US 2009/0155904 A1Published application

Method of inhibiting expression of target mrna using sirna consisting of nucleotide sequence complementary to said target mrna

AssigneeBioneer Corporation

InventorsYoung-Chul Choi, Han Oh Park, Sorim Choung, Young Joo Kim, Sang Soo Kim, Seong-min Park, Sang-Cheol Kim, Gyuman Yoon, Kyoung Oak Choi, Hyo Jin Kang

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Abstract

A inhibition method of target mRNA expression includes: (a) obtaining binding energy of a double combination section on a dsRNA sequence of all combination comprising complementary nucleotides to a random target mRNA; (b) dividing the binding energy into four sections on the dsRNA sequence of each combination to obtain a difference of the mean binding energy between each section and convert into a score of a relative combination energy pattern; (c) selecting siRNA whose inhibition efficiency to target mRNA is expected to be high by applying the converted score to the dsRNA sequence with other factors that affect the efficiency of siRNA; and (d) inhibiting target mRNA expression using the selected siRNA. As a result, a researcher or an experimenter can analyze patterns of a relative binding energy on base sequences of unknown siRNA without actual experiments to determine whether the siRNA is effective or ineffective rapidly, thereby design and production efficiency of siRNA can be maximized and target mRNA can be effectively inhibited with efficient siRNA to the target mRNA.

Claims20

Dependent claims

A method of inhibiting target mRNA expression using siRNA, comprising the steps of:

(1) obtaining all combinations of ds (double strand) RNA sequences each of which consists of n numbers of nucleotides complementary to a predetermined target mRNA (n is an integer);
(2) obtaining EA, EB, EC and ED with respect to each dsRNA, which are mean binding energy values of 1st-2nd section (A), 3rd-7th section (B), 8th-15th section (C) and 16th-18th section (D) in the base sequence of the dsRNA, respectively;
(3) allotting Y(A-B), Y(B-C), Y(C-D) and Y(A-D) to each section of (A) through (D) according to the following equation,
i) in case of −0.02<EA−EB<0.38, −0.29<EB−EC<−0.01, 0.00<EC−ED<0.35, 0.07<ED−EA<0.37, then each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 10 point,
ii) in case of −0.63<EA−EB<−0.21, 0.05<EB−EC<0.44, −0.47<EC−ED<−0.09, −0.67<ED−EA<−0.23, each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 0 point,
iii) in case of EA−EB, EB−EC, EC−ED and ED−EA being out of range defined in (i) and (ii), each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 5 point;
(4) allotting a relative binding energy value Y value with respect to each dsRNA according to the following Equation 4:
Y
=
W
(
A
-
B
)

Y
(
A
-
B
)
+
W
(
B
-
C
)

Y
(
B
-
C
)
+
W
(
C
-
D
)

Y
(
C
-
D
)
+
W
(
A
-
D
)

Y
(
A
-
D
)
10

(
W
(
A
-
B
)
+
W
(
B
-
C
)
+
W
(
C
-
D
)
+
W
(
A
-
D
)
)
×
100
[
Equation


4
]
wherein W(A-B), W(B-C), W(C-D) and W(A-D) are weights for sections (A-B), (B-C), (C-D) and (A-D) which ranges from 0.90 to 1.00, 0.2 to 0.4, 0.2 to 0.3 and 0.7 to 0.9, respectively;
(5) allotting Z value with respect to each dsRNA according to the following Equation 5:
Z
=
100
×
∑
i

W
i

Z
i
M
i
∑
i

W
i
[
Equation


5
]
wherein i is an integer representing a factor affecting siRNA's inhibition efficiency on the target mRNA, at least one of which is the relative binding energy of the siRNA,
Zi is a point given to each factor, provided that Z1=Y, representing a relative binding energy,
Mi is a predetermined maximum value allotted to each factor, and
Wi is a predetermined weight allotted to each factor based on W1;
(6) arranging Z values obtained from the step (5) in a descending order with respect to each dsRNA to select predetermined top % of dsRNAs;
and
(7) applying the selected dsRNAs to inhibit the target mRNA expression.

134 more elements

A method of inhibiting target mRNA expression using siRNA, comprising the steps of:

(1) obtaining all combination of ds (double strand) RNA sequences each of which consists of n numbers of nucleotides complementary to a predetermined target mRNA (n is an integer);
(2) obtaining EA, EB, EC and ED with respect to each dsRNA, which are mean binding energy values of 1st-2nd section (A), 3rd-6th section (B), 14th-16th section (C) and 16th-18th section (D) in the base sequence of the dsRNA, respectively;
(3) allotting Y(A-B), Y(B-C), Y(C-D) and Y(A-D) to each section of (A) through (D) according to the following equation
i) in case of 0.00<EA−EB<0.40, −0.41<EB−EC<−0.01, 0.07<EC−ED<0.39, 0.07<ED−EA<0.37, then each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 10 point,
ii) in case of −0.63<EA−EB<−0.21, 0.10<EB−EC<0.51, −0.47<EC−ED<−0.19, −0.67<ED−EA<−0.23, each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 0 point,
iii) in case of EA−EB, EB−EC, EC−ED and ED−EA being out of range defined in (i) and (ii), each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 5 point;
(4) allotting a relative binding energy Y value with respect to each dsRNA according to the following Equation 4:
Y
=
W
(
A
-
B
)

Y
(
A
-
B
)
+
W
(
B
-
C
)

Y
(
B
-
C
)
+
W
(
C
-
D
)

Y
(
C
-
D
)
+
W
(
A
-
D
)

Y
(
A
-
D
)
10

(
W
(
A
-
B
)
+
W
(
B
-
C
)
+
W
(
C
-
D
)
+
W
(
A
-
D
)
)
×
100
[
Equation


4
]
wherein W(A-B), W(B-C), W(C-D) and W(A-D) are individually weights for sections (A-B), (B-C), (C-D) and (A-D) which ranges from 0.5 to 0.7, 0.3 to 0.5, 0.3 to 0.5 and 0.9 to 1.0, respectively;
(5) allotting Z value with respect to each dsRNA according to the following Equation 5:
Z
=
100
×
∑
i

W
i

Z
i
M
i
∑
i

W
i
[
Equation


5
]
wherein i is an integer representing a factor affecting siRNA's inhibition efficiency on the target mRNA, at least one of which is the relative binding energy of siRNA,
Z1 is a point given to each factor, provided that Zi=Y, representing a relative binding energy point,
Mi is a predetermined maximum value allotted to each factor, and
Wi is a predetermined weight allotted to each factor based on W1;
(6) arranging Z values obtained from the step (5) in a descending order with respect to each dsRNA to select predetermined top % of dsRNAs;
and
(7) applying the selected dsRNAs to inhibit the target mRNA expression.

134 more elements

A method of optimizing siRNA design, comprising the steps of;

(1) obtaining all combinations of ds (double strand) RNA sequences each of which consists of n numbers of nucleotides complementary to a predetermined target mRNA (n is an integer);
(2) obtaining EA, EB, EC and ED with respect to each dsRNA, which are mean binding energy values of 1st-2nd section (A), 3rd-7th section (B), 8th-15th section (C) and 16th-18th section (D) in the base sequence of the dsRNA, respectively;
(3) allotting Y(A-B), Y(B-C), Y(C-D) and Y(A-D) to each section of (A) through (D) according to the following equation,
i) in case of −0.02<EA-EB<0.38, −0.29<EB−EC<−0.01, 0.00<EC−ED<0.35, 0.07<ED−EA<0.37, then each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 10 point,
ii) in case of −0.63<EA−EB<−0.21, 0.05<EB−EC<0.44, −0.47<EC−ED<−0.09, −0.67<ED−EA<−0.23, each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 0 point,
iii) in case of EA−EB, EB−EC, EC−ED and ED−EA being out of range defined in (i) and (ii), each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 5 point;
(4) allotting a relative binding energy value Y value with respect to each dsRNA according to the following Equation 4:
Y
=
W
(
A
-
B
)

Y
(
A
-
B
)
+
W
(
B
-
C
)

Y
(
B
-
C
)
+
W
(
C
-
D
)

Y
(
C
-
D
)
+
W
(
A
-
D
)

Y
(
A
-
D
)
10

(
W
(
A
-
B
)
+
W
(
B
-
C
)
+
W
(
C
-
D
)
+
W
(
A
-
D
)
)
×
100
[
Equation


4
]
wherein W(A-B), W(B-C), W(C-D) and W(A-D) are weights for sections (A-B), (B-C), (C-D) and (A-D) which ranges from 0.90 to 1.00, 0.2 to 0.4, 0.2 to 0.3 and 0.7 to 0.9, respectively;
(5) allotting Z value with respect to each dsRNA according to the following Equation 5:
Z
=
100
×
∑
i

W
i

Z
i
M
i
∑
i

W
i
[
Equation


5
]
wherein i is an integer representing a factor affecting siRNA's inhibition efficiency on the target mRNA, at least one of which is the relative binding energy of the siRNA,
Zi is a point given to each factor, provided that Z1=Y, representing a relative binding energy,
Mi is a predetermined maximum value allotted to each factor, and
Wi is a predetermined weight allotted to each factor based on W1;
and
(6) arranging Z values obtained from the step (5) in a descending order with respect to each dsRNA to select predetermined top % of dsRNAs.

133 more elements

A method of optimizing siRNA design, comprising the steps of:

(1) obtaining all combination of ds (double strand) RNA sequences each of which consists of n numbers of nucleotides complementary to a predetermined target mRNA (n is an integer);
(2) obtaining EA, EB, EC and ED with respect to each dsRNA, which are mean binding energy values of 1st-2nd section (A), 3rd-6th section (B), 14th-16th section (C) and 16th-18th section (D) in the base sequence of the dsRNA, respectively;
(3) allotting Y(A-B), Y(B-C), Y(C-D) and Y(A-D) to each section of (A) through (D) according to the following equation
i) in case of 0.00<EA−EB<0.40, −0.41<EB−EC<−0.01, 0.07<EC−ED<0.39, 0.07<ED−EA<0.37, then each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 10 point,
ii) in case of −0.63<EA−EB<−0.21, 0.10<EB−EC<0.51, −0.47<EC−ED<−0.19, −0.67<ED−EA<−0.23, each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 0 point,
iii) in case of EA−EB, EB−EC, EC−ED and ED−EA being out of range defined in (i) and (ii), each of Y(A-B), Y(B-C), Y(C-D) and Y(A-D) is 5 point;
(4) allotting a relative binding energy Y value with respect to each dsRNA according to the following Equation 4:
Y
=
W
(
A
-
B
)

Y
(
A
-
B
)
+
W
(
B
-
C
)

Y
(
B
-
C
)
+
W
(
C
-
D
)

Y
(
C
-
D
)
+
W
(
A
-
D
)

Y
(
A
-
D
)
10

(
W
(
A
-
B
)
+
W
(
B
-
C
)
+
W
(
C
-
D
)
+
W
(
A
-
D
)
)
×
100
[
Equation


4
]
wherein W(A-B), W(B-C), W(C-D) and W(A-D) are individually weights for sections (A-B), (B-C), (C-D) and (A-D) which ranges from 0.5 to 0.7, 0.3 to 0.5, 0.3 to 0.5 and 0.9 to 1.0, respectively;
(5) allotting Z value with respect to each dsRNA according to the following Equation 5:
Z
=
100
×
∑
i

W
i

Z
i
M
i
∑
i

W
i
[
Equation


5
]
wherein i is an integer representing a factor affecting siRNA's inhibition efficiency on the target mRNA, at least one of which is the relative binding energy of siRNA,
Z1 is a point given to each factor, provided that Z1=Y, representing a relative binding energy point,
Mi is a predetermined maximum value allotted to each factor, and
Wi is a predetermined weight allotted to each factor based on W1;
and
(6) arranging Z values obtained from the step (5) in a descending order with respect to each dsRNA to select predetermined top % of dsRNAs.

133 more elements

Dependencies are inferred from the claim text.

Facts

Application: 11/721,303Filed 8 Dec 2005
Priority: KR 10-2004-01032838 Dec 2004
Published: 18 Jun 2009
Term ends: Not estimatedThe source records don’t support an estimate.

Classification9 codes

C12N 15/111
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: general methods applicable to biologically…
C12N 15/113
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: non-coding nucleic acids modulating the…
G16B 20/00
Ict specially adapted for functional genomics or proteomics
G16B 20/20
ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations: allele or variant detection, e.g. single…