Dynamic and combinatorial synthesis of polymerase primers
View Patent ↗This invention relates to the field of nucleic acid chemistry, more specifically to compositions of matter that are nucleic acid analogs, and processes that use them. Still more specifically, these compositions comprise two fragments of DNA-like molecules, each having one or more ends modified to carry a reactive group, where the reactive group on one fragment can form a transient covalent bond with the reactive group on the other under conditions of dynamic equilibrium to form a composite, where the composite can then bind to a target oligonucleotide, such as a DNA or RNA molecule. Most specifically, once the transient covalent bond forms, the composite serves as a primer for a template-directed polymerization using a DNA polymerase, an RNA polymerase, or a reverse transcriptase. Once incorporated, the epimerization causes the base pair to be destabilized, the duplex containing the epimerized nucleoside to likewise be destabilized, and the double strand to then disassociate. This leaves the template available to template the synthesis of another complementary oligonucleotide containing the epimerizing base.
1. The process of preparing a nucleic acid analog that binds to a preselected template, said process comprising contacting a 3′-fragment molecule having a functional group at its 5′-terminus, at the same time as contacting a 5′-fragment molecule having a functional group at its 3′-terminus, where the 3′- and 5′-fragments are Watson-Crick complementary to and bind through Watson-Crick pairing to adjacent segments of a preselected linear template molecule, wherein said 5′- and 3′-fragments, bound to said template, form a product molecule by a reaction between the functional groups at their respective 3′- and 5′-terminii to form a covalent bond, wherein the formation of said covalent bond is reversible under the conditions where the contacting occurs.
2. The process of claim 1 wherein said product molecule serves as a primer for a DNA polymerase, an RNA polymerase, or a reverse transcriptase.
3. The process of claim 1 wherein said preselected template molecule is a DNA molecule or an RNA molecule.
4. The process of claim 1 , wherein said fragment molecules are nucleic acid analogs selected from the group consisting of ribonucleic acid, 2′-O-methylribonucleic acid, 2′-deoxyribonucleic acid, locked nucleic acid, bicyclonucleic acid, peptide nucleic acid, or glycol nucleic acid.
5. The process of claim 1 wherein said product molecule is described by the formula
wherein X is selected from the group consisting of —H, —OH, —OCH 3 , and —O-allyl, the B groups are heterocycles independently selected from the group consisting of adenine and the heterocycles in the structures shown in FIGS. 5 , 7 , and 9 , k is an integer from 1 to 10, and m and n are integers between 2 and 13.
6. The process of claim 1 , wherein said 3′- and 5′-fragment molecules are presented as libraries, each library comprising two or more fragments, which differentially join to form product molecules depending on the presence of a template, whose sequence is not preselected.
7. A process of preparing a nucleic acid analog that binds to a preselected template, said process comprising contacting a 3′-fragment molecule having a functional group at its 5′-terminus, at the same time as contacting a 5′-fragment molecule having a functional group at its 3′-terminus, where the 3′- and 5′-fragments are Watson-Crick complementary to and bind through Watson-Crick pairing to adjacent segments of a pre-selected linear template molecule, wherein said 5′- and 3′-fragments, bound to said template, form a product molecule by a reaction between the functional groups at their respective 3′- and 5′-terminii to form a covalent bond, wherein the formation of said covalent bond is reversible under the conditions where the contacting occurs, wherein said product molecule is
wherein X is selected from the group consisting of —H, —OH, —OCH 3 , and —O-allyl, the B groups are heterocycles independently selected from the group consisting of adenine and the heterocycles in the structures shown in FIGS. 5 , 7 , and 9 , k is an integer from 1 to 10, and m and n are integers between 2 and 13.
8. A process of preparing a nucleic acid analog that binds to a preselected template, said process comprising contacting a 3′-fragment molecule having a functional group at its 5′-terminus, at the same time as contacting a 5′-fragment molecule having a functional group at its 3′-terminus, where the 3′- and 5′-fragments are Watson-Crick complementary to and bind through Watson-Crick pairing to adjacent segments of a pre-selected linear template molecule, wherein said 5′- and 3′-fragments, bound to said template, form a product molecule by a reaction between the functional groups at their respective 3′- and 5′-terminii to form a covalent bond, wherein the formation of said covalent bond is reversible under the conditions where the contacting occurs, wherein said product molecule is
wherein X is selected from the group consisting of —H, —OH, —OCH 3 , and —O-allyl, the B groups are heterocycles independently selected from the group consisting of adenine and the heterocycles in the structures shown in FIGS. 5 , 7 , and 9 , k is an integer from 1 to 10, and m and n are integers between 2 and 13.
9. A process of preparing a nucleic acid analog that binds to a preselected template, said process comprising contacting a 3′-fragment molecule having a functional group at its 5′-terminus, at the same time as contacting a 5′-fragment molecule having a functional group at its 3′-terminus, where the 3′- and 5′-fragments are Watson-Crick complementary to and bind through Watson-Crick pairing to adjacent segments of a pre-selected linear template molecule, wherein said 5′- and 3′-fragments, bound to said template, form a product molecule by a reaction between the functional groups at their respective 3′- and 5′-terminii to form a covalent bond, wherein the formation of said covalent bond is reversible under the conditions where the contacting occurs, wherein said product molecule is selected from the group consisting of
wherein X is selected from the group consisting of —H, —OCH 3 , and —O-allyl, the B groups are heterocycles independently selected from the group consisting of adenine and the heterocycles in the structures shown in FIGS. 5 , 7 , and 9 , k is an integer from 1 to 10, and m and n are integers between 2 and 13.