nucleic acid having a base sequence that is substantially complementary to the region having a low numerical value is identified as an anti-nucleic acid. A method for preparing an antisense nucleic acid, which is prepared as a sense nucleic acid. 2. The method for preparing an antisense nucleic acid according to claim 1, wherein the numerical value of the possibility of forming a duplex is based on the distance between substantially complementary nucleotide sequence regions. 3. The method according to claim 1, wherein the possibility of forming a double strand is maximum when the distance between base sequence regions substantially complementary to each other sandwiches 3 to 10 bases between the regions. 3. The method for preparing an antisense nucleic acid according to claim 2, wherein the magnitude of the sex is quantified. 4. The method according to claim 1, wherein the possibility of forming a double strand is maximized when the distance between base sequence regions substantially complementary to each other sandwiches 4 to 6 bases between the regions. 3. The method for preparing an antisense nucleic acid according to claim 2, wherein the magnitude of the sex is quantified. 5. The method for preparing an antisense nucleic acid according to claim 1, wherein the quantification of the possibility of forming a duplex is based on the binding energy at the time of forming the duplex. 6. The method for preparing an antisense nucleic acid according to claim 5, wherein the binding energy at the time of forming a duplex is calculated based on a nearest base pair model. 7. A numerical value of the possibility of forming a double chain, assuming that the possibility of forming a double chain is caused by the distance between regions and the binding energy at the time of forming a double chain. 2. The method for preparing an antisense nucleic acid according to claim 1, wherein