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Abstract Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ−:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ−:dZ pairs join parallel strands in a four-stranded compact down–up–down–up fold. These have two possible structures: one with intercalated dZ−:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC+-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis.
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This content will become publicly available on November 6, 2026
Improving the Fidelity of Replication of a Six-Letter DNA Alphabet
The Watson−Crick-Franklin (WCF) rules describing nucleobase pairing in antiparallel strands of DNA and RNA can be exploited to create artificially expanded genetic information systems (AEGIS) with as many as 12 independently replicable nucleotides joined by six hydrogen bond pairing schemes. One of these additional pairs joins two nucleotides trivially designated as Z (6-amino-5-nitro-(1H)-pyridin-2-one) and P (2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one). The Z:P pair has supported 6- nucleotide PCR to give diagnostics products, in environmental surveillance kits, and for laboratory in vitro evolution (LIVE) that has generated, inter alia, molecules that inactivate toxins, antibody analogs that bind cancer cells, therapeutic candidates that deliver drugs to those cells, reagents to identify targets on those cells’ surfaces, reagents to move cargoes across the blood−brain barrier, and catalysts with ribonuclease activity. However, the Z nucleoside is acidic, with a pKa of ∼7.8. In its deprotonated form, Z− forms a WCF pair with G. This leads to the slow replacement of Z:P pairs by C:G pairs during PCR or, in the reverse process, their introduction. Here, we examine analogs of Z that retain the same donor:donor:acceptor hydrogen bonding pattern as earlier generations of the Z heterocycle, still form a WCF pair with P, but have a higher pKa. Experiments with Taq polymerase show that the rate of loss of Z:P pairs decreases markedly as the pKa of the Z heterocycle increases. This provides direct support for the hypothesis that Z:P pairs are in fact lost via deprotonated Z−:G mismatches. Further, it provides a Z:P system that can be replicated with very high fidelity, with >97% retention of the Z:P pairs over 10,000-fold amplification.
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- Award ID(s):
- 2108028
- PAR ID:
- 10648289
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- ACS Chemical Biology
- ISSN:
- 1554-8929
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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