Kevin Bradley's Publications
Synthesis and Enzymology of 2'-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System
Karalkar NB, Leal NA, Kim MS, Bradley KM, Benner SA
ACS Synthetic Biology
, American Chemical Society (2016) doi: 10.1021/acssynbio.5b00276
As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ~92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ~86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.
Aptamers against Cells Overexpressing Glypican 3 from Expanded
Genetic Systems Combined with Cell Engineering and Laboratory
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
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 selectionâ€“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
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
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.
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
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
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-
[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.
Detecting respiratory viral RNA using expanded genetic alphabets and
Lyudmyla G. Glushakova, Nidhi Sharma, Shuichi Hoshika, Andrea C. Bradley, Kevin M. Bradley, Zunyi Yang, Steven A. Benner
, Elsevier (2015) Nov 15;489:62-72. doi: 10.1016/j.ab.2015.08.015
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
Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This
makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological
studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic
acids have been reported. None has become established for the routine identification of multiple viruses
in a "single tube" setting. With increasing multiplexing, the detection of viral RNAs is complicated by
noise, false positives and negatives. In this study, an assay was developed that avoids these problems
by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a
"self-avoiding molecular recognition system" (SAMRS), which enables high levels of multiplexing. The
second is an "artificially expanded genetic information system" (AEGIS), which enables clean PCR amplification
in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon,
improving their efficiency of hybridization on Luminex beads. When Luminex "liquid microarrays" are
exploited for downstream detection, this combination supports single-tube PCR amplification assays that
can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The
assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the
California serological group. The performance and the sensitivity of the assay were evaluated with dengue
viruses and infected mosquitoes; as few as 6-10 dengue virions can be detected in a single mosquito.
Helicase Dependent Isothermal Amplification of DNA and RNA using Self-Avoiding Molecular Recognition Systems
Zunyi Yang, Chris McLendon, Daniel Hutter, Kevin M. Bradley, Shuichi Hoshika, Carole Frye, and Steven A. Benner
(2015) June 15; 16(9): 1365-1370. doi:10.1002/cbic.201500135.
Assays that target DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low resource environments, requiring equipment and power that may not be available in these environments. However, isothermal procedures that avoid thermal cycling are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts to give clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, something difficult to achieve with HDA using standard primers. This shows that SAMRS-HDA is a more versatile approach than standard HDA with a broader applicability for xNA-targeted diagnostics and research.
OligArch: A software tool to allow artificially expanded genetic information systems (AEGIS) to guide the autonomous self-assembly of long DNA constructs from multiple DNA single strands
Kevin M. Bradley and Steven A. Benner
Beilstein J. Org. Chem.
, Beilstein Institute (2014) 10, 1826-1833, doi:10.3762/bjoc.10.192
Synthetic biologists wishing to self-assemble large DNA (L-DNA) constructs from small DNA fragments made by automated synthesis need fragments that hybridize predictably. Such predictability is difficult to obtain with nucleotides built from just the four standard nucleotides. Natural DNA's peculiar combination of strong and weak G:C and A:T pairs, the context-dependence of the strengths of those pairs, unimolecular strand folding that competes with desired interstrand hybridization, and non-Watson–Crick interactions available to standard DNA, all contribute to this unpredictability. In principle, adding extra nucleotides to the genetic alphabet can improve the predictability and reliability of autonomous DNA self-assembly, simply by increasing the information density of oligonucleotide sequences. These extra nucleotides are now available as parts of artificially expanded genetic information systems (AEGIS), and tools are now available to generate entirely standard DNA from AEGIS DNA during PCR amplification. Here, we describe the OligArch (for "oligonucleotide architecting") software, an application that permits synthetic biologists to engineer optimally self-assembling DNA constructs from both six- and eight-letter AEGIS alphabets. This software has been used to design oligonucleotides that self-assemble to form complete genes from 20 or more single-stranded synthetic oligonucleotides. OligArch is therefore a key element of a scalable and integrated infrastructure for the rapid and designed engineering of biology.
Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS)
Nidhi Sharma, Shuichi Hoshika, Daniel Hutter, Kevin M. Bradley, and Steven A. Benner
(2014) DOI: 10.1002/cbic.201402250
Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.
Conversion strategy using an expanded genetic alphabet to assay nucleic acids
Yang, Z., Durante, M., Glushakova, L., Sharma, N., Leal, N., Bradley, K., Chen, F., Benner, S. A.
Methods to detect DNA and RNA (collectively
xNA) are easily plagued by noise, false positives, and false
negatives, especially with increasing levels of multiplexing in
complex assay mixtures. Here, we describe assay architectures
that mitigate these problems by converting standard xNA
analyte sequences into sequences that incorporate nonstandard
nucleotides (Z and P). Z and P are extra DNA building blocks
that form tight nonstandard base pairs without cross-binding
to natural oligonucleotides containing G, A, C, and T
(GACT). The resulting improvements are assessed in an
assay that inverts the standard Luminex xTAG architecture,
placing a biotin on a primer (rather than on a triphosphate).
This primer is extended on the target to create a standard
GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a
polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that
is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces
higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures
improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of
competition from any natural oligonucleotides, even in complex biological samples.
An SNP-Based Linkage Map for Zebrafish Reveals Sex Determination Loci
Bradley KM, Breyer JP, Melville DB, Broman KW, Knapik EW, Smith JR
, Genetics Society of America (2011) Jun 1;1(1):3-9
A surprising diversity of mechanisms controls sex determination of vertebrate organisms, even among closely related species. Both genetic and temperature-dependent systems of sex determination have been described in teleost fish. In the common zebrafish model organism, heteromorphic sex chromosomes are not observed, and the potential role of a genetic component of sex determination remains largely unknown. Here we report a genome-wide linkage study of sex determination in zebrafish using a novel SNP genetic map. We identified loci on zebrafish chromosomes 5 (LOD score 7.9) and 16 (LOD score 9.3) governing sex determination as a complex trait, rather than as an XY or ZW genetic system. Each of these loci contains a prominent candidate gene with a conserved role in sex determination across additional species that suggest potential mechanisms of sex determination in zebrafish. The chromosome 5 locus harbors dmrt1, a key gene in sex determination from fruit flies to humans; mutation of the human DMRT1 ortholog is a cause of complete sex reversal of XY individuals. The chromosome 16 locus harbors cyp21a2; mutation of the human CYP21A2 ortholog is one of the more common causes of pseudohermaphroditism. Mutation detection at each of these candidate genes within the zebrafish cross identified hypomorphic variants on the female-associated allele of each locus. The two loci together accounted for 16% of variance of the trait. Interacting environmental cues are likely to be an additional important component of sex determination in zebrafish.
A major zebrafish polymorphism resource for genetic mapping
Bradley KM, Elmore JB, Breyer JP, Yaspan BL, Jessen JR, Knapik EW, Smith JR
, BioMed Central (2007) 8(4):R55
We have identified 645,088 candidate polymorphisms in zebrafish and observe a single nucleotide polymorphism (SNP) validation rate of 71% to 86%, improving with polymorphism confidence score. Variant sites are non-random, with an excess of specific novel T- and A-rich motifs. We positioned half of the polymorphisms on zebrafish genetic and physical maps as a resource for positional cloning. We further demonstrate bulked segregant analysis using the anchored SNPs as a method for high-throughput genetic mapping in zebrafish.