-
Research
-
Publications
-
All publications
-
Benner, SA
-
Biondi, E
-
Bradley, K
-
Chen, C
-
Hoshika, S
-
Karalkar, N
-
Kim, HJ
-
Kim, MJ
-
Laos, R
-
Leal, NA
-
Li, Y
-
Shaw, RW
-
Spacek, J
-
Yang, ZY
-
People
-
Benner, Steven
-
Biondi, Elisa
-
Bradley, Kevin
-
Chen, Cen
-
Darling, April
-
Hoshika, Shuichi
-
Karalkar, Nilesh
-
Kim, Hyo-Joong
-
Kim, Myong-Jung
-
Laos, Roberto
-
Leal, Nicole
-
Li, Yubing
-
Shaw, Ryan
-
Spacek, Jan
-
Yang, Zunyi
-
News and Events
-
Press Coverage
-
Our Foundation
|
Shuichi Hoshika's 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.
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.
Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen
Biondi E, Lane JD, Das D, Dasgupta S, Piccirilli JA, Hoshika S, Bradley KM, Krantz BA, Benner SA
Nucl. Acids Res.
(2016) Oct 3. pii: gkw890. PubMed PMID: 27701076
<Abstract>
Reported here is a laboratory in vitro evolution (LIVE)
experiment based on an artificially expanded genetic
information system (AEGIS). This experiment delivers
the first example of an AEGIS aptamer that binds
to an isolated protein target, the first whose structural
contact with its target has been outlined and
the first to inhibit biologically important activities of
its target, the protective antigen from Bacillus anthracis.
We show how rational design based on secondary
structure predictions can also direct the use
of AEGIS to improve the stability and binding of the
aptamer to its target. The final aptamer has a dissociation
constant of ~35 nM. These results illustrate
the value of AEGIS-LIVE for those seeking to
obtain receptors and ligands without the complexities
of medicinal chemistry, and also challenge the
biophysical community to develop new tools to analyze
the spectroscopic signatures of new DNA folds
that will emerge in synthetic genetic systems replacing
standard DNA and RNA as platforms for LIVE.
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
<Abstract>
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-
nitro-3-(1'-B-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone
and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo-
[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.
Transcription, Reverse Transcription, and Analysis of RNA Containing Artificial Genetic Components
Nicole A. Leal, Hyo-Joong Kim, Shuichi Hoshika, Myong-Jung Kim, Matthew A. Carrigan, and Steven A. Benner
ACS Synthetic Biology
, American Chemical Society (2015) Apr 17;4(4):407-13. doi: 10.1021/sb500268n
<Abstract>
Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.
Detecting respiratory viral RNA using expanded genetic alphabets and
self-avoiding DNA
Lyudmyla G. Glushakova, Nidhi Sharma, Shuichi Hoshika, Andrea C. Bradley, Kevin M. Bradley, Zunyi Yang, Steven A. Benner
Anal Biochem
, Elsevier (2015) Nov 15;489:62-72. doi: 10.1016/j.ab.2015.08.015
<Abstract>
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
<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.
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
<Abstract>
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
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.
Ribonucleosides for an Artificially Expanded Genetic Information
System
Hyo-Joong Kim, Nicole A. Leal, Shuichi Hoshika, Steven A. Benner
J. Org. Chem.
(2014) 79 (7), pp 3194-3199
<Abstract>
Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2?-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson?Crick complement,imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this "second-generation" AEGIS Z:P pair to add amino acids encoded by mRNA.
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.
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.
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
<Abstract>
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.
Study of modification pattern-RNAi activity relationships by using siRNAs modified with 4'-thioribonucleosides
Hoshika, S; Minakawa, N; Shionoya, A; Imada, K; Ogawa, N; Matsuda, A
ChemBioChem
8 (17) 2133-2138 (2007)
<Abstract>
A detailed study of the modification pattern-RNAi activity relationships by using siRNAs that are modified with 4'-thioribonucleosides has been carried out against photinus luciferase and renilla luciferase genes in cultured mammalian NIH/3T3, HeLa, and MIA PaCa-2 cell lines. When the photinus luciferase gene was targeted, all of the modified siRNAs showed activity equal to, or less than the unmodifed siRNA. In contrast, all modified siRNAs that have a similar modification pattern showed activity equal to or much higher than the unmodified siRNA when tested with the renilla luciferase gene. These results indicated that siRNAs such as RNA33 and RNA53, which each have four residues of the 4'-thioribonucleoside unit on both ends of the sense strand and four residues on the 3'-end of the antisense strand, were the most effective. Accordingly, we succeeded in developing modified siRNAs that have the greatest number of 4'-thioribonucleosides without loss of RNAi activity, and that exhibit potent RNAi activity against two target genes in three different cell lines. Our findings also indicate the significance of target sequences and cell lines when RNAi activity is compared with that of the unmodified siRNA.
RNA interference induced by siRNAs modified with 4 '-thioribonucleosides in cultured mammalian cells
Hoshika, S; Minakawa, N; Kamiya, H; Harashima, H; Matsuda, A
FEBS Lett.
579 (14) 3115-3118 (2005)
<Abstract>
Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Oligodeoxynucleotides having a loop consisting of 3 '-deoxy-4 '-C-(2-hydroxyethyl)thymidines form stable hairpins
Yamamoto, Y; Shuto, S; Tamura, Y; Kodama, T; Hoshika, S; Ichikawa, S; Ueno, Y; Ohtsuka, E; Komatsu, Y; Matsuda, A
Biochemistry
43 (27) 8690-8699 (2004)
<Abstract>
Components that form stable hairpin loops are highly useful for the development of functional DNA and RNA molecules. We have designed and synthesized a sugar-modified thymidine analogue, 3'-deoxy-4'-C-(2-hydroxyethyl)thymidine (X), as a nucleosidic loop component stabilizing the hairpin structure. The ODNs I-1-4, 5'-d[CGAACG-X-n-CGTTCG]-3' (I-1, n = 1; I-2, n = 2; I-3, n = 3; I-4, n = 4), forming the hairpin loop structures, of which the loop moiety consisted of the analogue X, and also the corresponding unmodified ODNs II-1-4, 5'-d[CGAACG-T-n-CGTTCG]-3' (II-1, n = 1; II-2, n = 2; II-3, n = 3; II-4, n = 4), having a thymidine loop, were synthesized by the phosphoramidite method. The melting temperatures (T m) of the ODNs I-1-4 containing X in the loop moiety at 5 muM were 67.1, 68.1, 73.0, and 69.3 degreesC, respectively, and those of the control natural ODNs II-1-4 were 65.3, 67.0, 69.2, and 68.8 degreesC, respectively. Thus, the ODNs I-1-4 formed a more thermally stable hairpin than the corresponding unmodified ODNs II-1-4 having an equal number of loop residues. The hairpin structures of the modified ODNs I-1-4 and the unmodified ODNs II-1-4 were investigated by CD spectroscopy and molecular mechanics calculations. These results showed that the 4'-branched nucleoside X can stabilize hairpin structures when it is present in the loop moiety, probably due to the flexibility of the one-carbon-elongated 4'-branched structure.
Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 3 '-3 '-linked oligodeoxynucleotides composed of asymmetrical sequences
Hoshika, S; Ueno, Y; Kamiya, H; Matsuda, A
Bioorg. Med. Chem. Lett.
14 (12) 3333-3336 (2004)
<Abstract>
In this paper, we describe the synthesis of the 3'-3'-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiment. It was found that the 3'-3'-linked oligonucleotides composed of asymmetrical sequences formed the stable antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine-oligopurine sequences. (C) 2004 Elsevier Ltd. All rights reserved.
Synthesis and physical and physiological properties of 4 '-thioRNA: application to post-modification of RNA aptamer toward NF-kappa B
Hoshika, S; Minakawa, N; Matsuda, A
Nucl. Acids Res.
32 (13) 3815-3825 (2004)
<Abstract>
We report herein full details of the preparation of 4'-thiouridine, -cytidine, -adenosine and -guanosine phosphoramidites based on our synthetic protocol via the Pummerer reaction. Fully modified 4'-thioRNAs containing four kinds of 4'-thioribonucleoside units were prepared according to the standard RNA synthesis. The T,, values and thermodynamic parameters of a series of duplexes were determined by UV melting and differential scanning calorimetry (DSC) measurements. The resulting overall order of thermal stabilities for the duplexes was 4'-thioRNA:4'-thioRNA >> 4'-thioRNA:RNA > RNA:RNA > RNA:DNA > 4'-thioRNA:DNA. In addition, it was shown that the dominant factor in the stability of the duplexes consisting of 4'-thioRNA was enthalpic in character. The CD spectra of not only 4'-thioRNA:RNA and 4'-thioRNA:4'-thioRNA but also 4'-thioRNA:DNA were all similar to those of duplexes in the A-conformation. The stability of 4'-thioRNA in human serum was 600 times greater than that of natural RNA. Neither the RNA:RNA nor the 4'-thioRNA: 4'-thioRNA duplexes were digested under the same conditions. The first example of a post-modification of an RNA aptamer by 4'-thioribonucleoside units was demonstrated. Full modification of the aptamer thioRNA3 resulted in complete loss of binding activity. In contrast, modifications at positions other than the binding site were tolerated without loss of binding activity. The post-modified RNA aptamer thioRNA5 was thermally stabilized and resistant toward nuclease digestion. The results presented in this paper will, it is hoped, contribute to the development of 4'-thioRNA as a new generation of artificial RNA.
Nucleosides and nucleotides. 218. Alternate-strand triple-helix formation by the 3 '-3 '-linked oligodeoxynucleotides using a purine motif
Hoshika, S; Ueno, Y; Matsuda, A
Bioconjugate Chem.
14 (3) 607-613 (2003)
<Abstract>
In this paper, we describe the synthesis of the X-X-linked TFOs that can form the antiparallel triplexes with the duplex DNA target by reverse Hoogsteen hydrogen bonds. Stability of the alternate-strand triplexes between these TFOs and the target DNAs was investigated using the electrophoretic mobility shift assay (EMSA). It was found that the alternate-strand triplexes were significantly stabilized by linking the TFO fragments with the pentaerythritol linker. And, unlike the alternate-strand triplexes composed of the pyrimidine motif, the terminal ammonium ion of the aminobutyl-linker and the intercalator of the TFOs did not contribute to the stability of the alternate-strand triplex comprised of the purine motif. We also tested the ability of the X-X-linked TFOs to inhibit cleavage of the duplex DNA target 17 by the restriction enzyme EcoT14I and found that the 3'-3'-Iinked TFOs 12 and 13 inhibited the cleavage by the enzyme more effectively than the unlinked decamer S. Thus, the TFOs linked with pentaerythritol may be useful as the antigene oligonucleotide to the DNA targets, which have alternating oligopyrimidine-oligopurine sequences.
Nucleosides and nucleotides. 208. alternate-strand triple-helix formation by the 3 '-3 '-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point
Ueno, Y; Mikawa, M; Hoshika, S; Matsuda, A
Bioconjugate Chem.
12 (4) 635-642 (2001)
<Abstract>
The synthesis of 3 ' -3 ' -linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3 ' -3 ' -linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3 ' -3 ' -linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3 ' -3 ' -linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3 ' -3 ' -linked ODN 15 without the intercalator.
|
|
|