Synthetic genetics is an area of synthetic biology that aims to extend the properties of heredity and evolution to artificial genetic polymers, commonly known as xeno‐nucleic acids or XNAs. In addition to establishing polymerases that are able to convert genetic information back and forth between DNA and XNA, efforts are underway to construct XNAs with expanded chemical functionality. α‐L‐Threose nucleic acid (TNA), a type of XNA that is recalcitrant to nuclease digestion and amenable to Darwinian evolution, provides a model system for developing XNAs with functional groups that are not present in natural DNA and RNA. Here, we describe the synthesis and polymerase activity of a cytidine TNA triphosphate analog (6‐phenyl‐pyrrolocytosine, tCpTP) that maintains Watson‐Crick base pairing with guanine. Polymerase‐mediated primer extension assays show that tCpTP is an efficient substrate for Kod‐RI, a DNA‐dependent TNA polymerase developed to explore the functional properties of TNA by
The 4-letter DNA alphabet (A, T, G, C) as found in Nature is an elegant, yet non-exhaustive solution to the problem of storage, transfer, and evolution of biological information. Here, we report on strategies for both writing and reading DNA with expanded alphabets composed of up to 12 letters (A, T, G, C, B, S, P, Z, X, K, J, V). For writing, we devise an enzymatic strategy for inserting a singular, orthogonal xenonucleic acid (XNA) base pair into standard DNA sequences using 2′-deoxy-xenonucleoside triphosphates as substrates. Integrating this strategy with combinatorial oligos generated on a chip, we construct libraries containing single XNA bases for parameterizing kmer basecalling models for commercially available nanopore sequencing. These elementary steps are combined to synthesize and sequence DNA containing 12 letters – the upper limit of what is accessible within the electroneutral, canonical base pairing framework. By introducing low-barrier synthesis and sequencing strategies, this work overcomes previous obstacles paving the way for making expanded alphabets widely accessible.
more » « less- Award ID(s):
- 1939086
- NSF-PAR ID:
- 10471019
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract in vitro selection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tCpTP and 7‐deaza‐tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available forin vitro selection. -
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Abstract Background Three genes clustered together on chromosome 12 comprise a group of hydroxycarboxylic acid receptors (HCARs):
HCAR1 ,HCAR2 , andHCAR3 . These paralogous genes encode different G-protein coupled receptors responsible for detecting glycolytic metabolites and controlling fatty acid oxidation. Though better known for regulating lipid metabolism in adipocytes, more recently, HCARs have been functionally associated with breast cancer proliferation/survival;HCAR2 has been described as a tumor suppressor andHCAR1 andHCAR3 as oncogenes. Thus, we sought to identify germline variants inHCAR1 ,HCAR2, andHCAR3 that could potentially be associated with breast cancer risk.Methods Two different cohorts of breast cancer cases were investigated, the Alabama Hereditary Cancer Cohort and The Cancer Genome Atlas, which were analyzed through nested PCRs/Sanger sequencing and whole-exome sequencing, respectively. All datasets were screened for rare, non-synonymous coding variants.
Results Variants were identified in both breast cancer cohorts, some of which appeared to be associated with breast cancer BC risk, including
HCAR1 c.58C > G (p.P20A),HCAR2 c.424C > T (p.R142W),HCAR2 c.517_518delinsAC (p.G173T),HCAR2 c.1036A > G (p.M346V),HCAR2 c.1086_1090del (p.P363Nfs*26),HCAR3 c.560G > A (p.R187Q), andHCAR3 c.1117delC (p.Q373Kfs*82). Additionally,HCAR2 c.515C > T (p.S172L), a previously identified loss-of-function variant, was identified.Conclusions Due to the important role of HCARs in breast cancer, it is vital to understand how these genetic variants play a role in breast cancer risk and proliferation and their consequences on treatment strategies. Additional studies will be needed to validate these findings. Nevertheless, the identification of these potentially pathogenic variants supports the need to investigate their functional consequences.
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Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such “artificially expanded genetic information systems” are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, and catalysts; and to explore issues related to the early evolution of life. Further applications are limited by the inability to directly sequence DNA containing nonstandard nucleotides. Nanopore sequencing is well-suited for this purpose, as it does not require enzymatic synthesis, amplification, or nucleotide modification. Here, we take the first steps to realize nanopore sequencing of an 8-letter “hachimoji” expanded DNA alphabet by assessing its nanopore signal range using the MspA (Mycobacterium smegmatis porin A) nanopore. We find that hachimoji DNA exhibits a broader signal range in nanopore sequencing than standard DNA alone and that hachimoji single-base substitutions are distinguishable with high confidence. Because nanopore sequencing relies on a molecular motor to control the motion of DNA, we then assessed the compatibility of the Hel308 motor enzyme with nonstandard nucleotides by tracking the translocation of single Hel308 molecules along hachimoji DNA, monitoring the enzyme kinetics and premature enzyme dissociation from the DNA. We find that Hel308 is compatible with hachimoji DNA but dissociates more frequently when walking over C-glycoside nucleosides, compared to N-glycosides. C-glycocide nucleosides passing a particular site within Hel308 induce a higher likelihood of dissociation. This highlights the need to optimize nanopore sequencing motors to handle different glycosidic bonds. It may also inform designs of future alternative DNA systems that can be sequenced with existing motors and pores.more » « less
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Thermococcus kodakarensis ( T. kodakarensis ), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites. In this work, we demonsrate T. kodakarensis strains deleted for either or both R-M enzymes grow more slowly but display significantly increased competency compared to strains with intact R-M systems, suggesting that both TkoI and TkoII assist in maintenance of genomic integrity in vivo and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of T. kodakarensis strains containing both, one or neither R-M systems permitted assignment of the recognition sites for TkoI and TkoII and demonstrated that both R-M enzymes are TypeIIL; TkoI and TkoII methylate the N 6 position of adenine on one strand of the recognition sequences GTGA A G and TTCA A G, respectively. Further in vitro biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N) 20 /(N) 18 and TTCAAG(N) 10 /(N) 8 , respectively, away from the recognition sequences, while in vitro characterization of the MTase activities reveal transfer of tritiated S-adenosyl methionine by TkoI and TkoII to their respective recognition sites. Together these results demonstrate TkoI and TkoII restriction systems are important for protecting T. kodakarensis genome integrity from invading foreign DNA.more » « less
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Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in C•G to A•T “byproduct” introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle.more » « less
