By rearranging hydrogen bond donor and acceptor groups within a standard Watson-Crick geometry, DNA can add eight independently replicable nucleotides forming four additional not found in standard Terran DNA. For many applications, the orthogonal pairing of standard and nonstandard pairs offers a key advantage. However, other applications require standard and nonstandard nucleotides to communicate with each other. This is especially true when seeking to recruit high-throughput instruments (e.g., Illumina), designed to sequence standard 4-nucleotide DNA, to sequence DNA that includes added nucleotides. For this purpose, PCR workflows are needed to replace nonstandard nucleotides in (for example) a 6-letter DNA sequence by defined mixtures of standard nucleotides built from 4 nucleotides. High-throughput sequencing can then report the sequences of those mixtures to bioinformatic alignment tools, which infer the original 6-nucleotide sequence by analysis of the mixtures. Unfortunately, the intrinsic orthogonality of standard and nonstandard nucleotides often demand polymerases that violate pairing biophysics to do this replacement, leading to inefficiencies in this "transliteration" process. Thus, laboratory in vitro evolution (LIVE) using "anthropogenic evolvable genetic information systems" (AEGIS), an important "consumer" of new sequencing tools, has been slow to be democratized; robust sequencing is needed to identify the AegisBodies and AegisZymes that AEGIS-LIVE delivers. This work introduces a new way to connect synthetic and standard molecular biology: biversal nucleotides. In an example presented here, a pyrimidine analogue (pyridine-2-one, y) pairs with Watson-Crick geometry to both a nonstandard base (2-amino-8-imidazo-[1,2a]-1,3,5-triazin-[8H]-4-one, P, the Watson-Crick partner of 6-amino-5-nitro-[1H]-pyridin-2-one, Z) and a base that completes the Watson-Crick hydrogen bond pattern (2-amino-2 '-deoxyadenosine, amA). PCR amplification of GACTZP DNA with dyTP delivers products where Z:P pairs are cleanly transliterated to A:T pairs. In parallel, PCR of the same GACTZP sample at higher pH delivers products where Z:P pairs are cleanly transliterated to C:G pairs. By allowing robust sequencing of 6-letter GACTZP DNA, this workflow will help democratize AEGIS-LIVE. Further, other implementations of the biversal concept can enable communication across and between standard DNA and synthetic DNA more generally.

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.

With just four building blocks, low sequence information density, few functional groups, poor control over folding, and difficulties in forming compact folds, natural DNA and RNA have been disappointing platforms from which to evolve receptors, ligands, and catalysts. Accordingly, synthetic biology has created "artificially expanded genetic information systems" (AEGIS) to add nucleotides, functionality, and information density. With the expected improvements seen in AegisBodies and AegisZymes, the task for synthetic biologists shifts to developing for expanded DNA the same analytical tools available to natural DNA. Here we report one of these, an enzyme-assisted sequencing of expanded genetic alphabet (ESEGA) method to sequence six-letter AEGIS DNA. We show how ESEGA analyses this DNA at single base resolution, and applies it to optimized conditions for six-nucleotide PCR, assessing the fidelity of various DNA polymerases, and extending this to AEGIS components with functional groups. This supports the renewed exploitation of expanded DNA alphabets in biotechnology. The expansion of the genetic code with synthetic nucleotides has broadened our ability to evolve DNA as a functional material, but we lack analytical tools for the expanded alphabet. Here the authors demonstrate an enzyme-assisted method for the sequencing of six-letter DNA.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3β-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Adding nucleotide pairs to a DNA alphabet not only increases the linear information density of DNA molecules, but also may increase the diversity of folds available to the molecular system. Reported here is an example of this. A single additional nucleotide, with the 5-nitro-6-aminopyridone ("Z") heterocycle, selected from an artificially expanded genetic information system (AEGIS), was incorporated at twelve sites into a DNA strand 23 nucleotides in length. With a fluor at its 5'-end and a quencher at its 3'-end, this molecule is shown by fluorescence quenching to fold in mildly alkaline (pH 8-9) conditions. This folded structure is quite stable, melting at Tm = 66.5 ℃ at pH 8.5. Spectroscopic, gel, and 2-dimensional NMR (800 MHz) analyses show that the structure is held together by "reverse" Z-:Z base pairs, which have been previously seen in a crystal structure of the free heterocycle, 5-nitro-6-aminopyridone. This, in turn, requires that the strands joined by Z-:Z reverse pairs be oriented in parallel. This, in turn, requires the fold to have a four stranded compact "down-up-down-up" fold. The structure does not form when various Zs are replaced or scrambled. Of two possible structures, we also considered the possibility of "intercalated" Z-:Z base pairs, a fold that resemble the i-motif of standard DNA where an analogous "down-up-down-up" fold is held together by C:C+ pairing at lower pH between parallel strands. An alternative model has a "down-up-down-up" fold without intercalation.

Many efforts have sought to apply laboratory in vitro evolution (LIVE) to natural nucleic acid (NA) scaffolds to directly evolve functional molecules. However, synthetic biology can move beyond natural NA scaffolds to create molecular systems whose libraries are far richer reservoirs of functionality than natural NAs. For example, "artificially expanded genetic information systems" (AEGIS) add up to eight nucleotides to the four found in standard NA. Even in its simplest 6-letter versions, AEGIS adds functional groups, information density, and folding motifs that natural NA libraries lack. To complete this vision, however, tools are needed to sequence molecules that are created by AEGIS LIVE. Previous sequencing approaches, including approaches from our laboratories, exhibited limited performance and lost many sequences in diverse library mixtures. Here, we present a new approach that enzymatically transforms the target AEGIS DNA. With higher transliteration efficiency and fidelity, this Enzyme-Assisted Sequencing of Expanded Genetic Alphabet (ESEGA) approach produces substantially better sequences of 6-letter (AGCTZP) DNA than previous transliteration approaches. Therefore, ESEGA facilitates precise analysis of libraries, allowing 'next-generation deep sequencing' to accurately quantify the sequences of 6-letter DNA molecules at single base resolution. We then applied ESEGA to three tasks: (a) defining optimal conditions to perform 6-nucleotide PCR (b) evaluating the fidelity of 6-nucleotide PCR with various DNA polymerases, and (c) extending that evaluation to AEGIS components functionalized with alkynyl and aromatic groups. No other approach at present has this scope, allowing this work to be the next step towards exploiting the potential of expanded DNA alphabets in biotechnology.

Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological systems. Here, we show how six-letter GACTZP DNA contributes this property in two parts of a nanoassembly: 1) in an aptamer evolved from a six-letter DNA library to selectively bind liver cancer cells; and 2) in a six-letter self-assembling GACTZP nanotrain that carries the drug doxorubicin. The aptamer-nanotrain assembly, charged with doxorubicin, selectively kills liver cancer cells in culture, as the selectivity of the aptamer binding directs doxorubicin into the aptamer-targeted cells. The assembly does not kill untransformed cells that the aptamer does not bind. This architecture, built with an expanded genetic alphabet, is reminiscent of antibodies conjugated to drugs, which presumably act by this mechanism as well, but with the antibody replaced by an aptamer.

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how "self-avoiding molecular recognition systems" (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer-primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

Investigation of metal-organic frameworks (MOFs) for biomedical applications has attracted much attention in recent years. MOFs are regarded as a promising class of nanocarriers for drug delivery owing to well-defined structure, ultrahigh surface area and porosity, tunable pore size, and easy chemical functionalization. In this review, the unique properties of MOFs and their advantages as nanocarriers for drug delivery in biomedical applications were discussed in the first section. Then, state-of-the-art strategies to functionalize MOFs with therapeutic agents were summarized, including surface adsorption, pore encapsulation, covalent binding, and functional molecules as building blocks. In the third section, the most recent biological applications of MOFs for intracellular delivery of drugs, proteins, and nucleic acids, especially aptamers, were presented. Finally, challenges and prospects were comprehensively discussed to provide context for future development of MOFs as efficient drug delivery systems.

Here we are reporting, for the first time, a ligand-guided selection (LIGS) experiment using an artificially expanded genetic information system (AEGIS) to successfully identify an AEGIS-DNA aptamer against T cell receptor-CD3 epsilon expressed on Jurkat.E6 cells. Thus, we have effectively combined the enhanced diversity of an AEGIS DNA library with LIGS to develop a superior screening platform to discover superior aptamers. Libraries of DNA molecules from highly diversified building blocks will provide better ligands due to more functional diversity and better-controlled folding. Thus, a DNA library with AEGIS components (dZ and dP) was used in LIGS experiments against TCR-CD3 epsilon in its native state using two clinically relevant monoclonal antibodies to identify an aptamer termed JZPO-10, with nanomolar affinity. Multiple specificity assays using knockout cells, and competition experiments using monoclonal antibodies utilized in LIGS, show unprecedented specificity of JZPO-10, suggesting that the combination of LIGS with AEGIS-DNA libraries will provide a superior screening platform to discover artificial ligands against critical cellular targets.

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms. One advantage of H over the widely used inosine 'universal base' and 'mixed sequence' probes is seen in ligation-based assays to detect SNPs. The need to detect medically relevant SNPs within ambiguous sequences is especially important when probing RNA viruses, which rapidly mutate to create drug resistance, but also suffer neutral drift, the second obstructing simple methods to detect the first. Thus, H is being developed to detect variants of viruses that are rapidly mutating.

Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives.

One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK), which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) to the phosphorylation of an UBP (a pyrimidine analogue (6-amino-5-nitro-3-(1’-b-d-2’-deoxyribofuranosyl)-2(1H)-pyridone, Z) and its complementary purine analogue (2-amino-8-(1’-b-d-2’-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, P). The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo.

Noroviruses are the major cause of global viral gastroenteritis with short incubation times and small inoculums required for infection. This creates a need for a rapid molecular test for norovirus for early diagnosis, in the hope of preventing the spread of the disease. Non-chemists generally use off-the shelf reagents and natural DNA to create such tests, suffering from background noise that comes from adventitious DNA and RNA (collectively xNA) that is abundant in real biological samples, especially feces, a common location for norovirus. Here, we create an assay that combines artificially expanded genetic information systems (AEGIS, which adds nucleotides to the four in standard xNA, pairing orthogonally to A:T and G:C) with loop-mediated isothermal amplification (LAMP) to amplify norovirus RNA at constant temperatures, without the power or instrument requirements of PCR cycling. This assay was then validated using feces contaminated with murine norovirus (MNV). Treating stool samples with ammonia extracts the MNV RNA, which is then amplified in an AEGIS-RT-LAMP where AEGIS segments are incorporated both into an internal LAMP primer and into a molecular beacon stem, the second lowering background signaling noise. This is coupled with RNase H nicking during sample amplification, allowing detection of as few as 10 copies of noroviral RNA in a stool sample, generating a fluorescent signal visible to human eye, all in a closed reaction vessel.

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. In this work, we used cell engineering to place glypican 3 (GPC3), a possible marker for liver cancer theranostics, on the surface of a liver cell line. Libraries were then built from a six-letter genetic alphabet containing the standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a][1,3,5]triazin-4-one and 6-amino-5-nitropyridin-2-one), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against non-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3-overexpressing cells. This selection-counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low‐resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off‐target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase‐dependent isothermal amplification (SAMRS‐HDA). We also show that incorporating SAMRS into the 3′‐ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS‐HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS‐HDA is a more versatile approach than standard HDA, with a broader applicability for xNA‐targeted diagnostics and research.

As one of its goals, synthetic biology seeks to increase the number of building blocks in nucleic acids. While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present a crystal structure for an RNA riboswitch where a stem C:G pair has been replaced by a pair between two components of an artificially expanded genetic-information system (AEGIS), Z and P, (6-amino-5-nitro-2(1H)-pyridone and 2-amino-imidazo[1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNA molecule nor the details of its interaction with a hypoxanthine ligand. This was confirmed in solution by in-line probing, which also measured a 3.7 nM affinity of the riboswitch for guanine. These data show that the Z:P pair mimics the natural Watson-Crick geometry in RNA in the first example of a crystal structure of an RNA molecule that contains an orthogonal added nucleobase pair.

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists Succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not :found in natural DNA) but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribo-furanosyl)-2-(1H)-Pyridone and 2-amino-8- (1'-beta-D -2'-deoxyrib ofuranosyl)-imidazo [1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These Were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a "single tube" setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a "self-avoiding molecular recognition system" (SAMRS), which enables high levels of multiplexing. The second is an "artificially expanded genetic information system" (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex "liquid microarrays" are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6-10 dengue virions can be detected in a single mosquito. (C) 2015 Elsevier B.V. All rights reserved.