Assembly of long nucleic acids by ligation using indexed splint oligos
Inventors
Taussig, David • Sampas, Nicholas M.
Assignees
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Publication Number
US-12281302-B2
Publication Date
2025-04-22
Expiration Date
Abstract
The present disclosure relates to a method of assembling long nucleic acids by enzymatically ligating oligonucleotide molecules hybridized to an indexed splint oligonucleotide molecules. Also disclosed are oligonucleotide structures comprising an indexed splint oligonucleotide useful in performing the disclosed method.
Core Innovation
The disclosed approach assembles two nucleic acid components to produce a multicomponent ligation product in solution by using an indexed splint. A first single-stranded oligonucleotide and a second single-stranded oligonucleotide are hybridized to the indexed splint such that both the 5′ and 3′ ends of each oligonucleotide are hybridized to the indexed splint, and the 3′ end of the first single-stranded oligonucleotide is ligated to the 5′ end of the second single-stranded oligonucleotide.
The indexed splint includes index sequences, non-hybridizing spacer sequences, and a splint sequence comprising a 5′ element and a 3′ element. The 5′ element of the splint sequence hybridizes to the 5′ terminal sequence of the second single-stranded oligonucleotide, while the 3′ element of the splint sequence hybridizes to the 3′ terminal sequence of the first single-stranded oligonucleotide.
The disclosure further refines the method to support indexed, multiplexed assembly and improved discrimination among nucleic acid libraries. It also describes iterative and scarless or removable assembly schemes using removable Type IIS sites and/or cleavable linkers, along with optional blockers/tethers, and supports multi-part multicomponent assembly using restriction digestion and re-ligation.
Claims Coverage
The partial record contains one independent claim. The independent claim is directed to an indexed-splint ligation method in which two single-stranded oligonucleotides are hybridized at both ends to an indexed splint and then ligated to form a multicomponent ligation product; the indexed splint contains defined index sequences, non-hybridizing spacers, and splint 5′/3′ elements with specified terminal hybridization relationships.
Indexed-splint hybridization at both oligonucleotide ends
Hybridizing a first single-stranded oligonucleotide comprising a first DNA sequence component and a second single-stranded oligonucleotide comprising a second DNA sequence component to an indexed splint, wherein both the 5′ end and the 3′ end of the first single stranded oligonucleotide are hybridized to the indexed splint and both the 5′ end and the 3′ end of the second single-stranded oligonucleotide are hybridized to the indexed splint.
End-defined ligation to form a multicomponent ligation product
Ligating the 3′ end of the first single-stranded oligonucleotide to the 5′ end of the second single-stranded oligonucleotide, thereby producing a first multicomponent ligation product.
Indexed splint with index sequences, non-hybridizing spacer sequences, and splint 5′/3′ elements
Defining the indexed splint comprises from 5′ to 3′: a second index sequence, a first spacer sequence which does not hybridize to sequences within the first and the second single-stranded oligonucleotides, a splint sequence comprising a 5′ element and a 3′ element, a second spacer sequence which does not hybridize to sequences within the first and second single-stranded oligonucleotides, and a first index sequence, wherein the 5′ element of the splint sequence hybridizes to the 5′ terminal sequence of the second single-stranded oligonucleotide and the 3′ element of the splint sequence hybridizes to the 3′ terminal sequence of the first single-stranded oligonucleotide.
Overall claim coverage centers on forming a multicomponent ligation product by hybridizing two single-stranded oligonucleotides at both ends to a specifically structured indexed splint, with index sequences, non-hybridizing spacer sequences, and splint 5′/3′ elements defining the terminal hybridization relationships before 3′-to-5′ ligation.
Stated Advantages
Improved correct-construct assembly/discrimination and reduced dropout/chimeras across library types and splint designs, as supported by proof-of-concept examples.
High correct-construction percentages are described in the provided examples.
Documented Applications
Assembly and discrimination for homologous/low-homology libraries, including complex libraries, tiled libraries, and other library types evaluated in proof-of-concept examples.
Multi-part nucleic acid assembly in solution using restriction digestion and re-ligation, as supported by the disclosed examples.
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