FfAME:org :: Zunyi Yang's Publications

Zunyi Yang's Publications

Joining Natural and Synthetic DNA Using Biversal Nucleotides: Efficient Sequencing of Six-Nucleotide DNA
Bang Wang, Hyo-Joong Kim, Kevin M. Bradley, Cen Chen, Chris McLendon, Zunyi Yang, Steven A. Benner
J. Am. Chem. Soc.146(51) 35129-35138 (2024) doi: 10.1021/jacs.4c11043
<Abstract>

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.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers
Bang Wang, Xiaoshu Pan, I-Ting Teng, Xiaowei Li, Firas Kobeissy, Zo-Yu Wu, Jiepei Zhu, Guangzheng Cai, He Yan, Xin Yan, Mingwei Liang, Fahong Yu, Zunyi Yang, Elisa Biondi, William Haskins, Y. Charles Cao, Steven A. Benner, Weihong Tan, Kevin Wang
Angew. Chem. Int. Ed. (2024) e202402007, https://doi.org/10.1002/anie.202402007
<Abstract>

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.

Enzyme-assisted high throughput sequencing of an expanded genetic alphabet at single base resolution
Bang Wang, Kevin M. Bradley, Myong-Jung Kim, Roberto Laos, Cen Chen, Dietlind L. Gerloff, Luran Manfio, Zunyi Yang & Steven A. Benner
Nat. Commun.15(4057), Nature (2024) https://doi.org/10.1038/s41467-024-48408-9
<Abstract>

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.

A folding motif formed with an expanded genetic alphabet
Bang Wang, James R. Rocca, Shuichi Hoshika, Cen Chen, Zunyi Yang, Reza Esmaeeli, Jianguo Wang, Xiaoshu Pan, Jianrong Lu, Kevin K. Wang, Y. Charles Cao, Weihong Tan & Steven A. Benner
Nat. Chem., Nature (2024) https://doi.org/10.1038/s41557-024-01552-7
<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.

Ultra-rapid detection of SARS-CoV-2 in public workspace environments
Yaren, O., McCarter, J., Phadke, N., Bradley, K. M., Overton, B., Yang, Z., Ranade, S., Patil, K., Bangale, R., Benner, S. A.
PLOS One, Public Library of Science (2021) 10.1371/journal.pone.0240524, DOI:10.1101/2020.09.29.20204131
<Abstract>

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 10⁵ virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture.

An Aptamer-Nanotrain Assembled from Six-Letter DNA Delivers Doxorubicin Selectively to Liver Cancer Cells.
Zhang, L., Wang, S., Yang, Z., Hoshika, S., Xie, S., Li, J., Chen, X., Wan, S., Li, L., Benner, S.A., Tan, W.
Angew. Chem. Int. Ed. (2020) 59(2): 663-668, DOI:10.1002/anie.201909691
<Abstract>

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.

Ligand Guided Selection (LIGS) with Artificially Expanded Genetic Information Systems against TCR-CD3ε
Zumrut, H., Yang, Z., Williams, N., Arizala, J., Batool, S., Benner, S.A., Mallikaratchy, P.
Biochemistry, ACS (2020) 59(4):552-562, DOI:10.1021/acs.biochem.9b00919
<Abstract>

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-CD3ε 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-CD3ε 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.

Eliminating Primer Dimers and Improving SNP detection using Self-Avoiding Molecular Recognition Systems (SAMRS)
Yang, Z., Le, J.T., Hutter, D., Bradley, K.M., Overton, B.R., McLendon, C., Benner, S.A.
Biol. Methods Protoc., Oxford Academics (2020) 5(1):bpaa004, DOI:10.1093/biomethods/bpaa004
<Abstract>

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.

Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.
Yang, Z., Kim, H.-J., Le, J., McLendon, C., Bradley, K.M., Kim, M.-S., Hutter, D., Hoshika, S., Yaren, O., Benner, S.A.
Nucl. Acids Res. (2018) 46(12): 5902-10,DOI:10.1093/nar/gky392
<Abstract>

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.

Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant.
Chen, F., Zhang, Y., Daugherty, A.B., Yang, Z.Y., Shaw, R., Dong, M.X., Lutz, S. and Benner, S.A.
PLOS One, Public Library of Science (2017) 12(3): e0174163, DOI:10.1371/journal.pone.0174163
<Abstract>

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.

Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya
Yaren, O., Alto, B.W., Gangodkar, P.V., Ranade, S.R., Patil, K.N., Bradley, K.M., Yang, Z., Phadke, N., Benner, S.A
BMC Infect. Dis., BioMed Central Ltd. (2017) 17(1):293, DOI:10.1186/s12879-017-2382-0
<Abstract>

Background: 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.

Methods: Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration.

Results: The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India.

Conclusions: A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.

Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution
Zhang, L, Yang, Z, Trinh, TL, Teng, I-T, Wang, S, Bradley, KM, Hoshika, S, Wu, Q, Cansiz, S, Rowold, DJ, McLendon, C, Kim, M-S, Wu, Y, Cui, C, Liu, Y, Hou, W, Stewart, K, Wan, S, Liu, C, Benner, SA, Tan, W
Angew. Chem. Int. Ed.55 (2016) doi: 10.1002/anie.201605058
<Abstract>

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 sixletter 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 selectionâ€“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.

A norovirus detection architecture based on isothermal amplification and expanded genetic systems
Ozlem Yaren, Kevin M. Bradley, Patricia Moussatche, Shuichi Hoshika, Zunyi Yang,Shu Zhu, Stephanie M. Karst, Steven A. Benner
J Virol Methods(237), Elsevier 64-71 (2016) doi: 10.1016/j.jviromet.2016.08.012
<Abstract>

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.

Evolution of functional six-nucleotide DNA
Zhang, L., Yang, Z., Sefah, K., Bradley, K. M., Hoshika, S., Kim, M-J,. Kim, H-J., Zhu., Jimenez, E., Cansiz, S., Teng, I-T., Champanhac, C, McLendon, C., Liu, C., Zhang, W., Gerloff, D. L., Huang, Z., Tan, W., Benner, S. A.
J. Am. Chem. Soc. (2015) DOI: 10.1021/jacs.5b02251
<Abstract>

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 additional 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'-B-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo- [1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via strict Watson?Crick geometry. These were added to 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 systems, 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.

Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA
Lyudmyla G. Glushakova, Nidhi Sharma, Shuichi Hoshika, Andrea C. Bradley, Kevin M. Bradley, Zunyi Yang, Steven A. Benner
Anal Biochem, Elsevier (2015) Nov 15;489:62-72. doi: 10.1016/j.ab.2015.08.015
<Abstract>

Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-ml sample.

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology
Lyudmyla G. Glushakova, Andrea Bradley, Kevin M. Bradley, Barry W. Alto, Shuichi Hoshika, Daniel Hutter, Nidhi Sharma, Zunyi Yang, Myong-Jung Kim, Steven A. Benner
J Virol Methods214, Elsevier 60-74 (2015) doi: 10.1016/j.jviromet.2015.01.003
<Abstract>

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.

A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair
Armando R. Hernandez, Yaming Shao, Shuichi Hoshika, Zunyi Yang, Sandip A. Shelke, Julien Herrou, Hyo-Joong Kim, Myong-Jung Kim, Joseph A. Piccirilli, and Steven A. Benner
Angew. Chem. Int. Ed.54 9853-9856 (2015) doi: 10.1002/anie.201504731
<Abstract>

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-aminoimidazo[ 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 RNAmolecule 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.

Helicase Dependent Isothermal Amplification of DNA and RNA using Self-Avoiding Molecular Recognition Systems
Zunyi Yang, Chris McLendon, Daniel Hutter, Kevin M. Bradley, Shuichi Hoshika, Carole Frye, and Steven A. Benner
ChemBioChem (2015) June 15; 16(9): 1365-1370. doi:10.1002/cbic.201500135.
<Abstract>

Assays that target 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, requiring equipment and power that may not be available in these environments. However, isothermal procedures that 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 to give 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, something difficult to achieve with HDA using standard primers. This shows that SAMRS-HDA is a more versatile approach than standard HDA with a broader applicability for xNA-targeted diagnostics and research.

Conversion strategy using an expanded genetic alphabet to assay nucleic acids
Yang, Z., Durante, M., Glushakova, L., Sharma, N., Leal, N., Bradley, K., Chen, F., Benner, S. A.
Anal. Chem. (2013) 85(9):4705-12
<Abstract>

Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.

Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity.
Chen, F; Yang, ZY; Yan, M; Alvarado, JB; Wang, G; Benner, SA
Nucl. Acids Res.39(9) 3949-3961 (2011)
<Abstract>

To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1'-?-d-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-?-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor-donor-acceptor and acceptor-acceptor-donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells.

Synthetic Biology, Tinkering Biology, and Artificial Biology: A Perspective from Chemistry
Benner, SA; Chen, F; Yang, ZY
Chemical Synthetic Biology, ed. Pier Luigi Luisi and Cristiano Chiarabelli, Wiley 69-106 (2011)

Setting the Stage: The History, Chemistry, and Geobiology behind RNA
Benner, SA; Kim, HJ; Yang, ZY
RNA Worlds: From Life's Origins to Diversity in Gene Regulation, ed. John F. Atkins, Raymond F. Gesteland, Thomas R. Cech, Cold Spring Harbor Laboratory Press 7-19 (2011)

Amplification, Mutation, and Sequencing of a Six-Letter Synthetic Genetic System
Yang, Z; Chen, F; Alvarado, JB; Benner, SA
J. Am. Chem. Soc.133(38) 15105-15112 (2011) dx.doi.org/10.1021/ja204910n
<Abstract>

The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a sixletter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.

Expanded Genetic Alphabets in the Polymerase Chain Reaction
Yang, ZY; Chen, F; Chamberlin, SG; Benner, SA
Angew. Chem. Int. Ed.49(1) 177-180 (2010)

Design of a novel molecular beacon: modification of the stem with artificially genetic alphabet
Sheng, PP; Yang, ZY; Kim, YM; Wu, YR; Tan, WH; Benner, SA
Chem. Comm.(41), Royal Society of Chemistry 5128-5130 (2008)
<Abstract>

A molecular beacon that incorporates components of an artificially expanded genetic information system (AEGIS) in its stem is shown not to be opened by unwanted stem invasion by adventitious standard DNA; this should improve the "darkness" of the beacon in real-world applications.

Synthetic Biology for Improved Personalized Medicine
Benner, SA; Hoshika, S; Sukeda, M; Hutter, D; Leal, NA; Yang, ZY; Chen, F
Nucleic Acids Symp. Ser.52(1) 243-244 (2008) doi: 10.1093/nass/nrn123
<Abstract>

Tools to re-sequence the genomes of individual patients having well described medical histories is the first step required to connect genetic information to diagnosis, prognosis, and treatment. There is little doubt that in the future, genomics will influence the choice of therapies for individual patients based on their specific genetic inheritance, as well as the genetic defects that led to disease. Cost is the principle obstacle preventing the realization of this vision. Unless the interesting parts of a patient genome can be resequenced for less than $10,000 (as opposed to $100,000 or more), it will be difficult to start the discovery process that will enable this vision. While instrumentation and biology are important to reducing costs, the key element to cost-effective personalized genomic sequencing will be new chemical reagents that deliver capabilities that are not available from standard DNA. Scientists at the Foundation for Applied Molecular Evolution and the Westheimer Institute have developed several of these, which will be the topic of this talk.

Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Yang, ZY; Sismour, AM; Benner, SA
Nucl. Acids Res.35(9) 3118-3127 (2007)
<Abstract>

The use of DNA polymerases to incorporate phosphorothioate linkages into DNA, and the use of exonuclease III to determine where those linkages have been incorporated, are re- examined in this work. The results presented here show that exonuclease III degrades single- stranded DNA as a substrate and digests through phosphorothioate linkages having one absolute stereochemistry, assigned ( assuming inversion in the polymerase reaction) as S, but not the other absolute stereochemistry. This contrasts with a general view in the literature that exonuclease III favors double-stranded nucleic acid as a substrate and stops completely at phosphorothioate linkages. Furthermore, not all DNA polymerases appear to accept exclusively the ( R) stereoisomer of nucleoside alpha- thiotriphosphates [ and not the ( S) diastereomer], a conclusion inferred two decades ago by examination of five Family- A polymerases and a reverse transcriptase. This suggests that caution is appropriate when extrapolating the detailed behavior of one polymerase from the behaviors of other polymerases. Furthermore, these results provide constraints on how exonuclease III - thiotriphosphate - polymerase combinations can be used to analyze the behavior of the components of a synthetic biology.

Enzymatic incorporation of a third nucleobase pair
Yang, ZY; Sismour, AM; Sheng, PP; Puskar, NL; Benner, SA
Nucl. Acids Res.35(13) 4238-4249 (2007)
<Abstract>

DNA polymerases are identified that copy a nonstandard nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro3-(l'-p-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-standard 'small' donordonor-acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1-beta-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4 (8H)-one [dP) implements the 'large' acceptor-acceptor-donor (puAAD) pattern. These nucleobases were designed to present electron density to the minor groove, density hypothesized to help determine specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo(-)) and Deep Vent (exo-) polymerases, with 94.4%, 97.5% and 97.5%, respectively, retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson-Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, molecular-, systems- and synthetic-biology.

Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern
Yang, ZY; Hutter, D; Sheng, PP; Sismour, AM; Benner, SA
Nucl. Acids Res.34(21) 6095-6101 (2006)
<Abstract>

To support efforts to develop a 'synthetic biology' based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin -4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where 'py' indicates a pyrimidine analog and 'pu' indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

One-pot glycosylation (OPG) for the chemical synthesis of oligosaccharides
Yu, B; Yang, ZY; Cao, HZ
Curr. Org. Chem.9(2) 179-194 (2005)
<Abstract>

This review provides a comprehensive survey of the "one pot glycosylation" (OPG) strategy for the chemical synthesis of oligosaccharides, covering literatures from the first example reported by Kahne and Raghavan in 1993 through May 2003. The essence of the OPG is to distinguish the reactivity difference of a pair of the glycosylation donors or acceptors so as to carry out two glycosylation steps sequentially without purification of the first-step coupling product. Accordingly, the literature reports are grouped based on the major stereoelectronic factors causing the reactivity differences, those include the "armed-disarmed effect", "orthogonality of leaving groups", "distinguishable acceptors", and "the hybrid". "The hybrid" OPG procedure takes advantage of a combination of the reactivity disparity of a set of the armed-disarmed donors, orthogonal leaving groups, as well as acceptors so as to proceed three or more steps of glycosylation sequentially in one pot. Relevant conception and exploitation of the reactivity differences of the donors and acceptors in the synthesis of oligosaccharides, which finally evolve the OPG or advance parallelly, are briefly described at the beginning.

1 -> 2 Migration and concurrent glycosidation of phenyl 1-thio-alpha-mannopyranosides via 2,3-O-cyclic dioxonium intermediates
Yang, ZY; Cao, HZ; Hu, J; Shan, RL; Yu, B
Tetrahedron59(2) 249-254 (2003)
<Abstract>

Treatment of phenyl 2,3-O-cyclic ketene acetal- and 2,3-O-thionocarbonyl-1-thio-mannopyranosides with TMSOTf and MeOTf, respectively, gave the corresponding 2,3-O-cyclic dioxonium intermediates, which proceeded via 1-->2 migration and concurrent glycosidation in the presence of alcohols to provide the corresponding 2-S-phenyl glycosides stereoselectively. While the former donors were too labile, the latter donors have proved superior for the present purpose. The X-ray crystallographic structures of phenyl 4-O-methyl-2,3-O-thiocarbonyl-1-thio-alpha-L-rhamnopyranoside (1), a typical donor for the present reaction, and its anomeric azide analogue (6), which could not undergo the present reaction under similar conditions, are provided. (C) 2002 Elsevier Science Ltd. All rights reserved.

Stereoselective synthesis of 2-S-ethyl(phenyl)-2-thio-beta-glucopyranosides via 1,2-migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha-mannopyranosides
Yang, ZY; Yu, B
Carbohydr. Res.333(2) 105-114 (2001)
<Abstract>

1,2-Migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha -D- and L-mannopyranosides under the action of TMSOTf readily afforded the corresponding 2-S-ethyl(phenyl)-2-thio-beta -glucopyranosides ready precursors to 2-deoxy-arabino-hexopyranosides (2-deoxy-beta -glucopyranosides). (C) 2001 Elsevier Science Ltd. All rights reserved.

Stereoselective synthesis of 2-S-phenyl-2-deoxy-beta-glycosides using phenyl 2,3-O-thionocarbonyl-1-thioglycoside donors via 1,2-migration and concurrent glycosidation
Yu, B; Yang, ZY
Org. Lett.3(3) 377-379 (2001)
<Abstract>

[GRAPHICS] 1,2-Migration and concurrent glycosidation of phenyl 2,3-O-thionocarbonyl 1-thio-alpha -L-rhamnopyranosides under the action of methyl trifluoromethanesulfonate (MeOTf) afforded in high yields the 3-O-(methylthio)carbonyl-2-S-phenyl-2,6-dideoxy-beta -L-glucopyranosides ready precursors to the corresponding 2-deoxy-beta -glycosides.

Rearrangement of sugar 1,2-orthoesters to glycosidic products: a mechanistic implication
Yang, ZY; Lin, WB; Yu, B
Carbohydr. Res.329(4) 879-884 (2000)
<Abstract>

A novel and expeditious approach to the stereoselective synthesis of 2-S-ethyl(phenyl)-2-deoxy-beta-glycosides, ready precursors to 2-deoxy-beta-glycosides
Yu, B; Yang, ZY
Tet. Lett.41(16) 2961-2964 (2000)
<Abstract>

1,2-Migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha-1-rhamnopyranosides under the action of TMSOTf readily afforded the corresponding 2-S-ethyl(phenyl)-2-deoxy-beta-glycosides, ready precursors to 2-deoxy-beta-glycosides. (C) 2000 Elsevier Science Ltd. All rights reserved.