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Abstract Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3′-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson–Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.
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The XNA alphabet
Abstract Inspired by nature, chemists have spent the last 50 years systematically designing and synthesizing a vast array of sugar-modified nucleic acids, so-called xenonucleic acids (XNAs), collectively forming what we now describe as the XNA alphabet. Within the alphabet, systems can be categorized into two major groups: those capable of interacting with natural nucleic acids and those that do not cross-pair with DNA or RNA. The sugar component of XNAs plays a crucial role in defining their conformational space, which, in turn, influences their hybridization properties and potential applications across biotechnology and synthetic biology. This review provides an overview of sugar-modified XNA systems developed to date as well as the geometric parameters and physicochemical principles that have enhanced our understanding of XNA conformational behavior, particularly in relation to their orthogonality to (i.e. inability to cross-pair with) natural nucleic acids. These insights are essential for developing a more rational approach to key processes such as XNA replication and evolution, ultimately paving the way for applications in areas including synthetic genetics, nucleic acid therapeutics, diagnostics, and nanotechnology.
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- Award ID(s):
- 2433788
- PAR ID:
- 10614759
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 53
- Issue:
- 13
- ISSN:
- 0305-1048
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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