DNA nanotechnology has created nanostructures with astonishing complexity. However, with nanostructures becoming increasingly larger and more intricate, they have become more difficult to obtain in high yields and quality. Expanding the alphabet beyond the canonical base pairs can therefore be the key to push the technology to the next level. Here, we describe examples of DNA nanostructures built from an “anthropogenic evolvable genetic information system (AEGIS).” Because AEGIS uses the same backbone as DNA, the existing rules for designing DNA nanostructures can be readily applied to AEGIS nanostructures, which also show greater stability, both thermal and enzymatic, greater control over autonomous assembly, and good phase separation. AEGIS can have as many as 12 different units and six different pairs with Watson-Crick-Franklin geometry. Thus, if further developed, then these nanostructures may represent a previously unexplored frontier in DNA nanoscience and nanotechnology, expanding the space of “soft” biomaterial design.

Thermoanaerobacter uzonensis strain AK85 belongs to the Thermoanaerobacter genus, which comprises rod-shaped, Gram-positive, thermophilic, obligate anaerobic bacteria. Members of this genus exhibit unique fermentation qualities, such as prolific ethanol production, and can generate longer-chain alcohols from carbohydrate and amino acid sources. Here we present the draft genome sequence of Thermoanaerobacter uzonensis strain AK85, which was previously isolated from a hot spring in Graensdalur in Southwestern Iceland. The genome was sequenced with a 150 bp paired-end library on a MGISEQ-2000. The assembled genome comprises 2,577,794 bp and a GC ratio of 33.69 %. With an ANI of 96.9 % strain AK85 was determined to be a strain of Thermoanaerobacter uzonensis. Annotation was conducted with Prokka which revealed 41 enzymes related to carbohydrate, amino acid, and carboxylic acid metabolism. The genomic dataset establishes the biotechnological capacity and potential of strain AK85 for the production of alcohols and other bio-manufactured products. Further, the genomic dataset is coupled with a cofactor and substrate analysis of the three detected alcohol dehydrogenases. These enzymes were assessed via a lysate based colorimetric assay with NAD⁺ and NADP⁺. Under these conditions the native alcohol dehydrogenases are able to oxidize long chain primary alcohols such as 1-octanol and benzyl alcohol. The reads and assembled draft genome of AK85 were deposited into SRA and NCBI under Bioproject PRJNA1108289, Genbank JBDHNK000000000, and Biosample SAMN41233939.

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.

Models for prebiotic syntheses often have many steps, each separately validated by laboratory experiments. The challenge then asks whether these steps work together in natural geological environments, absent human intervention. Here, we analyze a six-step Discontinuous Synthesis Model (DSM) for the prebiotic formation of RNA, proposed to be the first informational molecule to support Darwinian evolution, and life, on Earth and/or Mars. DSM requires that borate in multiple steps guide the formation of pentoses from simple carbohydrates and control phosphorylation, in all cases by binding adjacent HO-groups on key intermediates. However, adjacent HO-groups must react in two other steps, which borate might inhibit. Experiments here show that borate does not inhibit these two other steps, but rather facilitates them. This makes the six-step DSM a “no human intervention” route from simple
precursors (1 to 3 carbons, 0 to 2 nitrogens) to oligomeric RNA with predominately 3’,5’-linkages at least 6 nucleotides long, but possibly much longer. The process i) exploits privileged chemistry in ii) intermittently irrigated aquifers constrained by basalt that iii) have borate iv) above a redox-neutral mantle v) having access to an atmosphere transiently reduced by a Vesta-sized impactor. In a possible coincidence, such an impact occurred most likely ca. 4.3 billion years ago (Ga), ~100 Mya before some molecular clocks date the divergence of the three kingdoms of life on Earth (4.2 Ga), and ca. 200 Mya before isotopically “light” carbon is reported in zircons dated at 4.1 Ga. This carbon may be the oldest trace of life ever proposed.

Recombinant adeno-associated viruses (AAVs) with precise genome editing and cell-virus interaction have become a promising delivery tool for gene therapy. A robust AAV purification process is crucial for ensuring therapeutic efficacy. The challenges of AAV purification process development encompass limited material availability during early-stage development, high cost-of-goods compared to traditional biologics, and short development timelines for the critical first-in-human stages. The key to overcoming these challenges is to leverage high throughput (HTP) methods. In this article, an integrated end-to-end HTP workflow is proposed, utilizing a resin tip as the purification module and incorporating an HTP analytical toolkit on one platform. Purification parameters, including binding capacity, resin selection, and buffer composition screening for AAV full/partial/empty capsids separation, are efficiently determined using a 25 μL resin tip and HTP analytical tools with only micro-volume sample requirements. The process parameters determined from the HTP workflow predict the trends of full capsid enrichment and partial capsid removal for the bench-scale purification. This HTP workflow is also applied for the assessment of the AAV quality attributes to accelerate early-stage cell line and cell culture development. Comparable AAV quality attributes are demonstrated to Robocolumn as the benchmark HTP purification method. By leveraging HTP analytical tools to instantly interpret the purification data, this integrated HTP workflow effectively accelerates AAV purification process development, with a 2% material volume requirement compared to the benchmark method, 96-well format screening, short turnaround time for analytical assays, and significant cost-of-goods savings for downstream process development.

ALternative Isoinformational ENgineered” (ALIEN) DNA is a biomimetic polymer composed of four entirely anthropogenic nucleotides. These alternative nucleosides form base pairs orthogonal to canonical bases and fold into the familiar B-form DNA double-helix, endowing ALIEN DNA with valuable biotechnological applications. The ability to sequence ALIEN DNA is essential for its continued development. However traditional sequencing approaches rely on chemical recognition of ACGT-DNA and cannot be easily adapted to ALIEN DNA. Here we demonstrate de novo nanopore sequencing of DNA comprised entirely of the four anthropogenic DNA bases. We show direct, label-free, single-molecule sequencing of such nucleic acids without the requirements of fluorescent labels, transliteration, amplification, or enzymatic synthesis. This paves the way for routine, accessible, and high-accuracy sequencing of DNA beyond A, C, G, and T.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.