Bridging top-down and bottom-up approaches to synthetic biology

My research focuses on developing and applying tools for readout of mammalian cell states and for control of cellular processes, achieved via combining top-down and bottom-up approaches to synthetic biology.

The top-down approach involves building on the biological ensemble, modifying existing cellular pathways to explore and control biology.
My works focuses on building gene expression quantification and control tools via engineering single strand RNA binding proteins (more).

The bottom-up approach involves building chemical systems capable of mimicking cellular processes, such as protocells with replicating RNA and minimal peptide enzymes that couple catalysis to protocell fitness.
We aim at using those systems to develop synthetic minimal cell technology, to process chemical signals between mammalian cells and the environment (more).

I am currently postdoctoral associate in Ed Boyden's Synthetic Neurobiology group at MIT.
In the summer of 2016 I am starting independent research group at the Department of Genetics, Cell Biology, and Development at the University of Minnesota.


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bottom-up synthetic biology
Building artificial chemical systems mimicking cells

I have developed the first known system capable of non-enzymatic RNA synthesis inside model protocells.
In this work, I have shown how citric acid can stabilize fatty acid liposomes in presence of divalent cations, allowing for non-enzymatic template director RNA primer extension inside the liposomes. This discovery bridges the RNA-world hypothesis or earliest life having RNA-based metabolism with the origin of compartmentalization based on fatty acid liposomes. (Adamala and Szostak, Science 2013)
I was also involved in discovering a chemically-driven replication mechanism for model protocells (Zhu, Adamala, Zhang and Szostak, PNAS 2012).
Together, those two projects allow for drawing a plausible, complete protocell cycle: inside the protocells, RNA is replicated by template-directed non-enzymatic synthesis, and the protocells grow into filamentous threads by absorbing fatty acid molecules from micelles and divide in response to shear stress.

RNA replication inside protocells division via threads RNA is encapsulated
in protocells
activated nucleotides enter
via semi-permeable bilayer
RNA template is copied Source: Adamala and Szostak, Science 2013


As part of the same overarching goal of building a chemical system capable of Darwinian evolution, I have shown that an encapsulated small peptide catalyst can impact the fitness of model protocells.
I have studies catalysis of Ser-His and other di- and tri- amino acid catalytic peptides. I have shown that the minimal serine protease analogue, Ser-His, can catalyze formation of peptide nucleic acids (Gorlero et.al FEBS Letters 2009). I have also demonstrated how the same dipeptide catalyst's activity can be enhanced by the presence of fatty acid protocell vesicles, and in turn the product of the reaction catalyzed by said dipeptide allows the protocell to enhance uptake of membrane building blocks, resulting in protocell growth.
This couples the activity of encapsulated catalyst with the fitness of the protocell, in a model system exploring the origin of Darwinian selection. (Adamala and Szostak, Nature Chemistry 2013).

Ser-His catalysed reaction Source: Adamala and Szostak, Nature Chemistry 2013


Work done with Pier Luigi Luisi from University Roma Tre and Jack Szostak from Harvard University.


Selected publications on my bottom-up synthetic biology projects

Collaboration between primitive cell membranes and soluble catalysts;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; Nature Communications, 2016, in press; *equal contribution

Collaboration between primitive cell membranes and soluble catalysts;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; Nature Communications, 2016, in press; *equal contribution

One widely-held model of early life suggests primitive cells consisted of simple RNA-based catalysts within lipid compartments. One possible selective advantage conferred by an encapsulated catalyst is stabilization of the compartment, resulting from catalyst-promoted synthesis of key membrane components.
Here, we show model protocell vesicles containing an encapsulated enzyme that promotes the synthesis of simple fatty acid derivatives become stabilized to Mg2+, which is required for ribozyme activity and RNA synthesis. Thus, protocells capable of such catalytic transformations would have enjoyed a selective advantage over other protocells in high Mg2+ environments. The synthetic transformation requires both the catalyst and vesicles, which solubilize the water-insoluble precursor lipid.
We suggest that similar modified lipids could have played a key role in early life, and that primitive lipid membranes and encapsulated catalysts, such as ribozymes, may have acted in conjunction with each other, enabling otherwise-impossible chemical transformations within primordial cells.

Physical Basis for Homeostasis in Primordial Cells;
A. E. Engelhart*, K. Adamala*, and J. W. Szostak; Nature Chemistry, 2016, in press; *equal contribution


Physical Basis for Homeostasis in Primordial Cells;
A. E. Engelhart*, K. Adamala*, and J. W. Szostak; Nature Chemistry, 2016, in press; *equal contribution

The emergence of homeostatic mechanisms that enabled maintenance of an intracellular steady-state during growth was critical to the advent of cellular life. Here, we show that concentration-dependent reversible binding of short oligonucleotides, of both specific and random sequence, can modulate ribozyme activity. In both cases, catalysis is inhibited at high concentrations, and dilution activates the ribozyme via inhibitor dissociation, thus maintaining near-constant ribozyme specific activity throughout protocell growth. To mimic the result of RNA synthesis within non-growing protocells, we co-encapsulated high concentrations of ribozyme and oligonucleotides within fatty acid vesicles; ribozyme activity was inhibited. Following vesicle growth, the resulting internal dilution produced ribozyme activation.
This simple physical system enables a primitive homeostatic behavior: the maintenance of constant ribozyme activity per unit volume during protocell volume changes. We suggest such systems, wherein short oligonucleotides reversibly inhibit functional RNAs, could have preceded sophisticated modern RNA regulatory mechanisms, such as those involving miRNAs.

Construction of a Liposome Dialyzer for preparation of high-value, small-volume liposome formulations;
K. Adamala, A. E. Engelhart, N. Kamat, L. Jin and J. W. Szostak; Nature Protocols, 2015, 10(6), pp 927-938;
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details, abstract

Construction of a Liposome Dialyzer for preparation of high-value, small-volume liposome formulations;
K. Adamala, A. E. Engelhart, N. Kamat, L. Jin and J. W. Szostak; Nature Protocols, 2015, 10(6), pp 927-938;

The liposome dialyzer is a small-volume equilibrium dialysis device, built from commercially available materials, that is designed for the rapid exchange of small volumes of an extraliposomal reagent pool against a liposome preparation. The dialyzer is prepared by modification of commercially available dialysis cartridges (Slide-A-Lyzer cassettes), and it consists of a reactor with two 300-μl chambers and a 1.56 - cm2 dialysis surface area. The dialyzer is prepared in three stages: (i) disassembling the dialysis cartridges to obtain the required parts, (ii) assembling the dialyzer and (iii) sealing the dialyzer with epoxy. Preparation of the dialyzer takes approx 1.5 h, not including overnight epoxy curing. Each round of dialysis takes 1 - 24 h, depending on the analyte and membrane used. We previously used the dialyzer for small-volume non-enzymatic RNA synthesis reactions inside fatty acid vesicles. In this protocol, we demonstrate other applications, including removal of unencapsulated calcein from vesicles, remote loading and vesicle microscopy.

Generation of Functional RNAs from Inactive Oligonucleotide Complexes by Non-enzymatic Primer Extension;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; J. Am. Chem. Soc., 2015, 137 (1), pp 483 - 489, DOI: 10.1021/ja511564d; *equal contribution
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details, abstract

Generation of Functional RNAs from Inactive Oligonucleotide Complexes by Non-enzymatic Primer Extension;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; J. Am. Chem. Soc., 2015, 137 (1), pp 483 - 489, DOI: 10.1021/ja511564d; *equal contribution

The earliest genomic RNAs had to be short enough for efficient replication, while simultaneously serving as unfolded templates and effective ribozymes. A partial solution to this paradox may lie in the fact that many functional RNAs can self-assemble from multiple fragments. Therefore, in early evolution, genomic RNA fragments could have been significantly shorter than unimolecular functional RNAs. Here, we show that unstable, nonfunctional complexes assembled from even shorter 3' - truncated oligonucleotides can be stabilized and gain function via non-enzymatic primer extension. Such short RNAs could act as good templates due to their minimal length and complex-forming capacity, while their minimal length would facilitate replication by relatively inefficient polymerization reactions. These RNAs could also assemble into nascent functional RNAs and undergo conversion to catalytically active forms, by the same polymerization chemistry used for replication that generated the original short RNAs. Such phenomena could have substantially relaxed requirements for copying efficiency in early nonenzymatic replication systems.

Non-enzymatic template-directed RNA synthesis inside model protocells;
K. Adamala and J.W. Szostak, Science 342 (2013) 1098 - 1100;
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details, abstract

Non-enzymatic template-directed RNA synthesis inside model protocells;
K. Adamala and J.W. Szostak, Science 342 (2013) 1098 - 1100;

Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg2+ on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg2+ ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg2+ - catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides.

Competition between model protocells driven by an encapsulated catalyst;
K. Adamala and J.W. Szostak, Nature Chemistry 5 (2013) 495 - 501;
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details, abstract

Competition between model protocells driven by an encapsulated catalyst;
K. Adamala and J.W. Szostak, Nature Chemistry 5 (2013) 495 - 501;

The advent of Darwinian evolution required the emergence of molecular mechanisms for the heritable variation of fitness. One model for such a system involves competing protocell populations, each consisting of a replicating genetic polymer within a replicating vesicle. In this model, each genetic polymer imparts a selective advantage to its protocell by, for example, coding for a catalyst that generates a useful metabolite. Here, we report a partial model of such nascent evolutionary traits in a system that consists of fatty-acid vesicles containing a dipeptide catalyst, which catalyses the formation of a second dipeptide. The newly formed dipeptide binds to vesicle membranes, which imparts enhanced affinity for fatty acids and thus promotes vesicle growth. The catalysed dipeptide synthesis proceeds with higher efficiency in vesicles than in free solution, which further enhances fitness. Our observations suggest that, in a replicating protocell with an RNA genome, ribozyme-catalysed peptide synthesis might have been sufficient to initiate Darwinian evolution.

Open questions in origin of life: experimental studies on the origin of nucleic acids and proteins with specific and functional sequences by a chemical synthetic biology approach;
Adamala K, Anella F, Wieczorek R, Stano P, Chiarabelli C, Luisi PL; Comput Struct Biotechnol J. 2014;9:e201402004
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Open questions in origin of life: experimental studies on the origin of nucleic acids and proteins with specific and functional sequences by a chemical synthetic biology approach;
Adamala K, Anella F, Wieczorek R, Stano P, Chiarabelli C, Luisi PL; Comput Struct Biotechnol J. 2014;9:e201402004 ;

In this mini-review we present some experimental approaches to the important issue in the origin of life, namely the origin of nucleic acids and proteins with specific and functional sequences. The formation of macromolecules on prebiotic Earth faces practical and conceptual difficulties. From the chemical viewpoint, macromolecules are formed by chemical pathways leading to the condensation of building blocks (amino acids, or nucleotides) in long-chain copolymers (proteins and nucleic acids, respectively). The second difficulty deals with a conceptual problem, namely with the emergence of specific sequences among a vast array of possible ones, the huge "sequence space", leading to the question "why these macromolecules, and not the others?" We have recently addressed these questions by using a chemical synthetic biology approach. In particular, we have tested the catalytic activity of small peptides, like Ser-His, with respect to peptide- and nucleotides-condensation, as a realistic model of primitive organocatalysis. We have also set up a strategy for exploring the sequence space of random proteins and RNAs (the so-called "never born biopolymer" project) with respect to the production of folded structures. Being still far from solved, the main aspects of these "open questions" are discussed here, by commenting on recent results obtained in our groups and by providing a unifying view on the problem and possible solutions. In particular, we propose a general scenario for macromolecule formation via fragment-condensation, as a scheme for the emergence of specific sequences based on molecular growth and selection.

Photochemically driven redox chemistry induces protocell membrane pearling and division;
T. F. Zhu, K. Adamala, N. Zhang, J. W. Szostak; PNAS, 109 (2012) 9828–9832;
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details, abstract

Photochemically driven redox chemistry induces protocell membrane pearling and division;
T. F. Zhu, K. Adamala, N. Zhang, J. W. Szostak; PNAS, 109 (2012) 9828–9832;

Prior to the evolution of complex biochemical machinery, the growth and division of simple primitive cells (protocells) must have been driven by environmental factors. We have previously demonstrated two pathways for fatty acid vesicle growth in which initially spherical vesicles grow into long filamentous vesicles; division is then mediated by fluid shear forces. Here we describe a different pathway for division that is independent of external mechanical forces. We show that the illumination of filamentous fatty acid vesicles containing either a fluorescent dye in the encapsulated aqueous phase, or hydroxypyrene in the membrane, rapidly induces pearling and subsequent division in the presence of thiols. The mechanism of this photochemically driven pathway most likely involves the generation of reactive oxygen species, which oxidize thiols to disulfide-containing compounds that associate with fatty acid membranes, inducing a change in surface tension and causing pearling and subsequent division. This vesicle division pathway provides an alternative route for the emergence of early self-replicating cell-like structures, particularly in thiol-rich surface environments where UV-absorbing polycyclic aromatic hydrocarbons (PAHs) could have facilitated protocell division. The subsequent evolution of cellular metabolic processes controlling the thiol:disulfide redox state would have enabled autonomous cellular control of the timing of cell division, a major step in the origin of cellular life.

Experimental systems to explore life origin: perspectives for understanding primitive mechanisms of cell division;
K. Adamala, P.L. Luisi; Results Probl. Cell. Differ. 53: 1-9.
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details, abstract

Experimental systems to explore life origin: perspectives for understanding primitive mechanisms of cell division;
K. Adamala, P.L. Luisi; Results Probl. Cell. Differ. 53: 1-9.

Compartmentalization is a necessary element for the development of any cell cycle and the origin of speciation. Changes in shape and size of compartments might have been the first manifestation of development of so-called cell cycles. Cell growth and division, processes guided by biological reactions in modern cells, might have originated as purely physicochemical processes. Modern cells use enzymes to initiate and control all stages of cell cycle. Protocells, in the absence of advanced enzymatic machinery, might have needed to rely on physical properties of the membrane. As the division processes could not have been controlled by the cell’s metabolism, the first protocells probably did not undergo regular cell cycles as we know it in cells of today. More likely, the division of protocells was triggered either by some inorganic catalyzing factor, such as porous surface, or protocells divided when the encapsulated contents reached some critical concentration.

Ser-His catalyses the formation of peptides and PNAs;
M. Gorlero, R. Wieczorek, K. Adamala, A. Giorgi, M.E. Schinina, P. Stano, P.L. Luisi; FEBS Letters, 583 (2009) 153 - 156;
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details, abstract

Ser-His catalyses the formation of peptides and PNAs;
M. Gorlero, R. Wieczorek, K. Adamala, A. Giorgi, M.E. Schinina, P. Stano, P.L. Luisi; FEBS Letters, 583 (2009) 153 - 156;

The dipeptide seryl-histidine (Ser-His) catalyses the condensation of esters of amino acids, peptide fragments, and peptide nucleic acid (PNA) building blocks, bringing to the formation of peptide bonds. Di-, tri- or tetra-peptides can be formed with yields that vary from 0.5% to 60% depending on the nature of the substrate and on the conditions. Other simpler peptides as Gly-Gly, or Gly-Gly-Gly are also effective, although less efficiently. We discuss the results from the viewpoint of primitive chemistry and the origin of long macromolecules by stepwise fragment condensations.

How to close the door leaving it open? On the origin of membrane transport system;
K. Adamala International Journal of Astrobiology, 7-1 (2008) 74;
details, abstract

How to close the door leaving it open? On the origin of membrane transport system;
K. Adamala International Journal of Astrobiology, 7-1 (2008) 74;

Evolution of cellular life, with cells as individual specimens, become possible only when various cycles of primitive metabolic reactions were separated from the environment and from each other. Thus, to the origin of life, encapsulation of proto-metabolic reactions inside vesicle compartments was necessary. However, encapsulation of primitive metabolism would be no advantage at all without immediate origin of selective membrane transport system.
Encapsulated metabolism must have been able to exchange matter and energy with the environment, uptake nutrients, remove waste and collect information about environmental changes. The cross-membrane gradients of concentration of various compounds must have been kept from the very beginning of life’s encapsulated history. To maintain these gradients, trans-membrane system of channels and pores must have been able to provide selective permeability of inorganic ions and small organic molecules.
There are three main groups of compound potentially able to provide transport across bilayer membrane. Peptides, nucleic acids and small organic compounds all can serve as efficient channels and carriers. However, there are various problems of availability of these compounds and conditions required to channel formation.


Broader impact

The effort towards elucidating the origin and earliest evolution of life has always been receiving interest from broad audience.
As an example: an external review detailing my work on RNA replication in protocells: Angewandte Chemie International Edtion Citric Acid and the RNA World 2014, 53, 5245 - 5247, external link

My work on protocells have been featured in science news outlets and editorials, including:
Science Focus: Robert Service, The Life Force 29 Nov 2013 external link
BioTechniques: How Cellular Life Evolved 06 Jan 2014 external link
Science News: To cook up life, just add citrate 185(1):15 external link
Science Daily: Researchers find missing component in effort to create primitive, synthetic cells, 28 Nov 2013 external link
Chemical & Engineering News: Lab-Made Protocells Show Hints Of Evolution, 91(21), 27 May 2013
The Panda's Thumb: New Szostak protocell is closest approximation to origin of life and Darwinian evolution so far, 13 Dec 2013 external link


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top-down synthetic biology
Engineering generalized RNA-protein interactions: a toolbox for regulation and readout of gene expression

We are developing and validating a protein architecture which binds to single stranded RNA. Using this protein technology, we are developing a tool for visualization and quantification of levels of expression of genes of interest.
This tool will work by following the reconstitution of a protein probe upon interaction of sequence-specific RNA binding proteins with the mRNA of the gene of interest.

We are also aiming to edit the transcriptome of the gene of interest, selectively decreasing the level of expression of one splice variant (as opposed to cutting the DNA of the gene, which targets all splice variants indiscriminately).
This technology could potentially help in studying non-ER translation events, elucidating mechanisms of synaptic plasticity, as well as studying healthy and diseased translational profiles of genes, e.g., those involved in oncogenesis and other disease processes.

The ability to monitor and perturb RNA in living neurons - which would open up the investigation of many processes that contribute to development, plasticity, and disease progression - would benefit greatly from a method of systematically targeting unmodified RNA sequences for observation and control. The currrent ssRNA targeting methods are based on the RNA binding protein aptamers, like MS2 or PP7; this require the introduction of aptamer binding sites into the RNA. My work shows that it is possible to develop a ssRNA binding protein that can be engineered to target arbitrary sequences of variable length, thus eliminating the need to engineer the target sites into the RNA of interest.

Work done in Synthetic Neurobiology lab with Ed Boyden.



©2016 kate adamala