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Articles and book chapters


Engineering genetic circuit interactions within and between synthetic minimal cells;
Katarzyna P. Adamala*, Daniel A. Martin-Alarcon*, Katriona R. Guthrie-Honea, Edward S. Boyden; Nature Chemistry, 2016, doi:10.1038/nchem.2644; *equal contribution
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Engineering genetic circuit interactions within and between synthetic minimal cells;
Katarzyna P. Adamala*, Daniel A. Martin-Alarcon*, Katriona R. Guthrie-Honea, Edward S. Boyden; Nature Chemistry, 2016, doi:10.1038/nchem.2644; *equal contribution

Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.


Astrobiology Primer 2.0.;
Domagal-Goldman and Wright et al. (includes chapter editor K.P.Adamala)
Astrobiology, 2016, 16(8):561-653. doi:10.1089/ast.2015.1460.
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Astrobiology Primer 2.0.;
Domagal-Goldman and Wright et al. (includes chapter editor K.P.Adamala) Astrobiology, 2016, 16(8):561-653. doi:10.1089/ast.2015.1460.
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Oligoarginine peptides slow strand annealing and assist non-enzymatic RNA replication;
Tony Z. Jia, Albert C. Fahrenbach, Neha P. Kamat, Katarzyna P. Adamala & Jack W. Szostak; Nature Chemistry, 2016, doi:10.1038/nchem.2551
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Oligoarginine peptides slow strand annealing and assist non-enzymatic RNA replication;
Tony Z. Jia, Albert C. Fahrenbach, Neha P. Kamat, Katarzyna P. Adamala & Jack W. Szostak; Nature Chemistry, doi:10.1038/nchem.2551

The non-enzymatic replication of RNA is thought to have been a critical process required for the origin of life. One unsolved difficulty with non-enzymatic RNA replication is that template-directed copying of RNA results in a double-stranded product. After strand separation, rapid strand reannealing outcompetes slow non-enzymatic template copying, which renders multiple rounds of RNA replication impossible. Here we show that oligoarginine peptides slow the annealing of complementary oligoribonucleotides by up to several thousand-fold; however, short primers and activated monomers can still bind to template strands, and template-directed primer extension can still occur, all within a phase-separated condensed state, or coacervate. Furthermore, we show that within this phase, partial template copying occurs even in the presence of full-length complementary strands. This method to enable further rounds of replication suggests one mechanism by which short non-coded peptides could have enhanced early cellular fitness, and potentially explains how longer coded peptides, that is, proteins, came to prominence in modern biology.


Programmable RNA-binding protein composed of repeats of a single modular unit;
Katarzyna P. Adamala*, Daniel A. Martin-Alarcon*, and Edward S. Boyden; PNAS, 2016, 10.1073/pnas.1519368113; *equal contribution
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Programmable RNA-binding protein composed of repeats of a single modular unit;
Katarzyna P. Adamala*, Daniel A. Martin-Alarcon*, and Edward S. Boyden; PNAS, 2016, 10.1073/pnas.1519368113; *equal contribution

The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby - RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.


Collaboration between primitive cell membranes and soluble catalysts;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; Nature Communications, 2016, doi:10.1038/ncomms11041; *equal contribution
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Collaboration between primitive cell membranes and soluble catalysts;
K. Adamala*, A. E. Engelhart* and J. W. Szostak; Nature Communications, 2016, doi:10.1038/ncomms11041; *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.


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; *equal contribution
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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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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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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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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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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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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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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;
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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.



Patents

Pumilio Domain-based Modular Protein Architecture for RNA Binding
ES Boyden, KP Adamala, DA Martin-Alarcon
US Patent App. 14/995,169
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Spatial multiplexing for multisignal cellular imaging
G Xu, E Boyden, KD Piatkevich, K Adamala
US Patent App. 15/099,232, 2016
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