Fellow
Daniel Hutter

Education

MS in Chemistry. Swiss Federal Institute of Technology (ETH), Zurich, Switzerland (1994)
PhD in Chemistry. ETH, Zurich, Switzerland (2001)

Research summary

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.

Recent Publications

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.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and and four serotypes of dengue viruses in mosquitoes.
Glushakova, L.G.. Alto, B.W., Kim, M.-S., Hutter, D., Bradley, A., Bradley, K.M., Burkett-Cadena, N.D., Benner, S.A.
BMC Infect. Dis. , BioMed Central Ltd. (2019) 19:418, DOI:10.1186/s12879-019-3998-z
<Abstract>

Background The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed.

Methods We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides).

Results The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels.

Conclusions Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.

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.

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
<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.

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.

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
ChemBioChem (2014) DOI: 10.1002/cbic.201402250
<Abstract>

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)
<Abstract>

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."

Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection
Chen, F; Gaucher, EA; Leal, NA; Hutter, D; Havemann, SA; Govindarajan, S; Ortlund, EA; Benner, SA
Proc. Natl. Acad. Sci. USA 107 (5) 1948-1953 (2010)

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)
<Abstract>

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.

Synthesis of pyrophosphates for in vitro selection of catalytic RNA molecules
Kim, HJ; Kim, MJ; Karalkar, N; Hutter, D; Benner, SA
Nuc. Nuc. Nuc. acids 27 , Taylor & Francis Group 43-56 (2008)

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Fellow
Daniel Hutter

Education

Research summary

Recent Publications

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Interests

Contact

Fellow Daniel Hutter

Education

Research summary

Recent Publications

Photo

Interests

Contact

Fellow
Daniel Hutter