- BS in Molecular Biophysics and Biochemistry. Yale University (1976)
- MS in Molecular Biophysics and Biochemistry. Yale University (1976)
- PhD in Chemistry. Harvard University (1979)
The Benner group has:
- Initiated synthetic biology as a field. The Benner group was the first to synthesize a gene for an enzyme, and used organic synthesis to prepare the first artificial genetic systems. These systems have been used to direct the synthesis of artificial proteins having unnatural amino acids, in FDA-approved clinical assays for HIV, hepatitis B and hepatitis C that improves the medical care of over 400,000 patients annually, and to support the first artificial chemical system capable of Darwinian evolution.
- Invented dynamic combinatorial chemistry, combining ideas from molecular evolution, enzymology, analytical chemistry, and organic chemistry to generate a strategy to discover small molecule therapeutic leads. A German company, Alantos, is today using this technology to develop drug leads.
- Established paleomolecular biology, where researchers resurrect ancestral proteins from extinct organisms for study in the laboratory, The strategy allows scientists to connect chemistry to function in biology, which is defined by an organism's fitness in a complex and changing environment.
- Helped found evolutionary bioinformatics, in 1991, launched one of the first web-based bioinformatics servers with Gaston Gonnet, generated the first naturally organized protein sequence databases, and helped develop the MasterCatalog that generated ca. $4 million in sales. This work also supported the first exhaustive matching of a modern protein sequence database, the first convincing tools to predict structure in proteins from sequence data, strategies to detect distant homologs using structure prediction, and "post-genomic" tools to detect changing protein function.
- National Science Foundation Graduate Fellow
- Junior Fellowship, Harvard Society of Fellows
- Dreyfus Award for Young Faculty, 1982
- Searle Scholar, 1984-86
- Sloan Foundation Fellow, 1984-86
- Anniversary Prize, Federation of European Biochemical Societies, 1993
- Nolan Summer Award, 1998
- Arun Gunthikonda Memorial Award, 1998
- Townes R. Leigh Commemorative Professor, 1999
- B. R. Baker Award, 2001
- Sigma Xi Senior Faculty Award 2005
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.
Structural basis for a six nucleotide genetic alphabet
Georgiadis, M. M., Singh, I., Kellett, W. F., Hoshika, S., Benner, S. A. Richards, N. G. J.
J. Am. Chem. Soc.
(2015) DOI: 10.1021/jacs.5b3482
Expanded genetic systems are most likely to work
with natural enzymes if the added nucleotides pair with
geometries that are similar to those displayed by standard duplex
DNA. Here, we present crystal structures of 16-mer duplexes
showing this to be the case with two nonstandard nucleobases (Z,
6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-
1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair
with a standard "edge on" Watson?Crick geometry, but joined by
rearranged hydrogen bond donor and acceptor groups. One
duplex, with four Z:P pairs, was crystallized with a reverse
transcriptase host and adopts primarily a B-form. Another
contained six consecutive Z:P pairs; it crystallized without a
host in an A-form. In both structures, Z:P pairs fit canonical
nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on
Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are
approximately 1 Angstrom wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise,
ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in
solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of
natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore
the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology.
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 (2014) doi:10.1021/sb500268n
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.
Ribonucleosides for an Artificially Expanded Genetic Information
Hyo-Joong Kim, Nicole A. Leal, Shuichi Hoshika, Steven A. Benner
J. Org. Chem.
(2014) 79 (7), pp 3194-3199
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.
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.
Directed Evolution of Polymerases To Accept Nucleotides with Nonstandard Hydrogen Bond Patterns
Laos R, Shaw R, Leal NA, Gaucher E, Benner S.
(2013) 52, 5288-5294
Artificial genetic systems have been developed
by synthetic biologists over the past two decades to include
additional nucleotides that form additional nucleobase pairs
independent of the standard T:A and C:G pairs. Their use in
various tools to detect and analyze DNA and RNA requires
polymerases that synthesize duplex DNA containing unnatural
base pairs. This is especially true for nested polymerase chain
reaction (PCR), which has been shown to dramatically lower noise in multiplexed nested PCR if nonstandard nucleotides are
used in their external primers. We report here the results of a directed evolution experiment seeking variants of Taq DNA
polymerase that can support the nested PCR amplification with external primers containing two particular nonstandard
nucleotides, 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (trivially called P) that pairs with
6-amino-5-nitro-3-(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z). Variants emerging from the directed
evolution experiments were shown to pause less when challenged in vitro to incorporate dZTP opposite P in a template.
Interestingly, several sites involved in the adaptation of Taq polymerases in the laboratory were also found to have displayed
"heterotachy" (different rates of change) in their natural history, suggesting that these sites were involved in an adaptive change
in natural polymerase evolution. Also remarkably, the polymerases evolved to be less able to incorporate dPTP opposite Z in the
template, something that was not selected. In addition to being useful in certain assay architectures, this result underscores the
general rule in directed evolution that "you get what you select for".
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.
The "strong" RNA world hypothesis. Fifty years old.
Neveu, Mark; Kim, Hyo-Joong; Benner, Steven A
13 (4) (2013) DOI: 10.1089/ast.2012.0868
This year marks the 50th anniversary of a proposal by Alex Rich that RNA, as a single biopolymer acting in two
capacities, might have supported both genetics and catalysis at the origin of life. We review here both published and
previously unreported experimental data that provide new perspectives on this old proposal. The new data include
evidence that, in the presence of borate, small amounts of carbohydrates can fix large amounts of formaldehyde that
are expected in an environment rich in carbon dioxide. Further, we consider other species, including arsenate,
arsenite, phosphite, and germanate, that might replace phosphate as linkers in genetic biopolymers. While linkages
involving these oxyanions are judged to be too unstable to support genetics on Earth, we consider the possibility
that they might do so in colder semi-aqueous environments more exotic than those found on Earth, where cosolvents
such as ammonia might prevent freezing at temperatures well below 273 K. These include the ammonia-water
environments that are possibly present at low temperatures beneath the surface of Titan, Saturn’s largest moon.
(View publication page for Steven Benner)
- Chemical genetics
- Synthetic biology
- Planetary biology
- Systems biology
- The connection of natural history to the physical sciences