Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides
Inventors
Romesberg, Floyd E. • MALYSHEV, Denis A. • Li, Lingjun • Lavergne, Thomas • LI, Zhengtao
Assignees
Scripps Research Institute • National Institutes of Health NIH • US Department of Health and Human Services
Publication Number
US-10626138-B2
Publication Date
2020-04-21
Expiration Date
2034-08-08
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Abstract
Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker than can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.
Core Innovation
Provided herein are analogs of unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs that form unnatural base pairs during DNA polymerase-mediated replication of DNA or RNA polymerase-mediated transcription of RNA. In this manner, the unnatural nucleobases can be introduced in a site-specific way into oligonucleotides (single or double stranded DNA or RNA), where they can provide for site-specific cleavage, or can provide a reactive linker that can undergo functionalization with a cargo-bearing reagent by means of reaction with a primary amino group or by means of click chemistry with an alkyne group of the unnatural nucleobase linker.
Oligonucleotides including DNA and RNA each include only the four natural nucleotides, which restricts the potential functions and applications of oligonucleotides. Current methods for site-specific nucleic acid derivatization include solid-support based chemical synthesis, combined chemical/enzymatic synthesis, and end-labeling procedures, but these are limited by length, site-specificity, and enzymatic recognition constraints.
The compositions and methods disclosed allow site-specific incorporation of unnatural nucleotides bearing reactive linkers into any position of a DNA or RNA sequence using standard PCR or isothermal transcription methodologies. These linkers can be attached with or without cargos either at the nucleotide triphosphate stage or after synthesis, enabling the site-specific modification of nucleic acids with a variety of cargo molecules.
Claims Coverage
The claims include a set of 19 claims, wherein claim 1 is independent and details the structure of the core compound, followed by dependent claims specifying structural and moiety variations; claims 18 and 19 also detail specific structures.
Compound comprising a pyrido[4,5-b]pyrrolo[2,3-d]pyrimidine structure with defined substituents
A compound of a specified formula comprising a nucleobase analog structure with a wavy line indicating bond to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, wherein substituent R2 is hydrogen or halogen, specifying fluoro as an example, and variations in phosphate attachment at the 5′-hydroxy group.
Incorporation into nucleic acids
The compound is incorporated into RNA or DNA oligonucleotide chains through the ribosyl or 2′-deoxyribosyl moiety with triphosphate groups.
Specific structural embodiments
Claims specify explicit structures of the compound, including various linked phosphate groups at specified positions, variants of R2, and bonding configurations to nucleic acid analogs.
The claims cover a compound with a defined nucleobase analog structure, variations of attachment to sugar moieties and phosphate groups, and inclusion in nucleic acid chains, focusing on their chemical nature and structural features.
Stated Advantages
Enables the site-specific incorporation of unnatural nucleotides with reactive linkers into DNA and RNA oligonucleotides with high efficiency and fidelity.
Expands the chemical and functional diversity of nucleic acids beyond the natural genetic alphabet.
Allows site-specific modification of nucleic acids either during or after enzymatic synthesis, facilitating applications such as selective attachment of cargos including fluorophores, affinity tags, and catalytic groups.
The unnatural base pairs described are replicated and transcribed by natural polymerases with efficiencies and fidelities approaching natural base pairs, suitable for biotechnological uses like SELEX and aptamer development.
Documented Applications
Site-specific cleavage or functionalization of nucleic acids in vitro by incorporation of unnatural nucleotides bearing reactive linkers.
Site-specific labeling of DNA or RNA oligonucleotides with cargo-bearing reagents via reactions with functionalized linkers, including NHS coupling and click chemistry.
Generation of DNA or RNA aptamers and libraries with expanded chemical diversity for SELEX, improving binding affinity and functionality.
Use in PCR and transcription processes for producing functionalized nucleic acids with unnatural base pairs.
Preparation of molecular beacons and nucleic acid-based sensors with site-specific fluorophore labeling.
Production of double stranded oligonucleotide duplexes containing unnatural base pairs for biotechnological applications.
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