- MS in Chemistry. Swiss Federal Institute of Technology (ETH), Zurich, Switzerland (1994)
- PhD in Chemistry. ETH, Zurich, Switzerland (2001)
Synthetic biology. I synthesized DNA oligonucleotides containing modified backbone linkers and studied their duplex conformation and stability. The results obtained have significant implications for gene therapy as well as for the search for life beyond Earth. I also synthesized nucleosides exhibiting hydrogen bonding patterns different from those found in natural DNA, giving rise to an artificially expanded genetic information system with more than just two base pairs. The vast variety of possible applications includes many medical diagnostic tools inaccessible through natural DNA alone.
Personalized medicine. Genetically based side effects from drugs could be managed if it would be possible to sequence the entire genome of an individual patient in a short time (a few days at most) and at low cost (a few thousand dollars). This would vastly increase the range of available drugs. Current methods for sequencing DNA are not able to match these goals. I am synthesizing modified nucleoside triphosphates for highly parallel sequencing technologies on micro arrays that would slash price and time of current methods by several orders of magnitude. In a related project I am developing an improved synthetic method for the fast, clean and selective transformation of nucleosides into their respective triphosphates.
Dynamic combinatorial synthesis. This concept should greatly increase the speed for finding leads in drug discovery. Instead of a scientist designing a drug to a particular enzymatic target, the enzyme itself synthesizes its preferred ligand from two combinatorial libraries of small molecules that are in dynamic equilibrium with each other. These libraries have to exhibit several specific properties, however, that render the successful application of this concept non-trivial. I am currently synthesizing different libraries that are promising candidates.
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.
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.
Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS)
Nidhi Sharma, Shuichi Hoshika, Daniel Hutter, Kevin M. Bradley, and Steven A. Benner
(2014) DOI: 10.1002/cbic.201402250
Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.
Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups
Hutter, D; Kim, MJ; Karalkar, N; Leal, NA; Chen, F; Guggenheim, E; Visalakshi, V; Olejnik, J; Gordon, S; Benner, SA
Nuc. Nuc. Nuc. acids
29 (11) , Taylor & Francis Group 879-895 (2010)
Nucleoside triphosphates having a 3'-ONH(2) blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH(2) group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH(2) blocking group in "next generation sequencing."
Incorporation of Multiple Sequential Pseudothymidines by DNA Polymerases and Their Impact on DNA Duplex Structure
Havemann, SA; Hoshika, S; Hutter, D; Benner, SA
Nuc. Nuc. Nuc. acids
27 (3) , Taylor & Francis Group 261-278 (2008)
In this article, we focus on the synthesis of aryl C-glycosides via Heck coupling. It is organized based on the type of structures used in the assembly of the C-glycosides (also called C-nucleosides) with the following subsections: pyrimidine C-nucleosides, purine C-nucleosides, and monocyclic, bicyclic, and tetracyclic C-nucleosides. The reagents and conditions used for conducting the Heck coupling reactions are discussed. The subsequent conversion of the Heck products to the corresponding target molecules and the application of the target molecules are also described.
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
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.
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)
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.
Expanding the genetic alphabet: Non-epimerizing nucleoside with the pyDDA hydrogen-bonding pattern
Hutter, D; Benner, SA
J. Org. Chem.
68 (25) 9839-9842 (2003)
6-Amino-3-(2'-deoxy-beta-D-ribofuranosyl)-5-nitro-1H-pyridin-2-one (4), a C-glycoside exhibiting the nonstandard pgammaDDA hydrogen-bonding pattern, was synthesized via Heck coupling. The nitro group greatly enhances the stability of the nucleoside toward acid-catalyzed epimerization without leading to significant deprotonation of the heterocycle at physiological pH. These results make nucleoside 4 a promising candidate for an expanded genetic alphabet.
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