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1. Synthetic Biology Pathway to Nucleoside Triphosphates for Expanded Genetic Alphabets

   https://doi.org/10.1021/acssynbio.3c00060  

   Li, Yubing; Abraham, Clay; Suslov, Oleg; Yaren, Ozlem; Shaw, Ryan W; Kim, Myong-Jung; Wan, Shuo; Marliere, Philippe; Benner, Steven A (June 2023, ACS Synthetic Biology)

   : One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson−Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution in vitro. To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that “polyphosphate kinases” can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway in vitro makes AEGIS triphosphates, including third-generation triphosphates having improved ability to survive in living bacterial cells. In α32P-labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third-generation AEGIS triphosphates perform better with natural enzymes than second-generation AEGIS triphosphates. 

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2. Recent Advances in Biomolecular Detection Based on Aptamers and Nanoparticles

   https://doi.org/10.3390/bios13040474  

   Xu, Ruiting; Ouyang, Leixin; Chen, Heyi; Zhang, Ge; Zhe, Jiang (April 2023, Biosensors)

   The fast, accurate detection of biomolecules, ranging from nucleic acids and small molecules to proteins and cellular secretions, plays an essential role in various biomedical applications. These include disease diagnostics and prognostics, environmental monitoring, public health, and food safety. Aptamer recognition (DNA or RNA) has gained extensive attention for biomolecular detection due to its high selectivity, affinity, reproducibility, and robustness. Concurrently, biosensing with nanoparticles has been widely used for its high carrier capacity, stability and feasibility of incorporating optical and catalytic activity, and enhanced diffusivity. Biosensors based on aptamers and nanoparticles utilize the combination of their advantages and have become a promising technology for detecting of a wide variety of biomolecules with high sensitivity, reliability, specificity, and detection speed. Via various sensing mechanisms, target biomolecules have been quantified in terms of optical (e.g., colorimetric and fluorometric), magnetic, and electrical signals. In this review, we summarize the recent advances in and compare different aptamer–nanoparticle-based biosensors by nanoparticle types and detection mechanisms. We also share our views on the highlights and challenges of the different nanoparticle-aptamer-based biosensors. 

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3. A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase

   https://doi.org/10.1038/s41467-023-43735-9  

   Oh, Juntaek; Shan, Zelin; Hoshika, Shuichi; Xu, Jun; Chong, Jenny; Benner, Steven A.; Lyumkis, Dmitry; Wang, Dong (December 2023, Nature Communications)

   Abstract Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show thatE. coliRNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition. 

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4. In Vitro Selection of an ATP-Binding TNA Aptamer

   https://doi.org/10.3390/molecules25184194  

   Zhang, Li; Chaput, John C. (September 2020, Molecules)

   Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability. 

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5. Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

   https://doi.org/10.1093/nar/gkaa849  

   Alkhamis, Obtin; Yang, Weijuan; Farhana, Rifat; Yu, Haixiang; Xiao, Yi (October 2020, Nucleic Acids Research)

   Abstract In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion. 

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