Associate
Shuichi Hoshika

Education

BS in Pharmaceutical Sciences. Hokkaido University, Japan (2001)
MS in Pharmaceutical Sciences. Hokkaido University, Japan (2003)
PhD in Pharmaceutical Sciences. Hokkaido University, Japan (2006)

Research summary

My research focuses on the development of rapid and cost-effective sequencing methods based on synthesis of nucleoside derivatives. This would be achieved by multiplex PCR using primers consisting of nucleoside derivatives which form base pairs with natural nucleobases and/or synthetic nucleobases in different hydrogen bonding patterns.

Recent Publications

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.

In vitro evolution of ribonucleases from expanded genetic alphabets
Jerome, C.A; Hoshika, S.; Bradley, K.M.; Benner, S.A.; Biondi, E.
Proc. Natl. Acad. Sci. USA (2022) 119(44). DOI: 10.1073/pnas.2208261119
<Abstract>

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucleotide building blocks is limited when compared with that of proteins. Here, we assess the evolutionary potential of libraries of nucleic acids with six nucleotide building blocks as reservoirs for catalysis. We compare the outcomes of in vitro selection experiments toward RNA-cleavage activity of two nucleic acid libraries: one built from the standard four independently replicable nucleotides and the other from six, with the two added nucleotides coming from an artificially expanded genetic information system (AEGIS). Results from comparative experiments suggest that DNA libraries with increased chemical diversity, higher information density, and larger searchable sequence spaces are one order of magnitude richer reservoirs of molecules that catalyze the cleavage of a phosphodiester bond in RNA than DNA libraries built from a standard four-nucleotide alphabet. Evolved AEGISzymes with nitro-carrying nucleobase Z appear to exploit a general acid–base catalytic mechanism to cleave that bond, analogous to the mechanism of the ribonuclease A family of protein enzymes and heavily modified DNAzymes. The AEGISzyme described here represents a new type of catalysts evolved from libraries built from expanded genetic alphabets.

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.

Confluence of Theory and Experiment Reveal the Catalytic Mechanism of the Varkud Satellite Ribozyme
Ganguly, A., Weissman, B.P., Giese, T.J., Li, N.-S. , Hoshika, S., Rao, S., Benner, S.A., Piccirilli, J.A., York, D.M.
Nat. Chem., Nature (2020) 12:193-201, DOI:10.1038/s41557-019-0391-x
<Abstract>

The Varkud satellite ribozyme catalyses site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here, we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free-energy relationship measurements with molecular dynamics, molecular solvation theory and ab initio quantum mechanical/molecular mechanical free-energy simulations to gain insight into the catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfer in the transition state. Further, we provide evidence for a critical Mg²⁺ in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg²⁺ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the Varkud satellite, hairpin and hammerhead ribozyme classes.

Hachimoji DNA and RNA: A genetic system with eight building blocks
Hoshika H, Leal N, Kim MJ, Kim MS, Karalkar NB, Kim HJ, Bates AM, Watkins Jr. NE, SantaLucia HA, Meyer AJ, DasGupta S, Piccirilli JA, Ellington AD, SantaLucia Jr. J, Georgiadis MM, Benner SA
Science (2019) 22 Feb 2019: Vol. 363, Issue 6429, pp. 884-887. DOI: 10.1126/science.aat0971
<Abstract>

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

"Skinny" and "Fat" DNA: Two New Double Helices
Hoshika S, Singh I, Switzer C, Molt RW Jr, Leal NA, Kim MJ, Kim MS, Kim HJ, Georgiadis MM, Benner SA
J. Am. Chem. Soc. (2018) Sep 19;140(37):11655-11660. doi: 10.1021/jacs.8b05042. Epub 2018 Sep 10
<Abstract>

According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide
Isha Singh, Roberto Laos, Shuichi Hoshika, Steven A. Benner, and Millie M. Georgiadis
Nucl. Acids Res.46(15) 7977-7988 (2018) doi: 10.1093/nar/gky552
<Abstract>

The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2- amino-8-(1-beta-D-2'- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2'-deoxyribofuranosyl)-5-nitro- 1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.

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.

Biophysics of Artificially Expanded Genetic Information Systems. Thermodynamics of DNA Duplexes Containing Matches and Mismatches Involving 2-Amino-3-nitropyridin-6-one (Z) and Imidazo[1,2?a]-1,3,5-triazin-4(8H)one (P)
Xiaoyu Wang, Shuichi Hoshika, Raymond J. Peterson, Myong-Jung Kim, Steven A. Benner, and Jason D. Kahn
ACS Synthetic Biology, American Chemical Society (2017) DOI: 10.1021/acssynbio.6b00224
<Abstract>

Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed. Here, we report the results of UV absorbance melting measurements and determine the energetics of binding of DNA strands containing Z and P to give short duplexes containing Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodynamic parameters (ΔH°, ΔS°, and ΔG°₃₇) for oligonucleotide hybridization were extracted. Consistent with the Watson-Crick model that considers both geometric and hydrogen bonding complementarity, the Z:P pair was found to contribute more to duplex stability than any mismatches involving either nonstandard nucleotide. Further, the Z:P pair is more stable than a C:G pair. The Z:G pair was found to be the most stable mismatch, forming either a deprotonated mismatched pair or a wobble base pair analogous to the stable T:G mismatch. The C:P pair is less stable, perhaps analogous to the wobble pair observed for C:O⁶-methyl-G, in which the pyrimidine is displaced into the minor groove. The Z:A and T:P mismatches are much less stable. Parameters for predicting the thermodynamics of oligonucleotides containing Z and P bases are provided. This represents the first case where this has been done for a synthetic genetic system.

Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase
Mariko F. Matsuura, Ryan W. Shaw, Jennifer D. Moses, Hyo-Joong Kim, Myong-Jung Kim, Myong-Sang Kim, Shuichi Hoshika, Nilesh Karalkar, and Steven A. Benner
ACS Synthetic Biology, American Chemical Society (2016) 5 (3), pp 234-240 DOI: 10.1021/acssynbio.5b00172
<Abstract>

One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3- (1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin- 4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

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Associate
Shuichi Hoshika

Education

Research summary

Recent Publications

Interests

Contact

Associate Shuichi Hoshika

Education

Research summary

Recent Publications

Interests

Contact

Associate
Shuichi Hoshika