Peptide synthesis

Abstract

To provide a simple, inexpensive and environmentally safe method for synthesizing peptides.SOLUTION: The present invention provides a method for synthesizing a peptide, the method comprising the steps of adding a quantity of a template peptide and amino acids capable of forming copies of the template peptide into an aqueous solution, and providing a low amount of energy to the solution in order to synthesise copies of the template peptide in the solution. The energy may be provided in the form of thermal energy. The energy may include infrared, visible light or ultraviolet radiation, or mixtures thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a simple, inexpensive and environmentally safe method for synthesizing peptides. [Background technology]

[0002] Many synthetic peptides are important commercial or pharmaceutical products, ranging from the dipeptide sugar-substituted aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. Peptides are also increasingly being used in cosmetic applications, for example for application to skin and hair.

[0003] Peptides are currently generally produced by recombinant means using bacterial or eukaryotic cell cultures, or by solid-phase peptide synthesis using FMOC- or FBOC-protected amino acids. Both methods are expensive, and purification of the peptides can be cumbersome. Furthermore, solid-phase synthesis methods require large amounts of toxic organic solvents for chemical reactions, and removal of the resulting waste products accounts for approximately 10% of the overall cost of peptide production.

[0004] Native peptide ligation of peptides has been performed to provide proteins from larger peptide fragments. Typically, this requires that one half of the peptide has a reactive thioester chemistry to allow for reaction. Furthermore, this technique relies on solid phase synthesis of the two parts or halves of the desired peptide before these parts or halves are ligated together. Summary of the Invention

[0005] The present invention is based on research into the synthesis of peptides without the use of complex chemistry, high temperature and / or pressure, or DNA templates to facilitate peptide synthesis. Unlike solid phase synthesis of two parts or halves of a desired peptide and then linking these parts or halves together, the template method described herein does not rely on pre-synthesizing most of the peptide; suitably, only the constituent amino acids are required.

[0006] Unexpectedly, it has been found that template-directed synthesis of peptides can occur by providing small amounts of energy to amino acids in solution.

[0007] For example, it was determined by the inventors that samples provided in the dark with a temperature difference of between 3-5 °C based on a base of approximately 21 °C were provided with enough energy to carry out the synthesis reaction, but the reaction took longer than the exposed samples, and the longer time resulted in smaller structures being produced.

[0008] Suitably, the template method used herein allows for the use of any peptide and places more emphasis on itself. Without wishing to be bound by theory, it is believed that providing a template (the selected peptide to be synthesized) to the synthesis reaction provides two advantages. First, the cross-sectional area of ​​a peptide molecule in a concentrated solution of amino acids is larger than that of a single amino acid. Sterically, this means that there are more interactions with the template, causing accumulation or aggregation of amino acids on the template peptide surface. Secondly, it is believed that the presence of the template peptide provides organization of amino acids by structural compatibility with similar amino acids, i.e., arginine "sits" on arginine in the template peptide due to the similar structure, possibly retarding its movement. Suitably, it is believed that sequence-selective molecular recognition on complementary surfaces has an important role in peptide self-replication. It is believed that this occurs with other amino acids in the sequence, bringing them into close proximity with each other. This system provides enough energy to allow peptide bond formation to occur between amino acids and to allow template synthesis of peptides to occur.

[0009] Advantageously, the present method is believed to be distinct from prior art methods that require synthetic reactive chemistry on the ends of molecules, peptides, DNA or RNA, or bulk pre-synthesis of reactive peptide moieties for linking together.

[0010] According to a first aspect, adding an amount of a template peptide and amino acids capable of forming copies of the template peptide to an aqueous solution; providing a small amount of energy to the solution to synthesize copies of a template peptide in the solution; A method of synthesizing a peptide is provided, comprising: adding a small amount of energy to the system, the small amount being defined as sufficient energy to exceed the energy required to form an amide bond. The energy may be provided, for example, by sunlight, e.g., the entire spectrum of light applied to the system, including UV and some of the IR wavelengths of the light spectrum.

[0011] Without wishing to be bound by theory, it is believed that the ionisation products It has been suggested that it is advantageous to synthesize peptide bonds in the absence of abiotic products. Furthermore, it has been proposed that the formation of a dipeptide in a non-biogenic scenario is approximately 1.2 kcal / mole. It has also been proposed that this is 8 times more difficult than adding an amino acid to a peptide of any length and 5 times more difficult than joining two peptides of at least dipeptide size. Suitably, the energy provided may be at least 0.15 kcal / mole, at least 0.24 kcal / mole, at least 0.3 kcal / mole, at least 0.6 kcal / mole, at least 1.2 kcal / mole. As will be appreciated, sufficient energy can be provided to provide multiple peptide bonds. In an embodiment, the energy provided may be energy that allows for 1 mole of dipeptide formation. In an embodiment, the energy provided may be energy that allows for at least 2 moles of dipeptide formation, at least 3 moles of dipeptide formation, at least 4 moles of dipeptide formation, at least 5 moles of dipeptide formation, at least 10 moles of dipeptide formation, at least 15 moles of dipeptide formation, at least 50 moles of dipeptide formation, or at least 100 moles of dipeptide formation. As will be appreciated, energy may be provided to provide the required peptide formation according to the present methods.

[0012] To accurately identify the difference between the energy that can be consumed in the dehydration reaction required to create a peptide bond, we used a controlled temperature environment and control samples of PBS, as well as PBS + amino acid solutions, along with ambient temperature measurements. Measurements were taken every 5 seconds to document the heating and cooling curves with high accuracy. The difference in the area under the curve was 28962.97 °C. 2 s, i.e. 170°Cs, and the area is smaller in the PBS amino acid sample. The peak maxima in the PBS + amino acid sample were always less than in the control, suggesting that energy was always consumed in this system compared to the control. The energy gained in the PBS + amino acid sample was 1.31 kcal, suggesting that more than one mole of dipeptide could be formed in one cycle of heating and cooling from 24°C to 34°C.

[0013] It is contemplated that any form of energy may be suitably provided to enable bond formation, for example light, heat, or other electromagnetic radiation.

[0014] In particular, it has been observed that providing a small amount of energy (e.g., in the form of increased temperature and / or full spectrum light) to a solution of amino acids in the presence of a template peptide can promote the synthesis of copies of that peptide. Thus, the present method can provide a simpler and more cost-effective process of synthesizing peptides that does not require the use of nucleic acids, enzymes or coenzymes, cells or cellular material and / or organic or environmentally harmful solvents. This method can be thought of as an amplification process, where the amount of template peptide present in the solution is amplified as additional copies of the template peptide are made.

[0015] Suitably, the method of synthesising the peptide may take place in at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours.

[0016] Suitably, synthesis of the peptide may occur both under light and dark conditions.

[0017] The present invention is based on providing a small amount of energy to an aqueous solution. Previous knowledge teaches that a large amount of energy is required to support chemical synthesis or transformation, and therefore the small amount of energy used according to the present method is unexpected. However, the following experiment clearly shows the production of peptides in the absence of bacteria or other sources of peptidase, thus providing evidence that peptide production can occur based on template reactions rather than enzymatic reactions.

[0018] Without wishing to be bound by theory, the inventors believe that protein production from only the 20 standard amino acids in solution utilizing energy input, for example from the sun, allows proteins to be the starting functional, structural, and replicative material of an organism, which allows later replenished nucleic acids to replicate themselves more efficiently.

[0019] As will be appreciated by those skilled in the art, large amounts of energy can be provided to the system by application of extreme heat, intense microwave, or radioactive energy.

[0020] Mass spectrometry-based proteomics has been used by the inventors as the primary mode of analysis. Assuming that peptide and protein generation is primarily stochastic, all spectra were first identified using De Novo Sequencing (Peaks software) and the generated database was used to quantitatively analyze the data using MaxQuant. Structural characterization was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Quantification of electron microscopy images was performed using Omero. The absence of DNA and RNA was tested using fluorescence quantitation (Qubit®, Life Technologies). Samples were sterilized by gamma irradiation (1000 Gy) followed by standard peptide synthesis conditions in sealed pouches to further confirm the absence of contribution of living organisms to peptide and protein synthesis.

[0021] The energy may be provided in the form of thermal energy or heat provided, for example, by application of electromagnetic radiation. This may include, for example, infrared, visible and ultraviolet light, or mixtures thereof. Alternatively, the heat may be associated with the use of a heat pump, or may simply result from the surroundings or ambient environment, which raises the temperature of the aqueous solution.

[0022] In one embodiment, the energy supply can be constant, for example, the energy supply can include exposing the solution to a constant full spectrum light source for the duration of peptide synthesis.

[0023] As used herein, "full spectrum light" may be understood to mean light covering the infrared to ultraviolet regions of the electromagnetic spectrum. For example, a full spectrum light source may provide light having a wavelength of about 300 nm to about 700 nm. Full spectrum light may be configured to mimic the composition of natural light.

[0024] The supply of energy may include maintaining the solution at a constant temperature. For example, the solution may be maintained in the range of about 10°C to 100°C, about 15°C to 70°C, about 20°C to 50°C, or about 30°C to 45°C. As a representative example, the solution may be maintained at a temperature of about 35°C or 40°C. Suitably, the present invention may be at room temperature, at least 21°C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31°C, at least 32°C, at least 33°C, at least 34°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, 40°C.

[0025] In some cases, the solution can be maintained at about 40° C. and exposed to a constant full spectrum light source for the duration of the peptide synthesis.

[0026] Alternatively, the supply of energy to the aqueous solution may be performed in a cyclic or repetitive manner rather than in a constant manner, such that energy may be supplied for a period of time, then stopped, and then energy may be supplied again for a further period of time.

[0027] The period of energy supply can be the same or different from the period of no energy supply. For example, energy or light can be provided for 2 hours, followed by another period of no energy / light for another 2 hours. Alternatively, the period of energy supply can be shorter or longer than the period of no energy supply. In one embodiment, the supply / non-supply of energy can be in the form of a diurnal rhythm. Thus, the supply / non-supply of energy follows a regular 24-hour cycle, as is commonly seen in the supply of light and dark throughout the day.

[0028] In accordance with the above, the cyclical manner of providing energy to the solution may include increasing the heat of the solution by, for example, at least 0.25°C, 0.5°C, 1°C, 5°C, or even 10°C, 15°C, 20°C, or 100°C. If necessary, the solution is cooled to ambient temperature before application of heat occurs again. The cyclical manner of providing energy to the solution may correspond to the increase and decrease of ambient temperature during a day / night cycle. This process can be repeated many, hundreds, or thousands of times.

[0029] In some cases, the solution may be exposed to a constant light source (e.g., a full spectrum light source) but may be exposed to periodic temperature oscillations as described above. Alternatively, the solution may be exposed to a periodic supply of light (e.g., full spectrum light) under constant temperature conditions.

[0030] The method can be carried out in an aqueous solution. The solvent of the aqueous solution can include pure or substantially pure water (substantially pure water) or can consist essentially of pure or substantially pure water. For example, the aqueous solution can include or consist essentially of pure or substantially pure water, a template peptide, and amino acids capable of forming copies of the template peptide.

[0031] The aqueous solution may be sterile. As used herein, sterile may mean that the solution is free of biological contaminants. In some instances, the aqueous solution may be sterilized through the use of irradiation, such as gamma irradiation. Thus, the method relates to abiotic peptide synthesis.

[0032] The aqueous solution is free or substantially free of nucleic acids, enzymes, coenzymes (e.g., adenosine triphosphate), cells, cellular material, and / or organic solvents. For example, the aqueous solution can be free or substantially free of components thereof, such as bacteria, viruses, eukaryotic cells, and / or organelles.

[0033] By way of example, pure or substantially pure water may be MilliQ® water, which is a form of Type 1 ultrapure water (as defined by ISO 3696 (1987)). Other types of pure or substantially pure water may be provided by capacitive deionization, reverse osmosis, carbon filtration, microfiltration, ultrafiltration, ultraviolet oxidation, and the like.

[0034] Typically, such pure or substantially pure water should have low levels of solids, low organic matter, and low conductivity. For example, pure or substantially pure water may have a solids content of less than 5 μg / ml, a solids content of less than 1 μg / ml, or a solids content of less than 0.1 μg / ml. Additionally, or alternatively, pure or substantially pure water may have an organic matter content of less than 100 μg / l, or less than 50 μg / l of total organic carbon. Additionally, or alternatively, pure or substantially pure water may have a conductivity of less than 1 μS·cm at 25° C. -1 Less than 0.1μS cm -1 Less than or equal to 0.01 μS cm -1 It may have a conductivity of less than 1000 nm.

[0035] Alternatively, the aqueous solution may contain a phosphate source. For example, the aqueous solution may contain a phosphate source other than ATP or ADP. Examples of such solutions may be water containing disodium hydrogen phosphate, or phosphate buffered saline. Phosphate buffered saline (PBS) may contain disodium hydrogen phosphate, sodium chloride. In some formulations, phosphate buffered saline may further contain potassium chloride and potassium dihydrogen phosphate. An exemplary PBS composition is shown below.

[0036] [Table 1]

[0037] However, this should not be construed as a limitation. Typically, phosphate will be present in an amount of at least 50 μM, for example 100 μM, or 1 mM.

[0038] The use of a phosphate source in solution can be useful when a significant proportion of acidic amino acids are involved in the synthesis. As an example, the presence of phosphate can help maintain the template peptide (and any copies of the template peptide) in solution for the duration of the synthesis.

[0039] All available amino acids may be provided in the aqueous solution. For example, the aqueous solution may contain a mixture of all proteinogenic amino acids. Alternatively, the amino acids provided in the solution may contain a mixture of amino acids including, but not limited to, those present in the template peptide. By way of example, the solution may contain a mixture of only those amino acids present in the template peptide (e.g., only those amino acids required to synthesize the desired peptide). The amino acids may be natural amino acids, i.e., as found in nature. Suitably, the amino acids may not be activated amino acids. For example, the amino acids may not be linked to a coupling reagent such as carbonyldiimidazole.

[0040] The relative amount of each amino acid provided or added to the solution may correspond to the relative amount of each amino acid in the template peptide. For example, the amino acids may be provided in a molar ratio or stoichiometric amount equal to the molar ratio or stoichiometric amount of each amino acid found in the template peptide (e.g., the peptide to be copied and synthesized). For example, if the peptide has the sequence GlyAlaGly, Gly may be provided in the solution at twice the concentration relative to Ala. In some cases, the stoichiometric amount of the amino acid provided to the aqueous solution may be approximately equal to the stoichiometric amount of the amino acid found in the template peptide (e.g., equal to within about ±10% or ±20% of the stoichiometric amount of the amino acid found in the template peptide). Alternatively, the amino acids may be provided in equimolar amounts.

[0041] The amino acid notation used herein is conventional and is as follows:

[0042] [Table 2]

[0043] The invention can be considered to extend to the proteinogenic amino acids, the 20 standard genetically encoded amino acids (as identified above), and three additional amino acids, selenocysteine, pyrrolysine, and N-formylmethionine. As well as the more conventional L-amino acids, the invention can extend to the use of D-amino acids and post-translationally modified amino acids such as phosphorylated, glycosylated, and methylated residues.

[0044] The methods described herein can be applied to a wide variety of template peptides. The template peptide copied in peptide synthesis can include any number of amino acids, for example, 2 to 200 amino acids. Suitably, the template peptide copied in peptide synthesis can include at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids. For example, it was possible to synthesize copies of relatively long and / or difficult peptide sequences using the methods described herein. The methods described herein may be effective for template synthesis of peptides with specific structural conformations, such as conformations determined by one or more disulfide bonds between amino acids in the peptide. In general, synthesis of peptides with specific structural conformations may be expected to occur only in the presence of molecular chaperones (e.g., proteins that can assist in folding and assembling peptides into specific structural conformations). However, it was surprisingly found that copies of such peptides could be synthesized using the methods described herein. For example, it was possible to synthesize insulin (a relatively long peptide with three disulfide bonds and a specific structural conformation) using the methods described herein. The template peptide does not have to be an amyloid peptide.

[0045] Suitably, the template peptide may have a secondary structure. Suitably, the secondary structure may be substantially helical. Suitably, the secondary structure may be substantially beta-sheet. Suitably, the secondary structure of the template structure may be a combination of helix and beta-sheet.

[0046] A quantity of template peptide may be added to the aqueous solution to provide a concentration of template peptide in the solution. Typically, a very small amount of template peptide is used to synthesize copies of the template peptide in the solution. Thus, the template peptide may be provided at any concentration. The concentration of the template peptide in the solution may also vary in relation to the length of the peptide. A higher concentration of template peptide may be used to increase the synthesis rate of copies of the template peptide.

[0047] Utilizing less template in the starting material is believed to result in longer synthesis times. By increasing the template present, the reaction can proceed more quickly.

[0048] The relative amounts of amino acids and template peptide added to the aqueous solution can also be varied. For example, the total weight of all amino acids and the weight of template peptide can be provided in any w / w (weight to weight) ratio between 20,000 to 1 and 10 to 1. For example, w / w ratios of 15,000 to 1, 10,000 to 1, 5,000 to 1, 1,000 to 1, 100 to 1 and 10 to 1 w / w. As a further example, the solution can contain about 21.5 to 1 w / w of amino acids to template peptide.

[0049] The total weight of amino acids may be provided in an amount to provide a solution having a concentration of about 0.001 g / mL to 10 g / mL, or about 0.005 g / mL to 5 g / mL, or about 0.01 g / mL to 1 g / mL. For example, the solution may have an amino acid concentration of about 0.02 g / mL. It should be understood that in some cases, the concentration of the solution may depend on the composition of the template peptide. For example, different amino acids have different water solubility, and therefore, if the template peptide contains a relatively high proportion of amino acids with higher water solubility, a more concentrated solution may be used in peptide synthesis.

[0050] The amino acids and template peptides can be added to the aqueous solution sequentially or simultaneously. This method can facilitate peptide synthesis, where all the required reagents (e.g., amino acids and template peptides) can be added in solution at the beginning of synthesis. As a result, the method described herein can eliminate the need to use complex chemistry and / or multi-step processing (such as the need to use protective group strategies and / or solid phase components).

[0051] By providing a template peptide in the present method, it is possible to obtain large quantities of high quality copies of the template peptide, typically at least 80%, 90%, 95%, 99%, 99.5% or more of the peptides produced are identical to the template peptide.

[0052] The progress of peptide synthesis can be monitored. For example, the amount of synthetic peptide in aqueous solution can be monitored using mass spectrometry (MS) techniques (e.g., matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion trap electrospray MS).

[0053] As will be appreciated, initially, an increase in the amount of synthesized peptide may be observed as amino acids react to form copies of the template peptide. The amount of synthesized peptide may then plateau (e.g., as all starting amino acids are consumed). Alternatively, after an initial increase, the amount of synthesized peptide may decrease (e.g., when additional amino acids begin to be added to the synthesized peptide, increasing the length of the peptide chain). Thus, at least in some cases, peptide synthesis may be terminated when the observed amount of synthesized peptide reaches a plateau or decreases.

[0054] The duration of peptide synthesis may depend on the length of the template peptide and / or the nature of the amino acids therein. For example, a shorter template peptide may be copied more quickly than a longer template peptide. Thus, monitoring peptide synthesis may be used to experimentally determine the duration of the process that provides a sufficient amount of the desired peptide. By way of example, the synthesis process may occur for several hours (e.g., 6, 12, 18 or 24 hours) or several days to provide a sufficient amount of the desired peptide. For example, the synthesis process may occur for at least 1, 2, 3, 4, or 5 days, or even 12, 20, 25, 30, or 50 days or more.

[0055] Termination of peptide synthesis can be performed by removing the small amount of energy from the solution. As a representative example, peptide synthesis can be terminated by cooling the aqueous solution (e.g., to below 10° C., below about 5° C., or below about 0° C.). Alternatively or additionally, peptide synthesis can be finally terminated by separating the synthesized peptide from the aqueous solution.

[0056] The peptides synthesized in the present methods may require minimal (or in some cases no) purification, for example, the peptides synthesized in the present methods may be reasonably homogeneous.

[0057] If further purification is required, the synthetic peptides may be purified from the aqueous solution in which the synthesis process takes place to remove the desired synthetic peptide from amino acids and shorter, longer, and / or miscopied peptides, as necessary.

[0058] Typically, purification can be performed by chromatographic means such as high performance liquid chromatography (HPLC). Examples of suitable HPLC methods include reversed phase HPLC, ion exchange HPLC and gel filtration HPLC, and such HPLC methods can be performed individually or in combination / tandem. The progress of peptide purification can be monitored by mass spectrometry (MS) techniques, including matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion trap electrospray MS. For example, sequencing by Edman degradation sequence analysis or in tandem with MS can be used to confirm the homogeneity of the peptide. Suitably, purification can occur using a binding member, such as an antibody or a fragment thereof, capable of selectively binding the peptide / protein of interest provided by the method. Suitably, the peptide / protein of interest provided by the method can be provided with a tag that allows it to be purified. Suitably, beads with a binding member specific for the peptide / protein of interest provided by the method or beads to a tag attached to the peptide / protein of interest provided by the method can be utilized.

[0059] The method of synthesizing peptides can be carried out at atmospheric pressure (e.g., 1 bar±0.2). Additionally or alternatively, the method can be carried out in air, e.g., in the presence of oxygen. For example, the method typically does not require a reducing gas environment and / or high pressure. Template synthesis of peptides can occur without the application of significant amounts of energy (e.g., as provided by an electric spark, etc.).

[0060] The method may be a batch process, or the process may be a continuous process in which the synthesized peptide is removed over time.

[0061] The method can be carried out in a vessel or reaction vessel. In some examples, the vessel or reaction vessel may be covered or sealed (e.g., to reduce the risk of contamination).

[0062] According to a further aspect of the invention, there is provided the use of a template peptide for amplification of itself (the template peptide itself), i.e. the use of the peptide as a template for peptide amplification, where the amplification involves overlapping of the peptide using the peptide as a template.

[0063] For example, amplification can include synthesis of additional copies of the template peptide. Amplification can be driven by the template peptide itself, in the absence of nucleic acids, enzymes or coenzymes, cells or cellular material. That is, amplification does not require an enzyme capable of forming a peptide bond, such as a peptidyl transferase. The amplification solution requires amino acids to incorporate into the growing double peptide chain.

[0064] Furthermore, throughout this specification the term "comprising" is used to indicate that an embodiment of the invention "comprises" a mentioned feature and may therefore include other features as well. However, in the context of the present invention the term "comprising" may also encompass embodiments in which the invention "consists essentially of" or "consists of" the relevant feature.

[0065] The applicant discloses each individual feature described herein, and any combination of two or more such features, independently, to the extent that such feature or combination can be implemented based on the specification as a whole, in light of the common general knowledge of a person skilled in the art, without being limited to the scope of the claims, regardless of whether such feature or combination solves any problem disclosed herein. The applicant indicates that an embodiment of the present invention may consist of any such individual feature or combination of features. In view of the above description, it will be apparent to a person skilled in the art that various modifications are possible within the scope of the present invention.

[0066] The invention will now be further described, by way of example only, with reference to the following drawings in which: [Brief description of the drawings]

[0067] [Figure 1A] Quantitative analysis of template peptide synthesis using mass spectrometry (Thermo Q Exactive Orbitrap) for synthetic peptide VR15 (SEQ ID NO: 1-VPDNLQQSLSDEAQR, this peptide does not occur in nature). The newly synthesized peptide is distinguished from the template peptide by the incorporation of heavy arginine (R, here denoted SILAC), meaning that this peptide can be unambiguously measured from its template peptide in mass spectrometry. [Figure 1B] The UV absorbance of the samples over time is shown, showing a significant increase in the signal of the novel peptide compared to time point 0. [Figure 1C] The completed chromatograms are shown. All samples were amplified with an internal standard (benzoic acid) that did not change throughout the process. This can be seen in Figures 1B and 1C. [Figure 1D] The intensities measured in the mass spectrometer are shown (intensity represents the ions detected in the mass spectrometer and is a direct indication of quantity). [Diagram 2]The amount by weight of template peptide before and after template peptide synthesis is shown for four different peptides; insulin (110 amino acids long), MRFA (4 amino acids long), VR9 (a synthetic peptide not found in nature, VMDSSYLSR, 9 amino acids long, SEQ ID NO:2), and WK20 SEQ ID NO:3 (a synthetic peptide not found in nature, WRWLEHNVVEGNAVNLMFSK). This shows that peptides of any length can be amplified and that structures and disulfide bonds do not present a problem in the amplification process. [Diagram 3] Quantitative analysis of MRFA peptide synthesis using mass spectrometry (Thermo Q Exactive Orbitrap) in the presence of MRFA template peptide ("Amplification") and in the absence of template peptide ("Control") is shown, demonstrating that addition of the template indeed catalyzes the production of the peptide. [Figure 4] The experimental design for subsequent experiments to demonstrate the lack of contamination present in the exemplified peptide synthesis process is shown. Additional control samples containing sodium azide (NaN3), chloramphenicol (Chlor) and D-amino acids (Daa's) were used to further ensure the absence of biological contamination. [Diagram 5] The results of the test for the presence of DNA and RNA are shown. Samples were tested for the presence of DNA and RNA using the high sensitivity Qubit® kit and an indication of the observed intensity is shown. After the sample changed color, no RNA was detected and DNA was detected. To verify if this was indeed DNA, benzonase (which digests DNA and therefore eliminates the signal of detectable DNA in the sample) was applied to the sample and incubated. A positive control was always used and the intensity of the positive control always dropped by more than half, while the peptide synthesis sample "DNA intensity" did not drop significantly, confirming the absence of "lifeforms" (i.e. DNA and RNA) in the peptide synthesis sample. [Figure 6A]Figure 1 shows the results of Marfey's reagent derivatization of D and L amino acids A. The chromatogram shows an overlay of a mixture of four commercially available L amino acids (black trace) with a mixture of the same four amino acids except for the D isomers (red trace). A delay in retention time can be seen for the D amino acids due to derivatization with Marfey's reagent (FDAA, 1-fluoro-2-4-dinitrophenyl-5-L-alaninamide). [Figure 6B] Figures 6B and 6C show a non-biological D-amino acid sample (red trace) taken on day 28, hydrolyzed to single amino acids, and derivatized with Marfey's reagent overlaid with four commercially available L-amino acid chromatograms (black trace). No conversion of D-amino acids to L-amino acids was observed (this would be expected to occur if biological organisms contaminating the sample were utilizing D-amino acids). [Figure 6C] Non-biotic D amino acid samples are shown. [Figure 7A] Results of a gamma irradiation experiment are shown (all samples were performed in triplicate). A positive control for sterilization was included in the form of an E. coli culture. The non-irradiated sample (Figure 7A, top) showed numerous colonies, while the irradiated sample (Figure 7A, bottom) showed no growth after 1000 Gy irradiation, demonstrating effective sterilization. [Figure 7B] 1 is a histogram showing peptide intensity distribution. The dark green population is the starting material, which has a lower peptide count (because this is a template-guided experiment, i.e., the only products are the peptides themselves and the proportion of miscopies occurring in this process is small) and a lower median intensity. The standard incubated sample produced more peptides (i.e., more copies of the peptide) with higher intensity, and the irradiated sample showed an increase in peptide production similar to the non-irradiated sample. The lack of clear differences between these two sample sets suggests that this is a chemical process that is not affected by any kind of external biological contamination. [Figure 7C]Venn diagram showing the colocalization of peptides produced between conditions. Templated production of peptides between irradiated and non-irradiated samples was comparable, demonstrating that the reaction is chemical, independent of bacterial / biological contamination. [Figure 8] A Venn diagram showing the coexistence of detected peptides between various conditions (chloramphenicol, 37°C, standard, sodium azide (NaN3), and D-amino acids) is shown. Chloramphenicol is a broad spectrum antibiotic and sodium azide is a Gram-negative bacterioside, and for D-amino acids, most living organisms require L-amino acids, and D-amino acids are converted to L-amino acids when utilized by living organisms, but at the concentrations used in these experiments, conversion does not occur in this case (see Marfey's reagent experiments), and bacterial growth should be eliminated. Peptide synthesis in all experiments was comparable, indicating a chemical basis for synthesis as opposed to a biological origin of peptides. [Figure 9A] Figures 9A-H show MALDI-TOF mass spectrometry of commercially available amino acids. These spectra show that the starting material is pure and does not contain any larger molecules that may contribute to the peptides seen in the experiment. Figure 9A shows the MALDI-TOF spectrum of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine and cysteine. [Figure 9B] Zoomed mass spectra of MALDI-TOF of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine and cysteine. [Figure 9C] Zoomed mass spectra of MALDI-TOF of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine and cysteine. [Figure 9D]1 is a MALDI-TOF mass spectrum of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine, and alanine. [Figure 9E] Zoomed mass spectra of MALDI-TOF of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine and alanine. [Figure 9F] Zoomed mass spectra of MALDI-TOF of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine and alanine. [Figure 9G] 1 is a HMALDI-TOF mass spectrum of glutamic acid and histidine. [Figure 9H] Zoomed mass spectrum of HMALDI-TOF of glutamic acid and histidine. [Figure 10] 1 shows a schematic diagram of the method of the present invention. [Figure 11A] 1 shows insulin synthesis using the method of the present invention. [Figure 11B] 1 shows insulin synthesis using the method of the present invention. [Figure 12A] 12A-12C show the experimental setup to test the requirement of sunlight and phosphate for the spontaneous polymerization of amino acids into peptides and proteins. All samples were performed in triplicate. [Figure 12B] The experimental set-up for all subsequent experiments with greater sterility is shown. Additional control samples containing sodium azide, chloramphenicol and D-amino acids were used to further ensure the absence of biological contamination. [Figure 12C] The experimental setup is shown. [Figure 13] The correlation (Log2 of peptide intensities) of all collected time points in the experiment is shown in FIG. 12, and the Venn diagram shows that the majority of peptides seen were shared between samples. [Figure 14A]Figures 14A-D show electron microscopy images of structures generated by protein synthesis by this method. The relative scale between TEM and SEM can be seen (Figure 14A), and the structures are very similar in appearance to viral capsids (right panel of Figure 14A). [Figure 14B] The structures were measured and counted using Omero software, and the results are graphed in Figure 14B. The PBS Sun sample produces larger, fewer structures, while PBS Dark and MilliQ Sun produce many, many smaller structures. [Figure 14C-1] Electron microscopy images are shown. The reproducibility of the biological replicates is highly remarkable and can be seen in the panels. [Figure 14C-2] Electron microscope images are shown. [Figure 14C-3] Electron microscope images are shown. [Figure 14C-4] Electron microscope images are shown. [Figure 14C-5] Electron microscope images are shown. [Figure 14C-6] Electron microscope images are shown. [Figure 14D] Electron microscope images are shown. [Figure 15A] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15B]Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15C] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15D] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15E]Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15F] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15G] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15H]Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15I] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15J] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15K]Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 15L] Ultrathin sectioning was used to visualize the interior of the spheres. In early samples (A, B, C, ~60 days), linear modified chains of proteins are prevalent. In later samples (J, K, L, 678 days), globular structures are the majority of those seen. These vary from completely empty spheres (F), to lightly filled spheres, and very rarely densely packed structures. Compared to traditional cross sections of cells, there is a clear lack of structure and noticeable voids, but these are nevertheless cell-like and could possibly represent prototypic cellular structures. [Figure 16A] Peptide pair analysis revealed the most common and least common amino acid pairs found in all identified peptides. It appears that non-polar amino acids (I, L, G) are favored to pair with acidic amino acids (E, D). The least favored pairings are non-polar amino acids with basic amino acids (as expected, the least abundant amino acids in the mixture). [Figure 16B] The amino acid utilization in the most stable peptides found in all samples is listed. There does not appear to be a trend in amino acid chemistry (polar, non-polar and acidic amino acids all have increased utilization). Aspartic acid and asparagine have increased utilization but are thought to destabilize the helix. [Figure 16C-1]Examples of some of the most stable peptides. Random coils are present, but there is a tendency towards helical conformations, suggesting that hydrogen bonding of the backbone NH groups to the backbone C=O groups of the amino acids four residues earlier is sufficient to stabilize in this situation. Structures predicted by PEP-FOLD (http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD). [Figure 16C-2] Examples of some of the most stable peptides. Random coils are present, but there is a tendency towards helical conformations, suggesting that hydrogen bonding of the backbone NH groups to the backbone C=O groups of the amino acids four residues earlier is sufficient to stabilize in this situation. Structures predicted by PEP-FOLD (http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD). [Figure 17A] Figures 17A and 17B show constant heat of samples in the dark at 37°C versus the same samples but set up with natural light source. Log2 intensity values ​​of standard set up proteins / peptides that showed an increasing trend over time (indicating stable peptide production and longevity) were subtracted from the values ​​of the 37°C samples and the resulting values ​​were clustered. [Figure 17B] Show the contrast. [Figure 18A]18A-18C show a proposed method for the initial polymerization of amino acids by the presence of amino acids in solution assisted by energy (e.g. provided by solar energy) to exceed the energy required to form an amide bond, where the subsequent addition of amino acids to an existing dipeptide is selected in two ways (B). (i) The enlarged cross section increases the probability of collision with an amino acid in the correct orientation. (ii) The energy required to add an amino acid to an existing dipeptide is 8 times less than the initial amide bond. The additional local chemical environment favors the presence of the peptide / protein and induces folding of the peptide structure excluding water molecules, forming secondary structures supported by hydrogen and disulfide bonds (C). This generates structural stability and therefore a long life of the formed peptide / protein. A reduction in entropy occurs by organizing the protein forming a globular cell-like structure (D). This also protects the formed structures and selects in favor of these structures. [Figure 18B] The proposed method is shown. [Figure 18C] The proposed method is shown. [Figure 19A]Figures 19A-C show the setup to measure temperature fluctuations on the window frame (A), then characterize the amino acid differences in the solutions created in a controlled heated environment without sunlight, and the amount of energy consumed in the setup (B), and finally the fully controlled environment used for all subsequent experiments (C). Using the Peltier chip, a solar replicating light bulb (with a wide range of wavelengths including UV) was added to heat and cool the system. Temperature was monitored with a medical grade temperature sensor. The setup was heated to 40°C continuously for 12 hours with the light bulb illuminated, and cooled to ambient temperature (~18°C) for 12 hours with the light bulb off to replicate the controlled circadian cycle. The data presented here, generated using the controlled environment, was a sterilization experiment (1000Gy irradiated sample). The temperature was measured on the window frame every 5 minutes with an accuracy of ±0.0625°C, and the temperature fluctuation on the window frame was 10.8125°C (2096.7 J°C-1g-1), while the temperature fluctuation of the "dark" sample was 0.4444°C (185.8 J°C-1g-1). To more accurately identify the difference between the energy that can be consumed in the dehydration reaction required to make peptide bonds, we used a controlled temperature environment and control samples of PBS and PBS + amino acid solutions with ambient temperature measurements. Measurements were taken every 5 seconds for the description of the heating and cooling curves with high accuracy, and the difference in the area under the curve was 28962.97°C2, i.e. 170°Cs, and the area was less for the PBS amino acid sample. The maximum value of the peak in the PBS + amino acid was always less than the control, suggesting that energy was always consumed in this system compared to the control. The resulting energy utilized in the PBS + amino acid sample was 1.31 kcal, suggesting that one cycle of heating from 24°C to 34°C and cooling was capable of forming more than one mole of dipeptide. It was determined that biological replicates for each condition were significantly correlated with each other (Pearson correlation up to 0.96) after 7 days of "growth," but that this correlation widened over the course of ~126 days of growth, but always remained positive.This is hypothesized to result from the formation of larger structures (as evidenced by TEM and SEM) and a tapering efficiency of peptide replication (through steric hindrance) as hydrophobic regions induce folding and reduce access to the protein surface. [Figure 19B] The device is shown. [Figure 19C] The device is shown. [Figure 20] The utilization of amino acids in the sample setup is shown. The percentage of amino acids in TrEMBL % 2013 represents the percentage of amino acids in the starting solution. The difference in the utilized amino acids compared to the starting material is likely due to several factors such as trypsin digestion, which introduces a bias (and therefore what can be measured by mass spectrometry), the chemical stability of the amino acid sequence combinations, and the affinity to form amide bonds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] [Method and Results] [Experiment 1] To distinguish template peptides from product peptides, synthesis experiments were performed utilizing heavy isotope-labeled amino acids, which were quantified using chromatographic analysis (an industry standard for purity) and mass spectrometry with an internal standard (benzoic acid) to quantify the peptides (both the light (template peptide) and heavy (newly synthesized) versions).

[0069] Eleven micrograms of synthetic peptide VR15 (VPDNLQQSLSDEAQR) SEQ ID NO: 1 was added to 1600 μl of an amino acid solution containing arginine, which is 6 daltons heavier than normal arginine, at a concentration of 0.2 g / ml. Samples were incubated at 37° C. with a full spectrum light source continuously for up to 4 hours.

[0070] Samples were taken at 2 and 4 hours of synthesis and run in triplicate on a reversed-phase C18 chromatography column measuring UV absorbance at 216 nm (chromatograms in Figure 1B and Figure 1C) and on a mass spectrometer to measure the intensities of the heavy and light versions of the peptide (Figure 1D).

[0071] 〔result〕 The consistent increase in signal after 2 hours in the chromatograms of the synthetic peptide (see FIG. 1A) and the lack of an increase in signal in the internal control (benzoic acid peak) clearly indicates that more VR15 was produced. The increase in signal was not due to instrumental variation or any other external factors.

[0072] SILAC quantification and labeling of newly synthesized peptides allows us to distinguish between old and new peptides, which is also evident in the resulting peptide intensities measured by mass spectrometry.

[0073] [Experiment 2] During initial experiments, it was found that proteins and peptides could be synthesized in the absence of cellular organelles and nucleic acids. Specific peptides were then selected to investigate the template amplification process.

[0074] The following peptides were selected for several factors. (1) They do not occur in nature (except for insulin). (2) They showed high copy numbers in the first experiments, suggesting that they were amplified. Some peptides were predicted to have long life spans and unstable structures that were not conducive to replication. However, over 8000 different sequences could be reproducibly generated. (3) They were of various lengths to demonstrate the utility of the template amplification process for a range of different peptides.

[0075] The peptide sequences initially investigated are shown in Table 1 below.

[0076] [Table 3]

[0077] The concentration of an amino acid represents the total weight of the amino acid per mL of solution. In each case, the relative amount of each amino acid present in the solution is proportional to the amount of the amino acid in the template peptide. Taking TR8 as an example, the composition of the amino acid mixture that constitutes a 0.01 g / mL solution is approximately: T-0.0013 g / ml, G-0.0013 g / ml, A-0.0013 g / ml, S-0.0026 g / ml, L-0.0013 g / ml, N-0.0013 g / ml, and R-0.0013 g / ml (no additional amino acids are present).

[0078] Table 1 shows a number of solutions used in the template peptide synthesis process.

[0079] The solutions shown in Table 1 were prepared in a sterile environment (such as a laminar flow hood) and filtered through a 0.22 μm filter. The filtered solutions were placed into autoclaved Schott bottles, sealed, and then placed into the amplification device.

[0080] The amplification device subjected the samples to a controlled environment with a consistent temperature of 40° C. and a constant full spectrum light source (note that this device can be configured in any manner).

[0081] Samples were taken daily under sterile conditions (100 μl) and analyzed using MS (10 ml injection, 15 min gradient, 2%–80% acetonitrile, 0.1% formic acid, Thermo Q Exactive Orbitrap) to determine peptide concentration over time.

[0082] If a decrease in intensity was observed, the amplification process was stopped by placing the samples in a refrigerator. Absolute termination of the amplification is achieved by chromatographic separation of the peptides from the reagents (amino acids in solution).

[0083] Chromatographic separation of products from reagents was achieved using reverse phase chromatography (mobile phases used were A: 2% acetonitrile, 0.1% formic acid vs. B: 80% acetonitrile, 0.1% formic acid) using a high speed, large capacity C18 column.

[0084] 〔result〕 The amount of peptide produced was measured after purification and the results are shown in Table 2 below.

[0085] [Table 4]

[0086] Table 2 shows the amount of template peptide before and after the amplification process.

[0087] In each case, the amount of template peptide increased after the amplification procedure, as shown in FIG.

[0088] It was observed that the shorter the peptide, the more rapid the amplification process, with the subsequent decrease in intensity being due to the addition of additional amino acids onto the product peptide, resulting in the loss of a large portion of the desired / template peptide.

[0089] Mass spectral analysis for the synthesis of MRFA in the absence of a template (negative control, Table 1), compared to templated synthesis (performed in the presence of the MRFA template), is shown in Figure 3. In contrast to templated synthesis, no appreciable difference in the intensity of the MRFA is observed in peptide syntheses performed in the absence of a template peptide.

[0090] [Experiment 3] [Excluding other causes of peptide amplification] To exclude the possibility of the presence of contamination (i.e. peptides present in the starting material, carryover in the chromatographic process, bacterial / viral contamination, or any kind of "lifeform"), the following experiment was performed. (1) Analysis of a sample for the presence of DNA and RNA. (2) The use of D-amino acids to amplify peptides (because most organisms cannot use them, or if they do, convert them to L-amino acids). (3) The use of gamma irradiation to sterilize samples and see if amplification is still occurring. (4) Conduct the experiment in the presence of chloramphenicol or sodium azide. (5) Test the purity of starting components.

[0091] Please refer to Figure 4.

[0092] 〔method〕 Commercially available amino acids were measured according to the proportions in Table 3 below to a total concentration of 1 g / 100 ml. Amino acids were solubilized in sterile PBS (gibco 1x DPBS 14190-094). The solution was mixed until complete solubilization occurred, then passed through a 0.22 μm stericup filter and 2 ml was decanted per autoclaved vial in a cell culture fume hood with sterilization equipment. Vials were not opened again until an aliquot was required at a specific time point (again, all done in a sterile environment). Samples were either analyzed that day (if prior to incubation on day 14) or immediately reduced, alkylated, followed by in-solution digestion (with trypsin). The remainder of the sample was frozen at -80°C. Aliquots were taken for DNA and RNA analysis and were also analyzed on the day of collection.

[0093] [Table 5]

[0094] Table 3 shows the total percentage of each of the 20 amino acids commonly found in the UniProtKB / TrEMBL protein database release October 2013 (http: / / www.ebi.ac.uk / uniprot / TrEMBLstats).

[0095] [DNA and RNA detection] Qubit commercial reagents were used according to the instructions for Qubit® dsDNA HS Assay Kit, Life Technologies, Q32851, and Qubit® RNA HS Assay Kit, Life Technologies, Q32855. 20 microliters of sample was used for the assay. Samples were measured on a Qubit® 2.0 fluorometer, Q32866.

[0096] Marfey's analysis of D amino acid composition One hundred microliters of each biological replicate of the D-amino acid sample (T28 = day 28) and standard sample (T28 = day 28) were pooled together to obtain one sample for each condition. The samples were dried on a rotary evaporator. 1 mL of 6 M HCl was then added to the samples and they were heated at 155 °C for 80 min. The samples were then dried on a rotary evaporator. The samples were reconstituted in 300 μl of MilliQ water and the pH was adjusted (to >5) with 3 μl of 10 M NaOH. One hundred microliters was combined with 200 μl of Marfey's reagent (10 mg / ml in acetone) and 40 μl of 1 M ammonium bicarbonate and gently shaken at 40 °C for 1 h. The reaction was terminated with 20 μl of 2 M HCl.

[0097] 100 microliters of each sample was added to the XBridge® BEH 130 Injections were performed on a C18 column, 130 Å, 3.5 μm, 4.6 mm × 250 mm. Amino acids were separated using a gradient of 20% to 65% B over 45 min (A: 0.1% formic acid, B: 80% acetonitrile, 0.1% formic acid) and UV absorbance was measured at 340 nm.

[0098] Mass acquisition was performed using a Thermo Orbitrap QEaxactive, top 10 ions selected for ms / ms, with ms resolution of 140,000 and ms / ms resolution of 17,500, scanning from 150 m / z to 2000 m / z.

[0099] [Gamma ray irradiation] Samples were prepared as described above (solubilization of amino acids, filtration and sterilization) except that VR15 was used as the template peptide and only the amino acids necessary to construct the peptide were used. As a positive control for sterilization (irradiation at 1000 Gy in a sealed plastic bag), samples were prepared in triplicate as well as a vial containing live E. coli. The positive control was then spread on an agar plate to ensure that sterilization had occurred, and the peptides in all the resulting samples were analyzed by mass spectrometry (as described above).

[0100] 〔result〕 [DNA and RNA detection] Referring to Figure 5, in a process testing for the presence of RNA and DNA in a sample, RNA was never detected (lower limit of detection < 20ng / ml), however, samples subjected to a full spectrum light source changed color over time, apparently resulting in a positive reading for DNA.

[0101] To determine if this was truly DNA, benzonase (an enzyme known to degrade DNA) was added to the sample, which was then incubated for 1 hour. A positive control with benzonase (which has a known bacterial contamination) was used. The DNA intensity was roughly halved in the positive control, whereas the DNA intensity was unchanged in the peptide synthesis samples. It was therefore concluded that some of the peptides / proteins formed in this manner autofluoresce at the same wavelengths as DNA (485 / 530 nm).

[0102] [D Amino Acid Analysis] Most naturally used amino acids are the L-chiral forms of amino acids, and most organisms cannot use them in standard protein production (although they are commonly found in peptidoglycan proteins in bacterial cell walls). The L-forms of methionine, serine, alanine, and tyrosine were substituted for the D-forms in the normal amino acid mixture above, but otherwise the same experimental conditions were used.

[0103] The samples were then tested to see if the D-amino acids had been converted to L-amino acids by an isomerase (e.g., such isomerases would be expected to be present if a contaminant / organism was present in the sample). A reagent known as Marfey's reagent (Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) was utilized. When reacting with D-amino acids, Marfey's reagent alters the retention times of these amino acids (when separated using chromatography) compared to the L-versions of the same amino acids.

[0104] The chromatograms shown in Figures 6A-6C show this shift in retention time from samples 28 days after synthesis. These data further indicate that no bacterial cell contamination was present in the samples.

[0105] [Gamma ray irradiation] Synthetic peptide synthesis experiments were performed in triplicate with 1000 Gy of radiation applied to samples that were then sealed to maintain the sterility achieved by radiation. A positive control of E. coli culture was included to ensure that sterilization had occurred. If peptide synthesis was still occurring, it indicated that the treatment was not the result of contamination in the form of life forms in the samples.

[0106] Positive controls for sterilization were included in the form of E. coli cultures: the non-irradiated sample (Figure 7A, top) showed numerous colonies, while the irradiated sample (Figure 7A, bottom) showed no growth after 1000 Gy irradiation, demonstrating effective sterilization.

[0107] Figure 7B is a histogram showing peptide intensity distribution. The dark green population is the starting material, which has a low peptide count (because not many peptides were produced) and a lower median intensity. The standard incubated sample produced more peptides (i.e., more copies of the peptide) with higher intensity, and the illuminated sample showed an increase in peptide production similar to the non-illuminated sample.

[0108] Figure 7C is a Venn diagram showing the colocalization of peptides generated between conditions. The stochastic generation of peptides between irradiated and non-irradiated samples is comparable.

[0109] The lack of clear differences between these two sample sets suggests that peptide synthesis is a chemical process that is not affected by external living contaminants in the samples.

[0110] Chloramphenicol and sodium azide-containing samples Chloramphenicol and sodium azide samples were performed in conjunction with a standard sample setup (sterile vials and amino acids solubilized in PBS) to allow for direct comparison with previously observed abiotic peptide syntheses. Peptides were analyzed in the same manner and compared to each other for significant differences.

[0111] The Venn diagram shown in Figure 8 indicates that the majority of peptides seen are identical in all samples. Furthermore, there was no significant deficit in peptide production in samples containing bacteriosides, which would be expected to be reduced if synthesis was the result of any bacterial or viral contamination. This result further demonstrates that the peptide synthesis process described is chemical.

[0112] [Starting material contamination test] All commercially available amino acids, solvents and digestion enzymes (trypsin) were independently analyzed by mass spectrometry and subjected to de novo peptide identification software to determine whether peptides were present in the first place.

[0113] In all cases, no unexpected masses greater than the stated reagents were observed.

[0114] [Example of spontaneous peptide formation] Consider the hypothesis that "spontaneous peptide formation requires sunlight and phosphate." Samples were set up in three different conditions (Figure 12A) based on two assumptions. The first assumption was that phosphate would be required if the unit of energy used by most life forms is ATP, and the transfer of a single phosphate molecule, and therefore a simple source of phosphate in the form of phosphate buffered saline was used. This had the additional property of being loose, similar to seawater (the environment in which life may have begun in the Archean Era 4 billion years ago). The second assumption was that energy from the sun was required to put in enough kilojoules to go beyond the amide bond formation step. Thus, the samples were: PBS Sun, PBS Dark, MilliQ Sun. The sample groups were intended to be "PBS Sun" a proven positive, "MilliQ Sun" a negative control, and "PBS Dark" another negative control.

[0115] However, protein formation was determined to be evident in all conditions. Thus, the inventors have determined that spontaneous, cell- and nucleic acid-independent protein synthesis occurs in solution, even with minimal temperature fluctuations, when the building blocks for life (amino acids) are present (Figure 13).

[0116] Example - Testing the hypothesis that "constant heating (37°C) is better for spontaneous peptide production than cycling heat for 1 day." Comparison of peptide / protein synthesis occurring in both standard samples (samples provided at room temperature on the window frame) and 37°C samples showed an increasing trend over time (indicating stable peptide production and longevity). The data showed that sunlight (variable wavelength) and cycling temperature were preferred over constant temperature (and no sunlight, Figures 17A and 17B). This effect is likely greater than the change seen here due to the fact that the energy input at 37°C was 24-7, whereas the energy and light input received on the window frame was at most half this.

[0117] [Conclusion] A method for peptide synthesis is described in detail above, based on the previously unknown property of proteins and peptides acting as templates to replicate themselves when placed in a solution of amino acids. Accordingly, it has been extensively demonstrated by several different means (gamma irradiation, D-amino acids, bactericides, and testing for the presence of DNA and RNA) that this process is a chemical reaction and proceeds in the absence of nucleic acids, enzymes, coenzymes, other cellular material, and / or cells. Rather, the inventors believe that the templated peptide synthesis described herein can be driven by the provision of small amounts of energy (e.g., in the form of full spectrum light (including IR and UV) and / or gentle, constant heat), reagent stoichiometry, and / or stereochemistry.

[0118] The spectra included in Figures 9A-9H are MALDI-TOF spectra verifying the purity of the commercially purchased starting amino acids. The inventors confirmed that they contained nothing but the specific amino acids (and also checked for polymerization of those amino acids into larger peptides). This was indeed the case, and the starting material was pure.

Claims

1. 1. A method for synthesizing a peptide, comprising the steps of: adding an amount of a template peptide and amino acids capable of forming copies of the template peptide to an aqueous solution; providing a small amount of energy to the solution to synthesize copies of a template peptide in the solution; A method comprising:

2. 10. The method of claim 1, wherein the energy is provided in the form of thermal energy.

3. 3. The method of claim 1 or 2, wherein the energy comprises infrared, visible or ultraviolet light, or a mixture thereof.

4. The supply of energy is constant, 4. The method according to any one of claims 1 to 3, characterized in that optionally, the supply of energy comprises exposing the solution to a constant full spectrum light source for the duration of the peptide synthesis.

5. 5. The method according to claim 1, wherein the energy is at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, at least 1.2 kcal / mol for peptide formation.

6. 6. The method of any one of claims 1 to 5, characterized in that the supply of energy comprises maintaining the solution at a constant temperature of about 10°C to 100°C, about 15°C to 70°C, about 20°C to 50°C, or about 30°C to 45°C.

7. 4. The method according to claim 1, wherein the supply of energy to the solution is periodic.

8. 8. The method of claim 7, wherein the periodic supply of energy comprises periodically increasing the heat of the solution and / or periodically exposing the solution to full spectrum light.

9. The method according to any one of claims 1 to 8, characterized in that the solvent of the aqueous solution is pure or substantially pure water, or the aqueous solution comprises phosphate buffered saline.

10. 10. The method of any one of claims 1 to 9, wherein the aqueous solution consists or consists essentially of pure or substantially pure water, a template peptide, and amino acids capable of forming copies of the template peptide.

11. The method according to any one of claims 1 to 10, characterized in that the aqueous solution is sterile.

12. The method according to any one of claims 1 to 11, characterized in that the aqueous solution contains only amino acids present in the template peptide.

13. 13. The method according to any one of claims 1 to 12, characterized in that the aqueous solution contains amino acids in stoichiometric amounts equal or approximately equal to the stoichiometric amounts of each amino acid found in the template peptide.

14. 14. The method according to any one of claims 1 to 13, characterized in that the total weight of all amino acids present in the aqueous solution and the weight of the template peptide are provided in a w / w (weight to weight) ratio of 20,000:1 to 10:1, or of 10,000:1 to 10:

1.

15. 15. The method according to any one of claims 1 to 14, characterized in that the total weight of the amino acids is provided in an amount such as to provide a solution having a concentration of about 0.001 g / mL to 10 g / mL, or about 0.005 g / mL to 5 g / mL, or about 0.01 g / mL to 1 g / mL.

16. A method according to any one of claims 1 to 15, characterized in that the peptide synthesis is terminated after a period of between 3 and 24 hours.

17. A method according to any one of claims 1 to 16, characterized in that the peptide synthesis is terminated after a period of 1 to 5 days.

18. Peptide synthesis, 18. The method according to any one of claims 1 to 17, characterized in that it is terminated by removing from the solution said small amount of energy sufficient to cause peptide bond formation and / or by separating the synthetic peptide from the aqueous solution.

19. The process according to any one of claims 1 to 18, characterized in that the process is carried out at atmospheric pressure and / or in the presence of oxygen.

20. 20. The method according to any one of claims 1 to 19, characterized in that the peptide synthesis is carried out in the absence of nucleic acids, enzymes, coenzymes, other cellular material and / or cells.

21. The use of a template peptide for the amplification of the template peptide itself in the absence of nucleic acids, enzymes or coenzymes, other cellular material, or cells.

22. 21. A peptide provided by the method of any one or more of claims 1 to 20.

23. A structure provided by a peptide according to claim 22 or by a method according to any one of claims 1 to 19.

24. 21. A kit for use in the method of any one of claims 1 to 20 for synthesizing copies of a template peptide in a solution comprising the template peptide and, optionally, at least one amino acid in the template peptide, each amino acid being necessary to form the template peptide.

Images