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Roberto Laos's Publications
Fluorinated oil-surfactant mixtures with the density of water: Artificial cells for synthetic biology
Roberto Laos, Steven Benner
17 (1) , Public Library of Science (2022) https://doi.org/10.1371/journal.pone.0252361
There is a rising interest in biotechnology for the compartmentalization of biochemical reactions in water droplets. Several applications, such as the widely used digital PCR, seek to encapsulate a single molecule in a droplet to be amplified. Directed evolution, another technology with growing popularity, seeks to replicate what happens in nature by encapsulating a single gene and the protein encoded by this gene, linking genotype with phenotype. Compartmentalizing reactions in droplets also allows the experimentalist to run millions of different reactions in parallel. Compartmentalization requires a fluid that is immiscible with water and a surfactant to stabilize the droplets. While there are fluids and surfactants on the market that have been used to accomplish encapsulation, there are reported concerns with these. Span® 80, for example, a commonly used surfactant, has contaminants that interfere with various biochemical reactions. Similarly, synthetic fluids distributed by the cosmetic industry allow some researchers to produce experimental results that can be published, but then other researchers fail to reproduce some of these protocols due to the unreliable nature of these products, which are not manufactured with the intent of being used in biotechnology. The most reliable fluids, immiscible with water and suitable for biochemical reactions, are fluorinated fluids. Fluorinated compounds have the peculiar characteristic of being immiscible with water while at the same time not mixing with hydrophobic molecules. This peculiar characteristic has made fluorinated fluids attractive because it seems to be the basis of their being biologically inert. However, commercially available fluorinated fluids have densities between 1.4 to 1.6 g/mL. The higher-than-water density of fluorinated oils complicates handling of the droplets since these would float on the fluid since the water droplets would be less dense. This can cause aggregation and coalescence of the droplets. Here, we report the synthesis, characterization, and use of fluorinated polysiloxane oils that have densities similar to the one of water at room temperature, and when mixed with non-ionic fluorinated surfactants, can produce droplets encapsulating biochemical reactions. We show how droplets in these emulsions can host many biological processes, including PCR, DNA origami, rolling circle amplification (RCA), and Taqman® assays. Some of these use unnatural DNA built from an Artificially Expanded Genetic Information System (AEGIS) with six nucleotide "letters".
The surprising pairing of 2-aminoimidazo[1,2-a]-
[1,3,5]triazin-4-one, a component of an expanded
Roberto Laos, Christos Lampropoulos, and Steven A. Benner
, Acta Crystallographica (2019) C75, 22-28, https://doi.org/10.1107/S2053229618016923
Synthetic biologists demonstrate their command over natural biology by
reproducing the behaviors of natural living systems on synthetic biomolecular
platforms. For nucleic acids, this is being done stepwise, first by adding replicable
nucleotides to DNA, and then removing its standard nucleotides. This challenge
has been met in vitro with 'six-letter' DNA and RNA, where the Watson-Crick
pairing 'concept' is recruited to increase the number of independently replicable
nucleotides from four to six. The two nucleobases most successfully added so far
are Z and P, which present a donor-donor-acceptor and an acceptor-acceptor-
donor pattern, respectively. This pair of nucleobases are part of an 'artificially
expanded genetic information system' (AEGIS). The Z nucleobase has been
already crystallized, characterized, and published in this journal [Matsuura et al.
(2016). Acta Cryst. C72, 952-959]. More recently, variants of Taq polymerase
have been crystallized with the pair P:Z trapped in the active site. Here we
report the crystal structure of the nucleobase 2-aminoimidazo[1,2-a][1,3,5]-
triazin-4-one (trivially named P) as the monohydrate, C5H5N5O-H2O. The
nucleobase P was crystallized from water and characterized by X-ray diffraction.
Interestingly, the crystal structure shows two tautomers of P packed in a
Watson-Crick fashion that cocrystallized in a 1:1 ratio.
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
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
Alternative Watson-Crick Synthetic
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
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.
engineered by directed evolution to incorporate non-standard nucleotides.
Frontiers in Microbiology
Laos, R., Thomson, J. M., & Benner, S. A.
Frontiers in Microbiology
(2014) 5, 565. http://doi.org/10.3389/fmicb.2014.00565
DNA polymerases have evolved for billions of years to accept natural nucleoside triphosphate substrates with high fidelity and to exclude closely related structures, such as the analogous ribonucleoside triphosphates. However, polymerases that can accept unnatural nucleoside triphosphates are desired for many applications in biotechnology. The focus of this review is on non-standard nucleotides that expand the genetic "alphabet." This review focuses on experiments that, by directed evolution, have created variants of DNA polymerases that are better able to accept unnatural nucleotides. In many cases, an analysis of past evolution of these polymerases (as inferred by examining multiple sequence alignments) can help explain some of the mutations delivered by directed evolution.
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".
Engineered DNA Polymerases
K. Murakami and M.A. Trakselis (eds.)
Nucleic Acid Polymerases, Nucleic Acids and Molecular Biology
, Springer-Verlag Berlin Heidelberg (2013)
Solution H-1 NMR confirmation of folding in short o-phenylene ethynylene oligomers
Jones, TV; Slutsky, MM; Laos, R; de Greef, TFA; Tew, GN
J. Am. Chem. Soc.
127 (49) 17235-17240 (2005)
Oligomers based on an o-phenylene ethynylene (oPE) backbone with polar substituents have been synthesized using Sonogashira methods. Folding of these extremely short oligomers was confirmed via 1D and 2D (NOESY) NMR methods. Utilizing electron-rich and electron-poor phenylene building blocks, variations of these oPE oligomers have been synthesized to determine the folded stability of pi-rich vs pi-poor vs pi-rich pi-poor systems. Slight variations in temperature offer a route, aside from solvent denaturation, to probe the stability of the folded structure. This is the first report of an NMR solution characterization of folding for a PE backbone without hydrogen bonds.