Method for improving loading and stability of nucleic acid

1. A method for improving loading of a nucleic acid nanoball on a solid support, comprising a) providing a solid support having a nucleic acid immobilized thereon, and b) allowing the solid support to contact with a poloxamer-containing reagent.

2. A group of partially double-stranded oligonucleotides, comprising at least two partially double-stranded oligonucleotides,

wherein the partially double-stranded oligonucleotide comprising a first oligonucleotide strand and a second oligonucleotide strand, wherein the first oligonucleotide strand comprises a region C, a region A and a region B from 5′ to 3′, and the second oligonucleotide strand comprises a region A′ and a region B′ from 5′ to 3′, and optionally, further comprises a region C′ at upstream of the region A′; wherein the regions A and A′ are reversely complementary to each other, and the regions B and B′ are not complementary to each other but each is complementary to a predetermined target sequence, wherein the first oligonucleotide strand and the second oligonucleotide strand hybridize to each other through the regions A and A′, thereby forming the partially double-stranded oligonucleotide;

wherein the region C of at least one partially double-stranded oligonucleotide is reversely complementary to the region C of at least one other partially double-stranded oligonucleotide such that at least two partially double-stranded oligonucleotides can hybridize to each other through the respective region C; and

wherein the regions A and A′ are 10-40 nucleotides in length; the regions B and B′ are 10-70 nucleotides in length; and, the regions C and C′ are 10-70 nucleotides in length.

3. The group of partially double-stranded oligonucleotides according to claim 2 , wherein the first oligonucleotide strand comprises a region C, a region A and a region B from 5′ to 3′, and the second oligonucleotide strand comprises a region C′, a region A′ and a region B′ from 5′ to 3′, wherein the regions A and A′ are reversely complementary to each other, and the regions B and B′ are not complementary to each other but each is complementary to a predetermined target sequence, wherein the first oligonucleotide strand and the second oligonucleotide strand hybridize to each other through the regions A and A′, thereby forming the partially double-stranded oligonucleotide, wherein at least one of the regions C and C′ of at least one partially double-stranded oligonucleotide is reversely complementary to at least one of the regions C and C′ of at least one other partially double-stranded oligonucleotide such that at least two partially double-stranded oligonucleotides are capable of hybridizing to each other through the respective regions C and/or C′.

4. The group of partially double-stranded oligonucleotides according to claim 3 , wherein the group of partially double-stranded oligonucleotides is characterized by one or more of the following:

(1) the regions C and C′ have the same or different sequences,

(2) the regions B and B′ are designed to be complementary to the same target sequence, or the regions B and B′ are designed to be complementary to different target sequences,

(3) the regions A and A′ are 15-30 nucleotides in length,

(4) the regions B and B′ are 30-42 nucleotides in length, and

(5) the regions C and C′ are 20-50 nucleotides in length.

5. A method for improving stability of a nucleic acid on a solid support, wherein,

the method comprises the steps of: a) providing a solid support having a nucleic acid molecule immobilized thereon; and b) allowing a partially double-stranded oligonucleotide or a group of partially double-stranded oligonucleotides to hybridize to the nucleic acid molecule on the solid support,

wherein, the partially double-stranded oligonucleotide comprises a first oligonucleotide strand and a second oligonucleotide strand, wherein the first oligonucleotide strand comprises a region C, a region A and a region B from 5′ to 3′; and the second oligonucleotide strand comprises a region A′ and a region B′ from 5′ to 3′, and optionally, further comprises a region C′ at upstream of the region A′; wherein the regions A and A′ are reversely complementary to each other, and the regions B and B′ are not complementary to each other but each is complementary to a predetermined target sequence; wherein the first oligonucleotide strand and the second oligonucleotide strand hybridize to each other through the regions A and A′, thereby forming the partially double-stranded oligonucleotide;

wherein the group of partially double-stranded oligonucleotides comprise at least two partially double-stranded oligonucleotides;

wherein the partially double-stranded oligonucleotide hybridizes to the nucleic acid molecule through the regions B and/or B′,

or

the method comprises the steps of: a) providing a nucleic acid molecule; b) allowing a partially double-stranded oligonucleotide or the group of partially double-stranded oligonucleotides to hybridize to the nucleic acid molecule,

wherein, the partially double-stranded oligonucleotide comprises a first oligonucleotide strand and a second oligonucleotide strand, wherein the first oligonucleotide strand comprises a region C, a region A and a region B from 5′ to 3′; and the second oligonucleotide strand comprises a region A′ and a region B′ from 5′ to 3′, and optionally, further comprises a region C′ at upstream of the region A′; wherein the regions A and A′ are reversely complementary to each other, and the regions B and B′ are not complementary to each other but each is complementary to a predetermined target sequence; wherein the first oligonucleotide strand and the second oligonucleotide strand hybridize to each other through the regions A and A′, thereby forming the partially double-stranded oligonucleotide;

wherein the group of partially double-stranded oligonucleotides comprise at least two partially double-stranded oligonucleotides;

wherein the partially double-stranded oligonucleotide hybridizes to the nucleic acid molecule through the regions B and/or B′; and c) immobilizing the nucleic acid molecule on a solid support.

6. A method for improving loading and stability of a nucleic acid on a solid support, wherein,

the method comprises the steps of: a) providing a solid support having a nucleic acid molecule immobilized thereon; and b) allowing the solid support to contact with a poloxamer-containing reagent; and c) allowing a partially double-stranded oligonucleotide or a group of partially double-stranded oligonucleotides to hybridize to the nucleic acid molecule on the solid support,

wherein, the partially double-stranded oligonucleotide comprises a first oligonucleotide strand and a second oligonucleotide strand, wherein the first oligonucleotide strand comprises a region C, a region A and a region B from 5′ to 3′; and the second oligonucleotide strand comprises a region A′ and a region B′ from 5′ to 3′, and optionally, further comprises a region C′ at upstream of the region A′; wherein the regions A and A′ are reversely complementary to each other, and the regions B and B′ are not complementary to each other but each is complementary to a predetermined target sequence; wherein the first oligonucleotide strand and the second oligonucleotide strand hybridize to each other through the regions A and A′, thereby forming the partially double-stranded oligonucleotide;

wherein the group of partially double-stranded oligonucleotides comprise at least two partially double-stranded oligonucleotides;

wherein the partially double-stranded oligonucleotide hybridizes to the nucleic acid molecule through the regions B and/or B′; wherein step c) is performed before or after step b);

or,

the method comprises the steps of: a) providing a nucleic acid molecule; b) allowing a partially double-stranded oligonucleotide or a group of partially double-stranded oligonucleotides to-hybridize to the nucleic acid molecule,

wherein, the partially double-stranded oligonucleotide comprises a first oligonucleotide strand and a second oligonucleotide strand, wherein the first oligonucleotide strand comprises a region C, a region A and a region B from 5′ to 3′; and the second oligonucleotide strand comprises a region A′ and a region B′ from 5′ to 3′, and optionally, further comprises a region C′ at upstream of the region A′; wherein the regions A and A′ are reversely complementary to each other, and the regions B and B′ are not complementary to each other but each is complementary to a predetermined target sequence; wherein the first oligonucleotide strand and the second oligonucleotide strand hybridize to each other through the regions A and A′, thereby forming the partially double-stranded oligonucleotide;

wherein the group of partially double-stranded oligonucleotides comprise at least two partially double-stranded oligonucleotides;

wherein the partially double-stranded oligonucleotide hybridizes to the nucleic acid molecule through the regions B and/or B′; c) immobilizing the nucleic acid molecule on a solid support; and d) allowing the solid support to contact with a poloxamer-containing reagent.

7. The method according to claim 1 , wherein the method is characterized by one or more of the following:

(1) the poloxamer is a hydrophilic poloxamer,

(2) the poloxamer is a hydrophilic poloxamer having a HLB of 20-29 or a mixture thereof,

(3) the poloxamer is selected from PF68, PF108, PF127, equivalents thereof, and mixtures thereof,

(4) the poloxamer-containing reagent contains 0.01% to 2% (w/v) poloxamer,

(5) the poloxamer-containing reagent contains 0.2% to 0.5% (w/v) poloxamer, and

(6) the nucleic acid nanoball is a DNA nanoball.

8. The method according to claim 6 , wherein the method is characterized by one or more of the following:

(1) the poloxamer is a hydrophilic poloxamer,

(2) the poloxamer is a hydrophilic poloxamer having a HLB of 20-29 or a mixture thereof,

(3) the poloxamer is selected from PF68, PF108, PF127, equivalents thereof, and mixtures thereof,

(4) the poloxamer-containing reagent contains 0.01% to 2% (w/v) poloxamer,

(5) the poloxamer-containing reagent contains 0.2% to 0.5% (w/v) poloxamer,

(6) the nucleic acid is a nucleic acid nanoball, and

(7) the nucleic acid nanoball is a DNA nanoball.

9. A solid support, wherein a nucleic acid molecule is immobilized on the solid support, and the nucleic acid molecule hybridizes to a partially double-stranded oligonucleotide or a group of partially double-stranded oligonucleotides,

wherein, the partially double-stranded oligonucleotide comprises a first oligonucleotide strand and a second oligonucleotide strand, wherein the first oligonucleotide strand comprises a region C, a region A and a region B from 5′ to 3′; and the second oligonucleotide strand comprises a region A′ and a region B′ from 5′ to 3′, and optionally, further comprises a region C′ at upstream of the region A′; wherein the regions A and A′ are reversely complementary to each other, and the regions B and B′ are not complementary to each other but each is complementary to a predetermined target sequence; wherein the first oligonucleotide strand and the second oligonucleotide strand hybridize to each other through the regions A and A′, thereby forming the partially double-stranded oligonucleotide;

wherein the group of partially double-stranded oligonucleotides comprise at least two partially double-stranded oligonucleotides;

wherein the partially double-stranded oligonucleotide hybridizes to the nucleic acid molecule through the regions B and/or B′.

10. The solid support according to claim 9 , wherein the nucleic acid is a nucleic acid nanoball or a DNA nanoball.

11. A method for nucleic acid analysis, comprising:

a) obtaining a solid support having a nucleic acid nanoball immobilized thereon according to the method according to claim 1 , and

b) analyzing the nucleic acid nanoball on the solid support.

12. The method according to claim 11 , wherein the method is characterized by one or more of the following:

(1) the nucleic acid analysis is selected from nucleic acid sequencing, nucleic acid hybridization tests, or enzyme-assisted nucleic acid tests, and

(2) the nucleic acid nanoball is a DNA nanoball.

13. A method for nucleic acid analysis, comprising:

a) obtaining a solid support having a nucleic acid molecule immobilized thereon according to the method according to claim 5 , and

b) analyzing the nucleic acid molecules on the solid support.

14. The method according to claim 13 , wherein the method is characterized by one or more of the following:

(1) the nucleic acid analysis is selected from nucleic acid sequencing, nucleic acid hybridization tests, or enzyme-assisted nucleic acid tests,

(2) the nucleic acid is a nucleic acid nanoball, and

(3) the nucleic acid nanoball is a DNA nanoball.

15. A method for nucleic acid analysis, comprising:

a) obtaining a solid support having a nucleic acid molecule immobilized thereon according to the method according to claim 6 , and

b) analyzing the nucleic acid molecules on the solid support.

16. The method according to claim 15 , wherein the method is characterized by one or more of the following:

(1) the nucleic acid analysis is selected from nucleic acid sequencing, nucleic acid hybridization tests, or enzyme-assisted nucleic acid tests,

(2) the nucleic acid is a nucleic acid nanoball, and

(3) the nucleic acid nanoball is a DNA nanoball.

17. The method according to claim 5 , wherein the nucleic acid is a nucleic acid nanoball or a DNA nanoball.

18. The group of partially double-stranded oligonucleotides according to claim 2 , wherein

the region C or C′ is designed such that when a first and a second partially double-stranded oligonucleotides are present, the region C or C′ of the first partially double-stranded oligonucleotide is reversely complementary to the region C or C′ of the second partially double-stranded oligonucleotide respectively, so that the first partially double-stranded oligonucleotide and the second partially double-stranded oligonucleotide can hybridize to each other through the respective region C or C′, or

the regions C and C′ is designed such that when a first and a second partially double-stranded oligonucleotides are present, at least one of the regions C and C′ of the first partially double-stranded oligonucleotide is reversely complementary to at least one of the regions C and C′ of the second partially double-stranded oligonucleotide, so that the first partially double-stranded oligonucleotide and the second partially double-stranded oligonucleotide can hybridize to each other through the respective region C and/or C′.

19. The method of claim 5 , wherein the group of the partially double-stranded oligonucleotides are subjected to hybridize to the nucleic acid molecule, wherein

the region C or C′ is designed such that when a first and a second partially double-stranded oligonucleotides are present, the region C or C′ of the first partially double-stranded oligonucleotide is reversely complementary to the region C or C′ of the second partially double-stranded oligonucleotide respectively, so that the first partially double-stranded oligonucleotide and the second partially double-stranded oligonucleotide can hybridize to each other through the respective region C or C′, or

the regions C and C′ is designed such that when a first and a second partially double-stranded oligonucleotides are present, at least one of the regions C and C′ of the first partially double-stranded oligonucleotide is reversely complementary to at least one of the regions C and C′ of the second partially double-stranded oligonucleotide, so that the first partially double-stranded oligonucleotide and the second partially double-stranded oligonucleotide can hybridize to each other through the respective region C and/or C′.

20. The method of claim 6 , wherein the group of the partially double-stranded oligonucleotides are subjected to hybridize to the nucleic acid molecule, wherein

the region C or C′ is designed such that when a first and a second partially double-stranded oligonucleotides are present, the region C or C′ of the first partially double-stranded oligonucleotide is reversely complementary to the region C or C′ of the second partially double-stranded oligonucleotide respectively, so that the first partially double-stranded oligonucleotide and the second partially double-stranded oligonucleotide can hybridize to each other through the respective region C or C′, or

the regions C and C′ is designed such that when a first and a second partially double-stranded oligonucleotides are present, at least one of the regions C and C′ of the first partially double-stranded oligonucleotide is reversely complementary to at least one of the regions C and C′ of the second partially double-stranded oligonucleotide, so that the first partially double-stranded oligonucleotide and the second partially double-stranded oligonucleotide can hybridize to each other through the respective region C and/or C′.