초록 ▼

The present invention relates to methods for the labeling of nucleic acid polymers in vitro and in vivo. In particular, the methods include a 3+2 cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Such methods do not require fixati

The present invention relates to methods for the labeling of nucleic acid polymers in vitro and in vivo. In particular, the methods include a 3+2 cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Such methods do not require fixation and denaturation and therefore can be applied to the labeling of nucleic acid polymers in living cells and in organisms. Also provided are methods for measuring cellular proliferation. In these methods, the amount of label incorporated into the DNA is measured as an indication of cellular proliferation. The methods of the invention can be used in a wide variety of applications including clinical diagnosis of diseases and disorders in which cellular proliferation is involved, toxicity assays, and as a tool for the study of chromosomes' ultrastructures.

대표청구항 ▼

1. A method of differentially labeling nucleic acid polymers, comprising steps of: providing a first nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group and a base, wherein the first reactive unsaturated group is directly covalentl

1. A method of differentially labeling nucleic acid polymers, comprising steps of: providing a first nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group and a base, wherein the first reactive unsaturated group is directly covalently attached to the base;providing a second nucleic acid polymer comprising at least one second nucleotide analogue that comprises a second reactive unsaturated group and a base, wherein the second reactive unsaturated group is directly covalently attached to the base;contacting the first nucleic acid polymer with a first reagent comprising a third reactive unsaturated group attached to a first label, such that a [3+2] cycloaddition occurs between the first and third unsaturated groups; andcontacting the second nucleic acid polymer with a second reagent comprising a fourth reactive unsaturated group attached to a second label, such that a [3+2] cycloaddition occurs between the second and fourth unsaturated groups;wherein the first reactive unsaturated group comprises a first 1,3-dipole and the third reactive unsaturated group comprises a first dipolarophile, the second reactive unsaturated group comprises a second dipolarophile and the fourth reactive unsaturated group comprises a second 1,3-dipole, orwherein the first reactive unsaturated group comprises a first dipolarophile and the third reactive unsaturated group comprises a first 1,3-dipole, and the second reactive unsaturated group comprises a second 1,3-dipole and the fourth reactive unsaturated group comprises a second dipolarophile. 2. The method of claim 1, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group and wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group. 3. The method of claim 1, wherein the first nucleic acid polymer is prepared by automated, solid-phase procedure using a phosphoramidite synthesis method, and at least one phosphoramidite used in the phosphoramidite synthesis method is a phosphoramidite form of 5-ethynyl-2′-deoxyuracil. 4. The method of claim 1, wherein the second nucleic acid polymer is prepared by automated, solid-phase procedure using a phosphoramidite synthesis method, and at least one phosphoramidite used in the phosphoramidite synthesis method is a phosphoramidite form of 5-ethynyl-2′-deoxyuracil. 5. The method of claim 1, wherein the first nucleic acid polymer, second nucleic acid polymer, or both is prepared by an in vitro extension or amplification method. 6. The method of claim 5, wherein at least one nucleoside analogue used in the in vitro extension or amplification method is a triphosphate form of 5-ethynyl-2′deoxyuracil or 5-azido-2′-deoxyuracil. 7. A nucleic acid polymer comprising at least one first nucleotide analogue attached to a first label and at least one second nucleotide analogue attached to a second label, wherein said nucleic acid polymer is prepared by a method of dually labeling a nucleic acid polymer, comprising steps of: providing a nucleic acid polymer comprising at least one first nucleotide analogue that comprises a first reactive unsaturated group and at least one second nucleotide analogue that comprises a second reactive unsaturated group;contacting the nucleic acid polymer with a first reagent comprising a third reactive unsaturated group attached to a first label, such that a [3+2] cycloaddition occurs between the first and third unsaturated groups; andcontacting the nucleic acid polymer with a second reagent comprising a fourth reactive unsaturated group attached to a second label, such that a [3+2] cycloaddition occurs between the second and fourth unsaturated groups. 8. The nucleic acid polymer of claim 7, wherein the at least one first nucleotide analogue comprises a first cycloadduct resulting from a [3+2] cycloaddition between a first ethynyl group and a first azide group and the at least one second nucleotide analogue comprises a second cycloadduct resulting from a [3+2] cycloaddition between a second ethynyl group and a second azide group. 9. The nucleic acid polymer of claim 7, wherein the first reactive unsaturated group is directly covalently attached to the base of the first nucleotide analogue, and the second reactive unsaturated group is directly covalently attached to the base of the second nucleotide analogue. 10. A kit for dually labeling a nucleic acid polymer, comprising at least one first nucleoside analogue that comprises a first reactive unsaturated group;at least one second nucleoside analogue that comprises a second reactive unsaturated group;a first reagent comprising a third reactive unsaturated group attached to a first label; anda second reagent comprising a fourth reactive unsaturated group attached to a second label;wherein the first reactive unsaturated group comprises a first 1,3-dipole, the third reactive unsaturated group comprises a first dipolarophile, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition and wherein the second reactive unsaturated group comprises a second dipolarophile, the fourth reactive unsaturated group comprises a second 1,3-dipole, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition, orwherein the first reactive unsaturated group comprises a first dipolarophile, the third reactive unsaturated group comprises a first 1,3-dipole, and the first and third reactive unsaturated groups can react via [3+2] cycloaddition and wherein the second reactive unsaturated group comprises a second 1,3-dipole, the fourth reactive unsaturated group comprises a second dipolarophile, and the second and fourth reactive unsaturated groups can react via [3+2] cycloaddition. 11. The kit of claim 10, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group or wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group. 12. The kit of claim 10 further comprising an aqueous medium and comprising Cu(I). 13. The kit of claim 10, wherein the first reactive unsaturated group is directly covalently attached to the base of the first nucleoside analogue, and the second reactive unsaturated group is directly covalently attached to the base of the second nucleoside analogue. 14. The kit of claim 10, wherein the first 1,3-dipole comprises a first azide group and the second 1,3-dipole comprises a second azide group and wherein the first dipolarophile comprises a first ethynyl group and the second dipolarophile comprises a second ethynyl group.