- BS in Chemistry. Northwest University, China (1997)
- PhD in Chemistry. Shanghai Institute of Organic Chemistry (2002)
- Postdoctoral Research Associate. University of Florida (2005)
My research focuses on the development of an efficient and accurate method for the detection of multiple nucleic acids in biological sample. This innovative technology is the multiplexed amplification of DNA and RNA and the orthogonal capture of oligonucleotides tagged with non-standard nucleobases in such a fashion where close mismatches do not compete. My research includes: 1) Synthesis of two components of a non-standard nucleobase pair, dZ:dP, along with a demonstration that the stability of the dZ:dP base pair is stronger than G:C base pair. Furthermore, a demonstration to show the ability of each base to effectively discriminate against mismatches in short duplex DNA. 2) PCR amplification of DNA containing dZ:dP base pair with sufficient fidelity and development of the methodology for the measurement of enzyme fidelity. 3) Synthesis of a novel molecular beacon containing dZ and dP to detect the level of the expression of viral genes in cancer cell. 4) Development of the technology of multiplexed PCR and microarray of dZ and dP, by combining the enzymology and chemistry of non-standard nucleobases. The goal of my research is to reduce the cost of personalized DNA sequencing, revolutionize the diagnosis, management, and treatment of human disease.
Aptamers against Cells Overexpressing Glypican 3 from Expanded
Genetic Systems Combined with Cell Engineering and Laboratory
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
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
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
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-
[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
Lyudmyla G. Glushakova, Nidhi Sharma, Shuichi Hoshika, Andrea C. Bradley, Kevin M. Bradley, Zunyi Yang, Steven A. Benner
, Elsevier (2015) Nov 15;489:62-72. doi: 10.1016/j.ab.2015.08.015
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 Methods
214 , Elsevier 60-74 (2015) doi: 10.1016/j.jviromet.2015.01.003
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
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
(2015) June 15; 16(9): 1365-1370. doi:10.1002/cbic.201500135.
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.
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)
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.
(View publication page for Zunyi Yang)
- Organic Chemistry
- Medicinal Chemistry
- Gene Therapy