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Self-replication in the genome in the seed

Self-replication in the genome in the seed



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Is the genome in the seeds of the plant turned off? That is, does the DNA in the seed not self-replicate? My second question is, what are the cells feeding on inside the seed before it is planted?


DNA in cells in general replicates when cell division occurs (unless it is a cell that contains many nuclei or multiple copies of its own genome, in which case DNA would replicate as copies are made. Multinucleate cells include things like the mycelium of fungi, and here is an example of a bacterium with thousands of copies of its own genome).

As long as a plant embryo is developing, then the cells are dividing and the DNA is replicated. You might be thinking of the stage of seed dormancy; insofar as during this stage the embryo is no longer growing, cells aren't dividing and therefore DNA is indeed not replicated. I don't know whether we could say the genome is "turned off" however, DNA's role isn't just being replicated, more importantly its role is to be transcribed into RNA which leads to the proteins the cell needs to function being created. I couldn't find whether DNA transcription happens during cell dormancy but it's plausible enough that it is at least slowed down as well. The cells presumably don't have much to do during this period and you wouldn't want them to do much, to conserve energy.

Seeds contain nutrients that can feed the embryo until it can feed itself (for plants that is the moment when they germinate enough to photosynthesize). That is where flour comes from!


Self-replication and self-reproduction

When the subject is self-replication, in the general context of life and nature, we think immediately to DNA. In fact, this is the driving force for the eternal flow from one generation to the other – being roses or elephants or microbes – and also, due to perturbations and mistakes in the replication mechanism itself, to the formation of new forms of life and new species – till the tree of life we know today.

The disclaimer, as we mentioned in the starting title of this first section, is important, and due to the fact that most common readers, but also simple-minded scientists, think that DNA is really able to self-replicate alone. DNA alone does not do anything. If you have DNA in a solution and provide all possible low molecular weight nutrients, salts, mononucleotides (the monomers of the nucleic acids) – nothing happens, there is no reproduction, no chemistry at all. DNA can give rise to self-reproduction only with the help of a family of enzymes, which are specific proteins which catalyze all various and several steps of the mechanism of self-replication. All self-replicating DNA of this world [1] is made by the living cells, or can even be done in vitro, but always and only with the help of that specific family of proteins – and of course in the presence of excess of nucleotides and energy bearing molecules like ATP (adenosine triphosphate). The bottom line is that DNA self-replication is a complex mechanism.

Now, all this is obvious to anyone who has followed a basic course of biochemistry, even at an undergraduate level. But in the majority of people, the idea that DNA is the only hero of all reproduction appears to be dominant in the collective thinking. This has to do with general ignorance in science, but it is also an interesting social phenomenon. As emphasized by Evelyne Fox-Keller in her well-known book, The Century of the Gene, and by several other open-mined scientists in this last period, we live in a world dominated by the notions of gene, genome, genetic engineering, a world in which DNA has become indeed the main protagonist. To the point that in many camps of the research biology – first of all the field of the origin of life – there is the unfortunate equation DNA = life. As if life would be due, or caused, by DNA alone or mostly by DNA.

Personally, I believe that this equation needs another strong disclaimer, and that actually this equation has been very detrimental to the research around the question “what is life?” and/or the complementary question “what is the origin of life?”

We are dealing with a reductionist, very restricted view, and I had the opportunity to discuss this in detail in my recent book, The Emergence of Life. And it has been emphasized in great detail that life and/or its origin cannot be ascribed to a single molecule – it is in fact a systems view, where many independent components have to interact with each other to produce a web, a wholeness, with the emergent property that we call life (see for example Capra and Luisi, 2014, or Noble, 2006).

Self-replication and self-reproduction

Not that he above disclaimers take away anything from the extraordinary beauty and molecular power of DNA, or RNA. DNA cannot reproduce by itself alone, but is the only molecular structure which has, encoded in its linear form, the information to reproduce itself not only, as in its linear structure it has also the information of how to construct proteins – two complementary functions which are, of course, of dramatic importance for all living organisms.

Let us proceed with order, starting with a semantic note about the two terms, self-replication and self-reproduction. Although often treated synonymously, there is in fact a difference. Self-replication (the word comes from the Latin term, replica) means a faithful molecular copy, while self-reproduction refers rather to a statistical process of making very similar things. Thus, cells self-reproduce, while molecules self-replicate. It is not simply a semantic question, as according to Dyson (1985), self-reproduction processes, being less precise, may have started first in the early evolution, and preceded self-replication processes, which require more complex control and editing.

Self-replication and non-linearity

The double-strand nature of DNA (see Figure 1) was discovered by Watson and Crick in 1953, a memorable year for biology indeed. There are four different nucleotides forming DNA, the famous bases Adenine (A), guanine (G), thymine (T) and cytosine (C), whereby the two right-handed strands of DNA are kept together by the so-called complementarity of the two base pairs, A with T, C with G, as can be seen more clearly from Figure 2. This complementarity is the most ingenious device for self-replication, in the sense that each of the two strands contains the information for making a complementary one. Suppose in fact to “lose” one of the two strands – you are left with only one! But you can then immediately reconstruct the missing one, by setting a T in face of an A of your strand, then a C facing a G, and so on – this is clearly shown in Figure 2. And this happens when DNA self-replicates.

Figures 3 and 4 illustrate the well-known semi-conservative mechanism of DNA replication, which is the basis of the meiosis and mitosis processes in cell reproduction. This process, as mentioned in the text, takes place with the help of several specialized enzymatic proteins. Here, semi-conservative means that the parent double stranded molecule is divided in two, and each old strand makes a novel double strand with fresh material. So, if the parent strand would be labeled somehow, this label would be diluted by one half at each step of the duplication.

And now notice the most important feature in this process: the non-linearity. What does this mean? A linear growth process is of the type: 1, 2, 3, 4, 5… but DNA grows 1, 2, 4, 8, 16, 32… To understand the dramatic relevance of this difference, consider a chemical process which makes A→B in one second – then to make one million of B, you need one million seconds. But if you have B→2B→4B→8B→16B…, to make one million B you need only 20 seconds. That’s incredible, isn’t?

The case of self-reproduction

We have spoken until now of self-replication of DNA, which is a linear polymer. Are there also examples in the case of models of cellular structures? The answer is yes, with micelles and vesicles. Let us recall in more detail the case of the micellar aqueous systems. The chemistry is based on fatty acids, see Figure 5, with the additional observation that fatty acids are considered possible candidates for the first prebiotic membranes. The experimental apparatus is particularly simple (see Figure 6), also a reminder of a possible prebiotic situation: the water-insoluble ethyl caprylate (a water-insoluble simple ester) is overlaid on an aqueous alkaline solution, so that at the macroscopic interphase there is a hydrolysis reaction that produces caprylate ions. The reaction is initially very slow, as shown in the Figure 6, but eventually the critical micelle concentration (namely, the concentration at which the micelles begin to form in solution) is reached, and then the caprylate micelles are rapidly formed.

Aqueous micelles can actually be seen as lipophilic spherical surfaces, and the main reason why soap takes away the grease from our hands, lies in the fact that grease is efficiently incorporated by the lipophilic core of the aqueous soap micelles. By the same mechanism, the lipophilic ethyl caprylate (EC) is avidly up-taken by the caprylate micelles. The efficient molecular dispersion of EC on the micellar surface speeds up its hydrolysis (a kind of physical micellar catalysis), and caprylate ions are rapidly formed. The result is the formation of more micelles, as illustrated in the Figure 6. However, more micelles determine more uptake of the water-insoluble EC, with the formation of more and more micelles: a typical autocatalytic behavior (Bachmann et al., 1992).

In the following years, this type of self-reproduction experiment was extended to vesicles. Let us emphasize again that the mechanism of self-reproduction of micelles and vesicles can be considered autopoietic, (see Luisi, 2016) since growth and eventually division comes from within the structure itself (in vesicles, initial reaction takes place on the bilayer, but the bilayer is part of the structure).

It is important to point out the main message of these experiments, which is the possibility of a spontaneous self-reproduction of spherical compartments. Since these can be considered as models and/or precursors of biological cells, the hypothesis was put forward, (Bachmann et al., 1992), that this autocatalytic self-reproduction process might have been of relevance for the origin of life.

Concluding

As we have emphasized, DNA does not replicate when alone, and chemists, particularly those studying the origin of life, have for several decades tried to mimic self-replication processes in the lab, without using enzymes. Two things should be said in general at this regard: that yes, chemists have succeeded to produce in the laboratory self-replication and self-reproduction processes without the use of enzymes however, these are not processes that occur spontaneously under prebiotic conditions, they have been brought to function thanks to the use of suitable reactive groups (nucleophiles, leaving groups, and the like… we cannot go here in the details).
Thus, from the one hand the notion of self-replication, that was considered a monopoly of nature, is now into the narrow space of the chemistry lab, and this should certainly be seen as a remarkable progress. However, in general, the origin of life is for science still an unsolved question, and prebiotic self-replication is one facet of this larger problem.

References
Bachmann, P. A., Luisi, P. L., and Lang, J. (1992). Autocatalytic self-replication of micelles as models for prebiotic structures. Nature, 357, 57-9.
Capra, F., and Luisi, P. L. (2014). The Systems View of Life: A Unifying Vision. Cambridge University Press.
Dyson, F. J. (1985). Origins of Life. Cambridge University Press.
Fox-Keller, E. (2002). The century of the gene, Harvard University Press.
Landenmark, H. K. E., Forgan, D. H., and Cockell, C. S. (2015). An Estimate of the Total DNA in the Biosphere. PLoS Biol, 13(6): e1002168. https://doi.org/10.1371/journal.pbio.1002168.
Luisi, P. L. (2016). The Emergence of Life: From Chemical Origins to Synthetic Biology. 2nd Edn., Cambridge University Press.
Noble, D. (2006). The Music of Life. Oxford University Press. Von Neumann, J., edited and completed by Burks, A. W. (1966). Theory of Self-Reproducing Automata, University of Illinois Press.

[1] By the way, have you attempted to calculate how much DNA we have in our world? What do you think? A few grams… or perhaps billions of tons?
Let us make a calculation, considering that each human body consists of 15-30 trillion cells, and that each cell (except for mature erythrocytes) contains ca. 6 pg (means pico-gram) of DNA. Now consider all people living on the Earth – but also all other species, including plants, bacteria and DNA viruses – a biomass which is approximately 2 × 1012 tons. How much DNA, then?
Well, the research group directed by Charles Cockell has calculated that the total DNA existing in the biosphere is ca. 50 billions of tons. Take account that more than 98% of this weight is due to plants (68%) and bacteria (30.2%), and only in a negligible way to animals (0.8%) and other living organisms (Landenmark et al., 2015).


The nature of seeds

In the typical flowering plant, or angiosperm, seeds are formed from bodies called ovules contained in the ovary, or basal part of the female plant structure, the pistil. The mature ovule contains in its central part a region called the nucellus that in turn contains an embryo sac with eight nuclei, each with one set of chromosomes (i.e., they are haploid nuclei). The two nuclei near the centre are referred to as polar nuclei the egg cell, or oosphere, is situated near the micropylar (“open”) end of the ovule.

With very few exceptions (e.g., the dandelion), development of the ovule into a seed is dependent upon fertilization, which in turn follows pollination. Pollen grains that land on the receptive upper surface (stigma) of the pistil will germinate, if they are of the same species, and produce pollen tubes, each of which grows down within the style (the upper part of the pistil) toward an ovule. The pollen tube has three haploid nuclei, one of them, the so-called vegetative, or tube, nucleus seems to direct the operations of the growing structure. The other two, the generative nuclei, can be thought of as nonmotile sperm cells. After reaching an ovule and breaking out of the pollen tube tip, one generative nucleus unites with the egg cell to form a diploid zygote (i.e., a fertilized egg with two complete sets of chromosomes, one from each parent). The zygote undergoes a limited number of divisions and gives rise to an embryo. The other generative nucleus fuses with the two polar nuclei to produce a triploid (three sets of chromosomes) nucleus, which divides repeatedly before cell-wall formation occurs. This process gives rise to the triploid endosperm, a nutrient tissue that contains a variety of storage materials—such as starch, sugars, fats, proteins, hemicelluloses, and phytate (a phosphate reserve).

The events just described constitute what is called the double-fertilization process, one of the characteristic features of all flowering plants. In the orchids and in some other plants with minute seeds that contain no reserve materials, endosperm formation is completely suppressed. In other cases it is greatly reduced, but the reserve materials are present elsewhere—e.g., in the cotyledons, or seed leaves, of the embryo, as in beans, lettuce, and peanuts, or in a tissue derived from the nucellus, the perisperm, as in coffee. Other seeds, such as those of beets, contain both perisperm and endosperm. The seed coat, or testa, is derived from the one or two protective integuments of the ovule. The ovary, in the simplest case, develops into a fruit. In many plants, such as grasses and lettuce, the outer integument and ovary wall are completely fused, so seed and fruit form one entity such seeds and fruits can logically be described together as “dispersal units,” or diaspores. More often, however, the seeds are discrete units attached to the placenta on the inside of the fruit wall through a stalk, or funiculus.

The hilum of a liberated seed is a small scar marking its former place of attachment. The short ridge (raphe) that sometimes leads away from the hilum is formed by the fusion of seed stalk and testa. In many seeds, the micropyle of the ovule also persists as a small opening in the seed coat. The embryo, variously located in the seed, may be very small (as in buttercups) or may fill the seed almost completely (as in roses and plants of the mustard family). It consists of a root part, or radicle, a prospective shoot (plumule or epicotyl), one or more cotyledons (one or two in flowering plants, several in Pinus and other gymnosperms), and a hypocotyl, which is a region that connects radicle and plumule. A classification of seeds can be based on size and position of the embryo and on the proportion of embryo to storage tissue the possession of either one or two cotyledons is considered crucial in recognizing two main groups of flowering plants, the monocotyledons and the eudicotyledons.


Self-replication of information-bearing nanoscale patterns

DNA molecules provide what is probably the most iconic example of self-replication--the ability of a system to replicate, or make copies of, itself. In living cells the process is mediated by enzymes and occurs autonomously, with the number of replicas increasing exponentially over time without the need for external manipulation. Self-replication has also been implemented with synthetic systems, including RNA enzymes designed to undergo self-sustained exponential amplification. An exciting next step would be to use self-replication in materials fabrication, which requires robust and general systems capable of copying and amplifying functional materials or structures. Here we report a first development in this direction, using DNA tile motifs that can recognize and bind complementary tiles in a pre-programmed fashion. We first design tile motifs so they form a seven-tile seed sequence then use the seeds to instruct the formation of a first generation of complementary seven-tile daughter sequences and finally use the daughters to instruct the formation of seven-tile granddaughter sequences that are identical to the initial seed sequences. Considering that DNA is a functional material that can organize itself and other molecules into useful structures, our findings raise the tantalizing prospect that we may one day be able to realize self-replicating materials with various patterns or useful functions.


Self-replication process holds promise for production of new materials

New York University scientists have developed artificial structures that can self-replicate, a process that has the potential to yield new types of materials. The work, conducted by researchers in NYU's Departments of Chemistry and Physics and its Center for Soft Matter Research, appears in the latest issue of the journal Nature.

In the natural world, self-replication is ubiquitous in all living entities, but artificial self-replication has been elusive. The discovery in Nature reports the first steps toward a general process for self-replication of a wide variety of arbitrarily designed seeds. The seeds are made from DNA tile motifs that serve as letters arranged to spell out a particular word. The replication process preserves the letter sequence and the shape of the seed and hence the information required to produce further generations.

This process holds much promise for the creation of new materials. DNA is a robust functional entity that can organize itself and other molecules into complex structures. More recently DNA has been used to organize inorganic matter, such as metallic particles, as well. The re-creation by the NYU scientists of this type of assembly in a laboratory raises the prospect for the eventual development of self-replicating materials that possess a wide range of patterns and that can perform a variety of functions. The breakthrough the NYU researchers have achieved is the replication of a system that contains complex information. Thus, the replication of this material, like that of DNA in the cell, is not limited to repeating patterns.

To demonstrate this self-replication process, the NYU scientists created artificial DNA tile motifs —short, nanometer-scale arrangements of DNA. Each tile serves as a letter—A or B—that recognizes and binds to complementary letters A' or B'. In the natural world, the DNA replication process involves complementary matches between bases—adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C) -- to form its familiar double helix. By contrast, the NYU researchers developed an artificial tile or motif, called BTX (bent triple helix molecules containing three DNA double helices), with each BTX molecule comprised of 10 DNA strands. Unlike DNA, the BTX code is not limited to four letters—in principle, it can contain quadrillions of different letters and tiles that pair using the complementarity of four DNA single strands, or "sticky ends," on each tile, to form a six-helix bundle.

In order to achieve self-replication of the BTX tile arrays, a seed word is needed to catalyze multiple generations of identical arrays. BTX's seed consists of a sequence of seven tiles—a seven-letter word. To bring about the self-replication process, the seed is placed in a chemical solution, where it assembles complementary tiles to form a "daughter BTX array"—a complementary word. The daughter array is then separated from the seed by heating the solution to

40 oC. The process is then repeated. The daughter array binds with its complementary tiles to form a "granddaughter array," thus achieving self-replication of the material and of the information in the seed—and hence reproducing the sequence within the original seed word. Significantly, this process is distinct from the replication processes that occur within the cell, because no biological components, particularly enzymes, are used in its execution—even the DNA is synthetic.

"This is the first step in the process of creating artificial self-replicating materials of an arbitrary composition," said Paul Chaikin, a professor in NYU's Department of Physics and one of the study's co-authors. "The next challenge is to create a process in which self-replication occurs not only for a few generations, but long enough to show exponential growth."

"While our replication method requires multiple chemical and thermal processing cycles, we have demonstrated that it is possible to replicate not just molecules like cellular DNA or RNA, but discrete structures that could in principle assume many different shapes, have many different functional features, and be associated with many different types of chemical species," added Nadrian Seeman, a professor in NYU's Department of Chemistry and a co-author of the study.


References

Von Neumann, J. Theory of Self-Reproducing Automata (Univ. Illinois Press, Champaign, Illinois 1966).

Freitas, R. A. Jr & Merkle, R. C. Kinematic Self-Replicating Machines (Landes Bioscience, Georgetown, Texas, 2004).

Penrose, L. S. & Penrose, R. Nature 179, 1183 (1957).

Alberts, B. et al. (eds) in Molecular Biology of the Cell 4th edn, 241 and 983 (Garland Science, New York, 2001).

Jacobson, H. Am. Sci. 46, 255–284 (1958).

Penrose, L. S & Penrose, S. V. Automatic Mechanical Self Replication (2) (Cresswell Film Unit, Galton Laboratory, University College London, December 1961).

Suthakorn, J., Cushing, A. B. & Chirikjian, G. S. Proc. 2003 IEEE/ASME Int. Conf. Adv. Intel. Mechatronic (AIM) (Kobe, Japan, 2003).

Zykov, V., Mytilinaios, E., Adams, B. & Lipson, H. Nature 435, 163–164 (2005).

Banks, E. R. Information Processing and Transmission in Cellular Automata. Thesis, Massachusetts Institute of Technology (1971).

Griffith, S. Growing Machines. Thesis, Massachusetts Institute of Technology (2004).


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Results and Discussion

The basic replication scheme remains the same as in ref. 20. A bath contains monomer DNA origami tiles functionalized with DNA single strands (sticky ends) for horizontal and vertical binding to other complementary monomer tiles. For the work described here, “horizontal” and “vertical” refer to directions in the plane of the cross-shaped origami tile. A seed dimer, a horizontally bonded set of monomers, is introduced into the bath. On cooling the system, the dimer vertically binds and holds in place two complementary monomers. While the system is cold the bound monomers bind horizontally. Four positions on the horizontal sticky ends are composed of the 3-cyanovinvylcarbazole nucleoside ( cnv K) (20, 22) which, when UV activated, can cross-link to a thymine nucleotide on the complementary sticky end, hence covalently linking monomers into daughter dimers. Upon heating the system, the vertical bonds are released and the seed/parent dimer and the daughter dimers are released. The daughter dimers can then act as parents for the next generation, ideally doubling the number of parent dimers each cycle/generation.

The demonstration of replication and exponential growth in ref. 20 was done with a rectangular origami molecule, essentially a raft of 12 double-stranded helices laced together with “staple strands.” It is known that interorigami binding is best accomplished along the axes of the double helices. Therefore, for the rectangular rafts we utilized the short edges of the rectangle for horizontal bonds and the top and bottom faces for vertical bonds. In the present work we make use of “cross-tile” origami (23) with double helices along both perpendicular directions of the cross. In this configuration we can have the more stable bonding along the outer four edges of the cross. This also leaves the two faces for additional binding for other uses and more flexible designs.

We first use our cross-tile system to repeat our previous self replication of a seed dimer with an amplification factor of 2. The process is illustrated in Fig. 1. The seed dimer (red tile in Fig. 1A) consists of two individual monomers connected by a set of 11-bp horizontal sticky ends. The melting temperature of the seed dimer is high enough that it remains intact as the template throughout the thermal annealing cycle. The set of six vertical sticky ends on each cross-tile of the seed dimer (Fig. 1A, α) allows it to bind to two next-generation tiles. The first-generation (FG) and second-generation (SG) tiles, shown in blue and green in Fig. 1A, contain vertical sticky ends, where α′ on FG is complementary to α on both SG and the seed dimer. These monomer tiles have a set of unique horizontal sticky ends: β and its complement β′ on FG, and γ and its complement γ′ on SG. There are six horizontal sticky ends in each set on both the left and right side of each tile. Four of the horizontal sticky ends contain CNV K. The CNV K units are positioned diagonally across from thymine on the complementary set, such that when the sample is subjected to UV light, the daughters are linked together covalently (Fig. 1B) (20, 23). When the seed dimer tiles capture two FG tiles by vertical sticky-end hybridization at a low temperature, a tetramer structure is formed. The FG tiles are then held close together, which increases the local concentration and results in their horizontal base pairing. In this sense the seed acts as a catalyst for binding the monomers to form the daughter dimer. The monomers in suspension are at a low concentration so that they will not bind to each other in the thermal annealing temperature range. Upon UV exposure, the hybridized horizontal sticky ends on the FG tiles are cross-linked to form dimers. After heating, the seeds and daughters separate, and the new FG dimers can now act as templates to produce later-generation dimers. The later-generation dimers will follow the same replication cycling, ideally doubling the number of dimers each cycle. A valuable feature of the cross-tiles is that they contain a thickened portion that appears as an “equal sign,” visible on the atomic force microscopy (AFM), thereby constituting an orientational label for each tile. The equal signs are visible as dark stippled areas in Fig. 1A and in other figures.

Self-replication of cross-shaped DNA origami tiles. (A) Schematics of seed dimer (red), FG monomer (blue), and SG monomer (green). The seed dimer (S) contains a set of six vertical sticky ends that extend from the top of each tile (α), and is held together by six 11-bp complementary horizontal sticky ends (blue lines on seed tile). Both FG and SG monomers have a unique set of six horizontal sticky ends on each side of the tile (β and its complement β′ for FG, and γ and its complement γ′ for SG), where there is a CNV K unit on each of the four strands (pink lines). The FG monomer has a set of six vertical sticky ends that extends from the bottom, α′, which are complementary to α. The SG monomer has an identical set of vertical sticky ends to the seed, α. (B) Schematic of FG dimer. When subject to UV light, the CNV K unit can photo–cross-link with thymine on the complementary strand to form a covalent bond. (C) Self-replication cycling. The seed dimer hybridizes with two next-generation monomers (FG) vertically to form a tetramer. After UV irradiation, the two next-generation monomers attached to the seed are chemically cross-linked via CNV K photo–cross-linking reaction. By heating the system to 48 °C, the tetramer will separate into the seed dimer and the FG dimer. The cross-linked dimers can then act as seeds to generate further generations.

To implement this approach experimentally, the seed dimer, daughter, and tetramer tiles were formed and characterized using AFM, as shown in Fig. 2A. Next, we used a thermal annealing protocol (Materials and Methods) to form the tetramer structure between seed dimers and FG tiles. A distinct tetramer band on a nondenaturing agarose gel run at 18 °C, which is the temperature that UV photo–cross-linking is carried out, suggests the stability of the tetramer structure (Fig. 2B, lane 3). Furthermore, the FG tile in lane 2 shows a single monomer band, demonstrating that there is no horizontal binding between the monomers. The tetramer structure can be separated vertically by heat (SI Appendix, Fig. S3). Fluorescence resonance energy transfer (FRET) results indicate that the melting temperature of the vertical sticky ends is around 35 °C (SI Appendix, Fig. S5).

Self-replication of cross-shaped DNA origami seeds. (A) AFM images of seed dimers, FG monomers, and seed-FG tetramers with their respective schematic drawings are shown from left to right. (Inset) The AFM image of seed dimers shows the orientation of equal sign in the target dimer structure. (Scale bars, 200 nm.) (B) A nondenaturing agarose gel (0.8%) shows the formation of the tetramer between a seed dimer and two FG monomers (lane 3). Lane 1 contains dimer seed, and lane 2 contains FG monomer. The gel was run at 18 °C. (C) A nondenaturing agarose gel showing the amplification and quantification of dimers in four cycles, with a starting ratio of 1:32:30 between seed:FG:SG. Lane 1 and lane 2 contain dimer seed and FG monomer, respectively. Lanes 3–5 contain the mixtures from cycles 0, 2 and 4, respectively. The intensity of the band showing dimers increases as the cycling proceeds. The gel was run at 48 °C. (D) Plot showing the total amplification factor at each cycle for different ratios between seed:FG:SG. The green, blue, and red curves represent the replication cycling containing seeds, FG tiles, and SG tiles with the ratios of 1:8:6, 1:16:14, and 1:32:30, respectively. The green and blue curves were obtained by evaluating AFM images, and the red curve was collected from quantification of the gel shown in C. Each curve shows an average replication rate of ∼1.7 per cycle.

We conducted the exponential growth experiments by using three different initial ratios (1:8:6, 1:16:14, and 1:32:30) between the seed dimer, FG monomers, and SG monomers, respectively. Ideally for the one-sided design the ratio seed:FG:SG is 1:2 M :2 M -2, producing 2 M dimers for M cycles, but typically we put in enough monomers for one additional cycle. The number of cycles that were run for each ratio was determined by when, theoretically, half of the monomers would be consumed. To quantify the amplification factor, we divided the fraction of dimers in each cycle by the initial fraction of seed dimmers: N = f n / f 0 .

fn: fraction of dimers after n cycle of self-replication

f0: fraction of dimers before self-replication

fn = number of dimers/(number of dimers + number of monomers/2).

For ratios 1:8:6 and 1:16:14, the amount of dimers and monomers was counted from AFM images (SI Appendix, Figs. S6 and S7). To calculate the dimer fraction, the total number of dimers was multiplied by two and then divided by the total number of monomers. At least 1,000 tiles were counted per cycle for both ratios. For ratio 1:32:30, the dimer percentage for each cycle was quantified by comparing the intensity of upper gel bands (dimer, trimer) to the total intensity of the bands in the entire lane (Fig. 2C). The fraction of monomers/dimers is determined by the integration of the gel intensity plot using ImageJ as discussed and illustrated in SI Appendix, Fig. S4. The plot in Fig. 2D shows the amplification factor over the course of self-replication cycling for the different ratios, with each ratio averaging ∼1.7 per cycle. Trimers begin to appear after the first cycle because it is then possible for the seed dimer to hybridize with both a single daughter monomer and a cross-linked daughter dimer.

After demonstrating the success of this cross-tile self-replication system, we proceeded to enhance the replication efficiency. Because the dimer tiles can only pick up two daughter tiles on one vertical side, the theoretical maximum amplification rate per cycle is 2. To exceed this limit, we extended the other vertical edge with the same set of vertical sticky ends on the tiles, as shown in Fig. 3A. In this ladder design, the seed dimer and SG tiles now have the vertical sticky-end set α extending from both the top and bottom, while the FG tile has vertical sticky-end set α′. When mixed together, they can hybridize to form railroad-track–like structures of a variable length. The seed dimer, which serves as the parent template in the initial stage, is absent from this schematic to emphasize that the new dimers formed from self-replication can serve as parents in the subsequent cycles. Successful formation of the ladder structure after hybridization and UV cross-linking was confirmed via AFM (Fig. 3B). Furthermore, to confirm that no replication occurs without the initial seed dimer, a sample containing only FG and SG monomer tiles was subject to replication cycling. The results show a faint dimer band on a nondenaturing agarose gel (SI Appendix, Fig. S10), which is substantially lower than when the seed dimer is used (SI Appendix, Fig. S13).

Ladder self-replication of cross-shaped DNA origami tiles using a serial transfer. (A) Schematic of the ladder self-replication. The seed dimer, FG (blue) and SG (green) monomer contain their respective vertical sticky-end set on both the top and bottom of each tile, detailed in SI Appendix, Fig. S1. The seed tile and SG monomer will have set α, and the FG monomer will have set α′, as labeled in Fig. 1A. The parent, as a cross-linked dimer, can hybridize with the daughter tiles in both vertical directions to form a ladder. When exposed to UV light, a cross-linked ladder is formed. After heating to 48 °C, the ladder separates vertically into individual cross-linked FG and SG dimers. These can then serve as parents as the cycle repeats. The seed dimer, not shown in this schematic, can act as a parent in the same way. (B) AFM images of the cross-linked ladder. (Scale bars, 200 nm.) These are taken from green-bar (1:32:30) experiments. (C) Column chart showing the amplification factors after each transfer and cycle of self-replication for both the ratio of 1:32:30 between seed:FG:SG (green) and the ratio of 1:64:62 (purple) from gel quantification. At transfer 0 cycle 1, the dimer percentage amplifies by ∼fourfold for the ratio of 1:32:30 and ∼sixfold for the ratio of 1:64:62, and the chart shows this repeated cycling until cycle 6. (D) Overall amplification of a serial transfer experiment. Overall amplification curves were obtained by six transfer cycles, resulting in an ∼2,700-fold amplification for the sample of the ratio 1:32:30 (green curve obtained from gel quantification, blue curve obtained from evaluating AFM images), and 270,000-fold amplification for the sample of the ratio of 1:64:62 (purple curve obtained from gel quantification, red curve obtained from evaluating AFM images).

To start the replication using the ladder design, we used two population pools: the starting ratio was 1:32:30 and 1:64:62 between seed: FG:SG, respectively. A serial transfer was conducted after each cycle, where a portion of the sample was transferred to a fresh pool of daughter tiles to dilute the dimer population to its initial percentage. This ensures that there are sufficient monomers for replication. A total of six cycles were run for each ratio, and the amplification was quantified by both AFM counting (SI Appendix, Figs. S11–S12) and agarose gel electrophoresis (SI Appendix, Fig. S13). For the two-sided ladder design, the amplification factor should be proportional to the ratio of monomers to seeds. The results from gel quantification are plotted in Fig. 3C, showing that the amplification factor per cycle averages ∼4 for the ratio of 1:32:30 and ∼8 for the ratio of 1:64:62. With the ladder design, the amplification factor is no longer limited to 2, and leads to a system that achieves a much greater amplification factor per cycle. The plot in Fig. 3D shows the overall amplification factor versus the cycle number, obtained from gel quantification and AFM counting data. After six cycles, the ratio of 1:32:30 (green, blue lines) results in an ∼2,700-fold overall amplification, while the ratio of 1:64:62 (red, purple lines) results in an ∼270,000-fold overall amplification.

In addition to the ladder design that exceeds the replication limit of 2 for each temperature–UV cycle, we also performed experiments to optimize yield by decreasing the annealing ramp time to accomplish more cycles within the same amount of time. The previous slow-annealing method (20) used a long annealing time, about 30 h, to assure “complete” specific self-assembly of the tetramer structure (parent dimer + two daughter monomers). However, subsequent experiments reported here show that the tetramer structure can be formed within a 40-min isothermal annealing period after heating the seed dimer and FG tiles at 48 °C for 30 min (Fig. 4A). Since the tetramer structure is the platform for self-replication, the kinetics of the binding between vertical sticky ends to form the tetramer structure and the pairing between the horizontal sticky ends for cross-linking are essential. FRET results show that the binding of vertical sticky ends occurs rapidly (Fig. 5 AC): 71% of the vertical sticky ends bind after 5 min of isothermal annealing at 16 °C, and 90% of vertical sticky-ended binding is completed after 40 min of isothermal annealing, compared with the slow-annealing method. Vertical sticky-end binding saturates at ∼91% after 40 min, indicating that the majority of vertical binding for tetramer structure formation is completed within 40 min. Furthermore, the FRET results of horizontal sticky-end binding show similar kinetics through isothermal annealing (Fig. 5 DF). As indicated by the FRET signal, binding increases rapidly at the beginning, and reaches a plateau of 97% after 40 min, indicating that 40 min of isothermal annealing is sufficient to form the tetramer structure for self-replication.

Self-replication of cross-shaped DNA origami using fast-annealing process. (A) AFM image of formation of seed-FG tetramer through 40-min fast annealing process. (B) Replication rate of DNA origami corresponded to the annealing time from 10 to 40 min, obtained from gel quantification. (C) Gel image of five-cycle fast-annealing self-replication of DNA origami using a serial transfer. T0C0 in the image represents transfer 0 cycle 0. The gel was run at 48 °C (D) Plot showing the change of the dimer percentage and replication rate of each cycle of fast-annealing self-replication, obtained from gel quantification of C.

Kinetic study of self-assembly of seed and FG during the fast-annealing process through FRET. (A) Schematics of formation of cy3–cy5 vertical pair through hybridization between seed–cy3 (donor) and FG–cy5 (acceptor). (B) Fluorescence spectra of seed–cy3 (donor alone) and seed-FG tetramer (cy3–cy5 vertical pair). (C) Fraction of the cy3–cy5 vertical pair formed through the hybridization between the seed–cy3 and FG–cy5 over the first 60 min after annealing at 16 °C. (D) Schematics of formation of cy3–cy5 horizontal pair through hybridization between cy5-FG–cy3. (E) Fluorescence spectra of cy5-FG–cy3 (donor–acceptor separated) and seed-FG tetramer (cy3–cy5 horizontal pair). (F) Fraction of the cy3–cy5 horizontal pair formed through the hybridization between the cy5-FG–cy3 over the first 60 min after annealing at 16 °C.

Upon UV exposure for 20 min, a systematic study shows that the replication rate increases from 1.28 to 1.50 for the prolonged isothermal annealing time from 10 to 40 min the data were collected from gel quantification (Fig. 4B). Then, a total of five self-replication cycles, by a series transfer after each cycle, were run using this fast-annealing process of 40-min isothermal annealing, and the amplification was quantified by agarose gel electrophoresis (Fig. 4 C and D). The replication rate of each cycle was consistent, indicating this is a reproducible procedure, and the average replication rate was 1.51. Although we obtained a smaller replication rate per cycle compared with the current slow-annealing method, we were able to accomplish 20 cycles within the same amount of time. In this manner, the fast-annealing method results in an ∼4,400-fold overall amplification in the time it takes for one cycle of the current method.


Reproductive genome from the laboratory

The field of synthetic biology does not only observe and describe processes of life but also mimics them. A key characteristic of life is the ability to ability for replication, which means the maintenance of a chemical system. Scientists at the Max Planck Institute of Biochemistry in Martinsried generated a system, which is able to regenerate parts of its own DNA and protein building blocks.

In the field of synthetic biology, researchers investigate so-called "bottom-up" processes, which means the generation of life mimicking systems from inanimate building blocks. One of the most fundamental characteristics of all living organism is the ability to conserve and reproduce itself as distinct entities. However, the artificial "bottom-up" approach to create a system, which is able to replicate itself, is a great experimental challenge. For the first time, scientists have succeeded in overcoming this hurdle and synthesizing such a system.

Hannes Mutschler, head of the research group "Biomimetic Systems" at the Max Planck Institute for Biochemistry, and his team are dedicated to imitate the replication of genomes and protein synthesis with a "bottom-up" approach. Both processes are fundamental for the self-preservation and reproduction of biological systems. The researchers now succeeded in producing an in vitro system, in which both processes could take place simultaneously. "Our system is able to regenerate a significant proportion of its molecular components itself," explains Mutschler.

In order to start this process, the researchers needed a construction manual as well as various molecular "machines" and nutrients. Translated into biological terms, this means the construction manual is DNA, which contains the information to produce proteins. Proteins are often referred to as "molecular machines" because they often act as catalysts, which accelerate biochemical reactions in organisms. The basic building blocks of DNA are the so-called nucleotides. Proteins are made of amino acids.

Modular structure of the construction manual

Specifically, the researchers have optimized an in vitro expression system that synthesizes proteins based on a DNA blueprint. Due to several improvements, the in vitro expression system is now able to synthesize proteins, known as DNA polymerases, very efficiently. These DNA polymerases then replicate the DNA using nucleotides. Kai Libicher, first author of the study, explains: "Unlike previous studies, our system is able to read and copy comparatively long DNA genomes.

The scientists assembled the artificial genomes from up to eleven ring-shaped pieces of DNA. This modular structure enables them to insert or remove certain DNA segments easily. The largest modular genome reproduced by the researchers in the study consists of more than 116,000 base pairs, reaching the genome length of very simply cells.

Regeneration of proteins

Apart from encoding polymerases that are important for DNA replication, the artificial genome contains blueprints for further proteins, such as 30 translation factors originating from the bacterium Escherischia coli. Translation factors are important for the translation of the DNA blueprint into the respective proteins. Thus, they are essential for self-replicating systems, which imitate biochemical processes. In order to show that the new in vitro expression system is not only able to reproduce DNA, but is also able to produce its own translation factors, the researchers used mass spectrometry. With this analytic method, they determined the amount of proteins produced by the system.

Surprisingly, some of the translation factors were even present in larger quantities after the reaction than added before. According to the researchers, this is an important step towards a continuously self-replicating system that mimics biological processes.

In the future, the scientists want to extend the artificial genome with additional DNA segments. In cooperation with colleagues from the research network MaxSynBio, they want to produce an enveloped system that is able to remain viable by adding nutrients and disposing of waste products. Such a minimal cell could then be used, for example, in biotechnology as a tailor-made production machine for natural substances or as a platform for building even more complex life-like systems.


NYU scientists' creation of self-replication process holds promise for production of new materials

New York University scientists have developed artificial structures that can self-replicate, a process that has the potential to yield new types of materials. The work, conducted by researchers in NYU's Departments of Chemistry and Physics and its Center for Soft Matter Research, appears in the latest issue of the journal Nature.

In the natural world, self-replication is ubiquitous in all living entities, but artificial self-replication has been elusive. The discovery in Nature reports the first steps toward a general process for self-replication of a wide variety of arbitrarily designed seeds. The seeds are made from DNA tile motifs that serve as letters arranged to spell out a particular word. The replication process preserves the letter sequence and the shape of the seed and hence the information required to produce further generations.

This process holds much promise for the creation of new materials. DNA is a robust functional entity that can organize itself and other molecules into complex structures. More recently DNA has been used to organize inorganic matter, such as metallic particles, as well. The re-creation by the NYU scientists of this type of assembly in a laboratory raises the prospect for the eventual development of self-replicating materials that possess a wide range of patterns and that can perform a variety of functions. The breakthrough the NYU researchers have achieved is the replication of a system that contains complex information. Thus, the replication of this material, like that of DNA in the cell, is not limited to repeating patterns.

To demonstrate this self-replication process, the NYU scientists created artificial DNA tile motifs --short, nanometer-scale arrangements of DNA. Each tile serves as a letter--A or B--that recognizes and binds to complementary letters A' or B'. In the natural world, the DNA replication process involves complementary matches between bases--adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C) -- to form its familiar double helix. By contrast, the NYU researchers developed an artificial tile or motif, called BTX (bent triple helix molecules containing three DNA double helices), with each BTX molecule comprised of 10 DNA strands. Unlike DNA, the BTX code is not limited to four letters--in principle, it can contain quadrillions of different letters and tiles that pair using the complementarity of four DNA single strands, or "sticky ends," on each tile, to form a six-helix bundle.

In order to achieve self-replication of the BTX tile arrays, a seed word is needed to catalyze multiple generations of identical arrays. BTX's seed consists of a sequence of seven tiles--a seven-letter word. To bring about the self-replication process, the seed is placed in a chemical solution, where it assembles complementary tiles to form a "daughter BTX array"--a complementary word. The daughter array is then separated from the seed by heating the solution to

40 oC. The process is then repeated. The daughter array binds with its complementary tiles to form a "granddaughter array," thus achieving self-replication of the material and of the information in the seed--and hence reproducing the sequence within the original seed word. Significantly, this process is distinct from the replication processes that occur within the cell, because no biological components, particularly enzymes, are used in its execution--even the DNA is synthetic.

"This is the first step in the process of creating artificial self-replicating materials of an arbitrary composition," said Paul Chaikin, a professor in NYU's Department of Physics and one of the study's co-authors. "The next challenge is to create a process in which self-replication occurs not only for a few generations, but long enough to show exponential growth."

"While our replication method requires multiple chemical and thermal processing cycles, we have demonstrated that it is possible to replicate not just molecules like cellular DNA or RNA, but discrete structures that could in principle assume many different shapes, have many different functional features, and be associated with many different types of chemical species," added Nadrian Seeman, a professor in NYU's Department of Chemistry and a co-author of the study.

The research was supported by grants from the W.M. Keck Foundation, the MRSEC Program of the National Science Foundation, the National Institute of General Medical Sciences, the Army Research Office, NASA, and the Office of Naval Research.

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Watch the video: Self Replication: How molecules can make copies of themselves (August 2022).