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Benner, SA
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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
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.
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.
Alternative Watson-Crick Synthetic
Genetic Systems
Steven A. Benner, Nilesh B. Karalkar, Shuichi Hoshika, Roberto Laos, Ryan W. Shaw, Mariko Matsuura, Diego Fajardo, and Patricia Moussatche
Cold Spring Harb Perspect Biol
, Cold Spring Harbor Laboratory Press (2016) doi: 10.1101/cshperspect.a023770
<Abstract>
In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is
substantially beyond current theoretical and technological capabilities. In pursuit of this
goal, scientists are forced across uncharted territory, where they must answer unscripted
questions and solve unscripted problems, creating new theories and new technologies in
ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery
and paradigm changes in ways that analysis cannot. Described here are the products
that have arisen so far through the pursuit of one grand challenge in synthetic biology:
Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using
genetic and catalytic biopolymers different from those that have been delivered to us by
natural history on Earth. The outcomes in technology include new diagnostic tools that have
helped personalize the care of hundreds of thousands of patients worldwide. In science, the
effort has generated a fundamentally different view of DNA, RNA, and how they work.
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 selection–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.
Standard and AEGIS nicking molecular beacons detect amplicons from the Middle East respiratory syndrome coronavirus
Ozlem Yaren, Lyudmyla G. Glushakova, Kevin M. Bradley, Shuichi Hoshika,Steven A. Benner
J Virol Methods
(236) , Elsevier 54-61 (2016) doi:10.1016/j.jviromet.2016.07.008
<Abstract>
This paper combines two advances to detect MERS-CoV, the causative agent of Middle East Respiratory Syndrome, that have emerged over the past few years from the new field of "synthetic biology". Both are based on an older concept, where molecular beacons are used as the downstream detection of viral RNA in biological mixtures followed by reverse transcription PCR amplification. The first advance exploits the artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four found in standard DNA and RNA (xNA); AEGIS nucleotides pair orthogonally to the A:T and G:C pairs. Placing AEGIS components in the stems of molecular beacons is shown to lower noise by preventing unwanted stem invasion by adventitious natural xNA. This should improve the signal-to-noise ratio of molecular beacons operating in complex biological mixtures. The second advance introduces a nicking enzyme that allows a single target molecule to activate more than one beacon, allowing "signal amplification". Combining these technologies in primers with components of a self-avoiding molecular recognition system (SAMRS), we detect 50 copies of MERS-CoV RNA in a multiplexed respiratory virus panel by generating fluorescence signal visible to human eye and/or camera.
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.
Polymerase Interactions with Wobble Mismatches in Synthetic
Genetic Systems and Their Evolutionary Implications
Christian B. Winiger, Myong-Jung Kim, Shuichi Hoshika, Ryan W. Shaw, Jennifer D. Moses, Mariko F. Matsuura, Dietlind L. Gerloff, and Steven A. Benner
Biochemistry
55 (28) 3847-3850 (2016) DOI: 10.1021/acs.biochem.6b00533
<Abstract>
In addition to completing the Watson-Crick nucleobase matching "concept" (big pairs with small,
hydrogen bond donors pair with hydrogen bond acceptors),
artificially expanded genetic information systems
(AEGIS) also challenge DNA polymerases with a
complete set of mismatches, including wobble mismatches.
Here, we explore wobble mismatches with AEGIS with
DNA polymerase 1 from Escherichia coli. Remarkably, we
find that the polymerase tolerates an AEGIS:standard
wobble that has the same geometry as the G:T wobble that
polymerases have evolved to exclude but excludes a wobble
geometry that polymerases have never encountered in
natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools
to help synthetic biologists expand DNA alphabets.
(View publication page for Shuichi Hoshika)
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