Peptide synthesis

Abstract

Methods of synthesizing peptides based on amino acid sequences of template peptides, peptides provided by the methods, and uses of peptides are provided.

Description

[0001] This invention relates to a simple, inexpensive, and environmentally safe method for synthesizing peptides.

[0002] background Many synthetic peptides are important commercially available or pharmaceutical products, ranging from dipeptide-substituted aspartame to clinically used hormones such as oxytocin, corticosteroids, and calcitonin. Peptides are also increasingly used in cosmetic applications, such as for skin and hair.

[0003] Currently, peptides are typically synthesized using bacterial or eukaryotic cell cultures or solid-phase peptide synthesis, employing recombinant methods with amino acids protected by FMOC or FBOC. Both methods are expensive, and peptide purification is arduous. Furthermore, for solid-phase synthesis methods, the chemical reactions require large amounts of toxic organic solvents, and the removal of the resulting waste materials accounts for approximately 10% of the total cost of peptide production.

[0004] Natural peptide linking of peptides has been performed to provide proteins from large peptide fragments. Typically, this requires half of the peptide to have reactive thioester chemistry to enable the reaction. Furthermore, this technique relies on the solid-phase synthesis of the desired peptides before linking the two parts or half-parts together.

[0005] Overview This invention is based on research into peptide synthesis that facilitates peptide synthesis without the use of complex chemical components, high temperatures and / or pressures, or DNA templates. Unlike the solid-phase synthesis of two or half portions of a desired peptide and the subsequent linking of these portions or half portions together, the templated method described herein does not rely on having a pre-synthesized majority of the peptide, but rather appropriately requires only the constituent amino acids.

[0006] Surprisingly, it has been discovered that the templated synthesis of peptides can occur by providing a small amount of energy to amino acids in solution.

[0007] For example, the inventors determined that providing sufficient energy for the synthesis reaction to the sample in the dark at approximately 21°C with a temperature difference of 3°C to 5°C resulted in a reaction that took longer than that of an exposed sample, and long-term results showed that smaller structures were produced.

[0008] Appropriately, the templated approach used herein allows for the use of any peptide to make fuller use of it. Not wishing to be bound by theory, it is considered that providing a template (the peptide of choice to be synthesized) to the synthetic reaction offers two benefits. First, in a concentrated solution of amino acids, the cross-section of a peptide molecule is larger than that of a single amino acid. Spatially, this is thought to mean more interactions with the template and cause the amino acids to accumulate or aggregate on the template peptide surface. Second, the presence of the template peptide provides organization of the amino acids due to structural compatibility with similar amino acids; that is, due to similar structures, it is thought that arginine would 'sit' on top of arginine in the template peptide, potentially slowing its movement. Appropriately, sequence-selective molecular recognition on complementary surfaces is considered to play a crucial role in the self-replication of peptides. This is thought to occur together with other amino acids in the sequence and to bring them close together. The system provides sufficient energy for peptide bond formation to occur between amino acids and for the templated synthesis of peptides to take place.

[0009] Advantageously, this method is considered to differ from prior art methods, which require the pre-synthesis of synthetic reactive chemicals at the molecular ends of peptides, DNA, or RNA, or of a large number of reactive peptide moieties for linking together.

[0010] According to the first aspect, a method for synthesizing peptides is provided, the method comprising: A certain amount of template peptide and amino acids capable of forming copies of the template peptide are added to an aqueous solution. A small amount of energy is provided to the solution to synthesize a copy of the template peptide in solution. A small amount of energy can be defined as sufficient to overcome the energy required to form the amide bond. This energy can be provided, for example, by sunlight, or light of the entire spectrum, including UV and a portion of the IR wavelengths, applied to the system.

[0011] Without being bound by theory, it has been shown that peptide bond synthesis is advantageous in the absence of ionized products. Furthermore, it has been proposed that the formation of dipeptides in a spontaneously occurring scenario requires approximately 1.2 kcal / mol. This has also been proposed to be 8 times more difficult than adding amino acids to peptides of any length, and 5 times more difficult than binding two peptides of at least dipeptide size. Appropriately, the energy provided can be at least 0.15 kcal / mol, at least 0.24 kcal / mol, at least 0.3 kcal / mol, at least 0.6 kcal / mol, or at least 1.2 kcal / mol. As will be understood, sufficient energy can be provided for multiple peptide bonds. In embodiments, the energy provided can be the energy that allows 1 mol of dipeptide to form. In the implementation scheme, the energy provided can be the energy that allows for the formation of at least 2 mol of dipeptide, at least 3 mol of dipeptide, at least 4 mol of dipeptide, at least 5 mol of dipeptide, at least 10 mol of dipeptide, at least 15 mol of dipeptide, at least 50 mol of dipeptide, or at least 100 mol of dipeptide. As will be understood, energy can be provided to provide the peptide formation required by the method.

[0012] To accurately identify the differences in energy consumption during the dehydration reaction required to form peptide bonds, the inventors used a controlled temperature environment and a control sample of PBS, along with a PBS + amino acid solution measured at ambient temperature. Measurements were performed every 5 seconds for highly accurate description of the heating and cooling curves. The difference in the area under the curves was determined to be 28962.97°C. 2 The peak area in the PBS amino acid sample was smaller at 170°C or s. The maximum value of the peak in the PBS+ amino acid sample was always smaller than that in the control, indicating that energy was always consumed in this system compared to the control. The energy utilized by the PBS+ amino acid sample was 1.31 kcal, indicating that a cycle of heating from 24°C to 34°C and cooling to form more than 1 mol of dipeptide is possible.

[0013] Appropriately, any form of energy that can provide suitable conditions to allow bond formation may be considered, such as 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 elevated 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. Therefore, this method provides a simpler and more cost-effective process for synthesizing peptides that does not require the use of nucleic acids, enzymes or coenzymes, cells or cellular materials, and / or organic or environmentally harmful solvents. This method can be considered an amplification process in which the amount of template peptide present in solution is amplified as more copies of the template peptide are prepared.

[0015] Appropriately, the method for synthesizing peptides can occur within 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, or at least 5 hours.

[0016] Peptide synthesis can occur under both light and dark conditions, if appropriate.

[0017] This invention is based on providing a small amount of energy to an aqueous solution. Previous knowledge has taught that large amounts of energy are required to support chemical synthesis or conversion, so the small amount of energy used in this method is unexpected. However, the following experiments clearly demonstrate the production of peptides in the absence of bacterial or other peptidase sources, thus providing evidence that peptides can be produced based on template reactions rather than enzymatic reactions.

[0018] Without wanting to be bound by theory, the inventors believe that, for example, using energy input from the sun to produce proteins in solution consisting of only 20 standard amino acids would allow proteins to become the initial functional, structural, and replicating material of life, which would then recruit nucleic acids to replicate themselves more efficiently.

[0019] As those skilled in the art will understand, a large amount of energy can be supplied to the system by applying extreme heat, strong microwaves, or radiation.

[0020] The inventors have used mass spectrometry-based proteomics as the primary analytical modality. Given that peptide and protein production is primarily random, all spectra were initially identified using De Novo sequencing (Peaks software), and the resulting database was used for quantitative analysis of the data using MaxQuant. Structural characterization was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Electron microscopy images were quantified using Omero. The absence of DNA and RNA was detected using quantitative fluorescence (Qubit®, LifeTechnologies). Samples were sterilized using gamma irradiation (1000 Gy), followed by standard peptide synthesis conditions, with additional verification in sealed bags that living organisms do not contribute to peptide and protein synthesis.

[0021] For example, energy can be provided in the form of heat or thermal energy through, for example, the application of electromagnetic radiation. This can include, for example, infrared, visible, and ultraviolet radiation, or a combination thereof. Alternatively, the heat can be associated with the use of a heat pump, or simply due to external or ambient conditions that raise the temperature of the aqueous solution.

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

[0023] As used herein, "full-spectrum light" can be considered to refer to light covering the infrared to ultraviolet region of the electromagnetic spectrum. For example, a full-spectrum light source can provide light with wavelengths from approximately 300 nm to approximately 700 nm. Full-spectrum light can be configured to mimic the composition of natural light.

[0024] The provision of energy may include maintaining the solution at a constant temperature. For example, the solution may be maintained at temperatures 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. By a representative example, the solution may be maintained at temperatures of about 35°C or 40°C. Suitably, the synthesis of the peptide may occur 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, or at least 40°C.

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

[0026] Alternatively, energy can be supplied to an aqueous solution in a periodic or repetitive manner, rather than in a constant manner. Therefore, energy can be supplied for a period of time and then stopped before supplying energy again for another period.

[0027] The periods of energy provision can be the same as or different from the periods of no energy provision. For example, there may be a period of 2 hours with energy or light provided, followed by another 2 hours without energy / light. Alternatively, the periods of energy provision can be shorter or longer than the periods without energy provision. In one implementation, the provision / absence of energy can take the form of a circadian rhythm. Thus, the provision / absence of energy follows a regular 24-hour cycle, as typically seen in a day with light and darkness.

[0028] As described above, the cyclical method of providing energy to a solution can include periodically increasing the heat of the solution, for example, by at least 0.25°C, 0.5°C, 1°C, 5°C, or even 10°C, 15°C, 20°C, or 100°C. Optionally, the solution can be allowed to cool to ambient temperature before applying heat again. The cyclical method of providing energy to the solution can correspond to the rise and fall of ambient temperature during a day / night cycle. This process can be repeated many times, hundreds of times, or even thousands of times.

[0029] In some cases, the solution can be exposed to a constant light source (e.g., full-spectrum light) while simultaneously undergoing the periodic temperature oscillations described above. Alternatively, the solution can be exposed to periodically supplied light (e.g., full-spectrum light) under constant temperature conditions.

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

[0031] The aqueous solution can be sterile. As used herein, sterile can mean that the solution is free of biological contaminants. In some cases, the aqueous solution can be sterilized by irradiation, such as gamma irradiation. Therefore, this method involves the synthesis of non-biological peptides.

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

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

[0034] Generally, any such pure or substantially pure water should have low levels of solids, low organic matter, and low electrical conductivity. For example, pure or substantially pure water may have less than 5 µg / ml of solids, less than 1 µg / ml of solids, or even less than 0.1 µg / ml of solids. Alternatively or additionally, 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. Alternatively or additionally, pure or substantially pure water may have an organic matter content of less than 1 μS·cm at 25°C. 1 <0.1 μS·cm 1 or even less than 0.01 μS·cm 1 The electrical conductivity.

[0035] Alternatively, the aqueous solution may include a phosphate source. For example, the aqueous solution may include a phosphate source other than ATP or ADP. An example of such a solution may be water containing disodium hydrogen phosphate or a phosphate buffered saline solution. Phosphate buffered saline (PBS) may contain disodium hydrogen phosphate and sodium chloride. In some formulations, the phosphate buffered saline may also contain potassium chloride and potassium dihydrogen phosphate. Exemplary PBS compositions are shown below:

[0036] However, this should not be interpreted as limiting. Typically, phosphates can be present in amounts of at least 50 µM, such as 100 µM, or 1 µM.

[0037] The use of a phosphate source in solution can be useful when a large proportion of acidic amino acids are involved in the synthesis. By way of example, the presence of phosphate can help keep the template peptide (and any copies of the template peptide) in solution during synthesis.

[0038] All available amino acids can be provided in an aqueous solution. For example, the aqueous solution may contain a mixture of all proteogenic amino acids. Alternatively, the amino acids provided to the solution may be 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, that is, those found in nature. Appropriately, the amino acids may not be activated amino acids. For example, the amino acids may not be linked to any coupling agent such as carbonyl diimidazole.

[0039] The relative amount of each amino acid provided or added to the solution can correspond to the relative amount of each amino acid in the template peptide. For example, amino acids can be provided in a molar ratio or stoichiometric amount equal to the molar ratio or stoichiometric amount of each amino acid present in the template peptide (e.g., the peptide to be replicated and synthesized). For example, if the peptide has the sequence GlyAlaGly, double the concentration of Gly (Ala) can be provided in the solution. In some cases, the stoichiometric amount of amino acids provided to the aqueous solution can be approximately equal to the stoichiometric amount of amino acids present in the template peptide (e.g., within approximately ±10% or ±20%). Alternatively, amino acids can be provided in equimolar amounts.

[0040] The amino acid notation disclosed in this article is conventional and is shown below:

[0041] This invention can be considered to extend to proteogenic amino acids, which contain 20 standard gene-encoded amino acids (as shown above) and 3 additional amino acids—selenocysteine, pyrrolidone, and N-formylmethionine. Similar to the more conventional L-amino acids, this invention can be extended to use D-amino acids and post-translational modified amino acids, such as phosphorylated, glycosylated, and methylated residues.

[0042] The methods described herein can be applied to many template peptides. The template peptide to be replicated in peptide synthesis can contain any number of amino acids, for example, 2 to 200 amino acids. Suitablely, the template peptide to be replicated in peptide synthesis can contain 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, or at least 220 amino acids. For example, using the methods described herein, copies of relatively long and / or challenging peptide sequences can already be synthesized. The methods described herein are efficient for the templated synthesis of peptides with specific structural conformations, such as those defined by one or more disulfide bonds between amino acids in the peptide. Typically, the synthesis of peptides with specific structural conformations can only be expected to occur in the presence of molecular chaperones (e.g., proteins capable of folding and assembling peptides into specific structural conformations). However, it has been surprisingly found that the methods described herein can be used to synthesize copies of such peptides. For example, insulin (a relatively long peptide with three disulfide bonds and a specific structural conformation) has already been synthesized using the methods described herein. The template peptide does not have to be an amyloid peptide.

[0043] Suitablely, the template peptide may have a secondary structure. Suitablely, the secondary structure may be substantially helical. Suitablely, the secondary structure may be substantially β-sheet. Suitablely, the secondary structure of the template structure may be a combination of helical and β-sheet.

[0044] A certain amount of template peptide can be added to an aqueous solution to provide a specific concentration of the template peptide in the solution. Typically, even very low amounts of the template peptide can be used to synthesize copies of the template peptide in solution. Therefore, the template peptide can be provided at any concentration. The concentration of the template peptide in solution can also vary depending on the length of the peptide. Higher concentrations of template peptide can be used to increase the rate of template peptide copy synthesis.

[0045] It is believed that using fewer templates in the starting materials will lead to longer synthesis times. By increasing the amount of template present, the reaction can proceed more quickly.

[0046] The relative amounts of amino acids and template peptides added to the aqueous solution can also vary. For example, the total weight of all amino acids and the weight of the template peptide can be provided at a w / w ratio (weight to weight) of any value from 20,000:1 to 10:1. For example, w / w ratios of 15,000:1; 10,000:1; 5,000:1; 1,000:1; 100:1 to 10:1; by another example, the solution can contain approximately 21.5:1 w / w of amino acids and template peptides.

[0047] The total weight of the amino acids can be provided in the form of a solution with 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 a concentration of about 0.02 g / mL of amino acids. In some cases, it should be understood that the concentration of the solution may depend on the composition of the template peptide. For example, different amino acids have different water solubilities, and therefore, where the template peptide contains a relatively high proportion of amino acids with high water solubility, a more concentrated solution can be used in peptide synthesis.

[0048] Amino acids and template peptides can be added to an aqueous solution sequentially or simultaneously. This method facilitates peptide synthesis, where all necessary reagents (e.g., amino acids and template peptides) can be added to the solution at the start of synthesis. Therefore, the method described herein can be implemented without the use of complex chemicals and / or multi-step processes (such as those requiring protecting group strategies and / or solid-phase components).

[0049] By providing a template peptide in this method, copies of the template peptide can be obtained in high quantity and quality. Typically, the resulting peptide is at least 80%, 90%, 95%, 99%, 99.5%, or even higher in quantity than the template peptide.

[0050] The progress of peptide synthesis can be monitored. For example, mass spectrometry (MS) techniques (e.g., matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion trap electrospray MS) can be used to monitor the amount of synthesized peptides in aqueous solutions.

[0051] As will be understood, an initial increase in the amount of synthesized peptide can be observed as amino acids react to form a copy of the template peptide. The amount of synthesized peptide can then be stable (e.g., as all starting amino acids are consumed). Alternatively, after the initial increase, the amount of synthesized peptide can decrease (e.g., if additional amino acids begin to be added to the synthesized peptide, increasing the length of the peptide chain). Therefore, in at least some cases, peptide synthesis can be terminated when the observable amount of synthesized peptide stabilizes or decreases.

[0052] The duration of peptide synthesis can depend on the length of the template peptide and / or the properties of its internal amino acids. For example, a shorter template peptide can be replicated faster than a longer template peptide. Therefore, monitoring peptide synthesis can be used to experimentally determine the duration of the process that will provide a sufficient quantity of the desired peptide. By way of example, the synthesis process can occur over a period of hours (e.g., 6, 12, 18, or 24 hours) or days to provide a sufficient quantity of the desired peptide. For example, the synthesis process can occur for at least 1, 2, 3, 4, or 5 days, or even up to 12, 20, 25, 30, or 50 days or more.

[0053] Peptide synthesis can be terminated by removing a small amount of energy from the solution. By way of representative examples, peptide synthesis can be terminated by cooling the aqueous solution (e.g., to a temperature below 10°C, below about 5°C, or below about 0°C). Alternatively or additionally, peptide synthesis can be ultimately terminated by isolating the synthetic peptide from the aqueous solution.

[0054] The peptides synthesized in this method may require minimal purification (and in some cases, no purification at all). For example, the peptides synthesized in this method can be of a reasonable class.

[0055] If further purification is required, the synthesized peptide can be purified in an aqueous solution in which the synthesis process takes place, in order to remove the desired synthetic peptide from the amino acids and, if necessary, remove shorter, longer, and / or mis-copied peptides.

[0056] Purification can typically be performed using chromatography (e.g., high-performance liquid chromatography (HPLC)). Examples of suitable HPLC methods include reversed-phase HPLC, ion-exchange HPLC, and gel filtration HPLC, which can be performed individually or in combination / tandem. Progress in peptide purification can be monitored, for example, using mass spectrometry (MS) techniques including matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or ion trap electrospray ionization MS. Sequencing, for example, using Edman degradation sequence analysis or in tandem with MS, can be used to confirm peptide homogeneity. Suitably, purification can be performed using binding elements capable of selectively binding to the target peptide / protein provided by the method, such as antibodies or fragments thereof. Suitably, the target peptide / protein provided by the method can be tagged to allow its purification. Suitably, beads with elements specifically binding to the target peptide / protein provided by the method, or to a tag attached to the target peptide / protein provided by the method, can be used.

[0057] The method for synthesizing peptides can be carried out at atmospheric pressure (e.g., 1 bar ± 0.2). Alternatively or additionally, 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. Templated synthesis of peptides can occur without the application of large amounts of energy (e.g., as provided, for example, by an electric spark).

[0058] This method can be a batch process or a continuous process in which the synthesized peptides are removed over time.

[0059] This method can be implemented in containers or reactors. In some cases, containers or reactors can be covered or sealed (e.g., to reduce the risk of contamination).

[0060] According to another aspect of the invention, the use of a template peptide in its own amplification is provided. That is, the use of the peptide as a template for peptide amplification. Amplification includes replicating the peptide using the peptide as a template.

[0061] For example, amplification can include synthesizing additional copies of the template peptide. Amplification can be facilitated by the template peptide itself in the absence of nucleic acids, enzymes or coenzymes, cells, or cellular material. That is, amplification does not require enzymes capable of forming peptide bonds, such as peptidyl transferases. The amplification solution does require amino acids to be incorporated into the growing, replicating peptide chain.

[0062] Furthermore, throughout this specification, the term "comprising" is used to indicate that embodiments of the invention "comprise" the features mentioned, and may similarly include other features. However, in the context of this invention, the term "comprising" may also cover embodiments of the invention that "consist substantially of the relevant features" or "comprise the relevant features".

[0063] The applicant hereby discloses in isolation each individual feature described herein, as well as any combination of two or more such features, enabling the implementation of such features or combinations based on the entire specification in accordance with the common knowledge of those skilled in the art, regardless of whether such features or combinations of features solve any problem disclosed herein, and without limitation to the scope of the claims. The applicant indicates that aspects of the invention can consist of any such individual feature or combination of features. In view of the foregoing description, it will be apparent to those skilled in the art that various modifications can be made within the scope of the invention. Invention Details The invention will now be further described by way of example and with reference to the following figures, which show: Figure 1A The quantitative analysis of templated peptide synthesis using mass spectrometry (Thermo Q Exactive Orbitrap) for the synthesis of peptide VR15 (SEQ ID NO: 1 - VPDNLQQSLSDEAQR - this peptide does not exist in nature) is shown. The newly synthesized peptide was distinguished from the template peptide by incorporation of arginine (R, here representing SILAC), meaning that the peptide could be clearly measured from its template peptide in mass spectrometry analysis. B shows the UV absorbance of the sample over time; the signal of the new peptide increased significantly compared to time point 0. C shows the complete chromatogram. All samples were amplified using an internal standard (benzoic acid) that remained unchanged throughout the process—as seen in B and C. D shows the intensities measured in the mass spectrometer (intensity represents the ions detected in the mass spectrometer and is a direct indication of quantity).

[0065] Figure 2 The amounts of template peptides for four different peptides before and after templated peptide synthesis are shown by weight: insulin (110 amino acids long), MRFA (4 amino acids long), VR9 (VMDSSYLSR, a synthetic peptide not found in nature, 9 amino acids long, SEQ ID NO: 2), and WK20 (WRWLEHNVVEGNAVNLMFSK, a synthetic peptide not found in nature). This demonstrates the ability to amplify peptides of any length without issues with structure and disulfide bonds during amplification.

[0066] Figure 3 The quantitative analysis of MRFA peptide synthesis using mass spectrometry (Thermo Q Exactive Orbitrap) is shown in the presence of the MRFA template peptide (“Amplification”) and in the absence of the template peptide (“Control”). This demonstrates that the addition of the template does indeed catalyze peptide production.

[0067] Figure 4 The experimental design for subsequent experiments is shown to demonstrate the absence of contamination during the exemplary peptide synthesis process. Additional control samples contained sodium azide (NaN3), chloramphenicol, and D-amino acids (Daa's) to further ensure the absence of biological contamination.

[0068] Figure 5 The results of testing for the presence of DNA and RNA are shown. The presence of DNA and RNA in the sample was tested using a high-sensitivity Qubit® kit, and the observed intensity is shown. No RNA was detected after the sample changed color, but DNA was detected. To verify that this was indeed DNA, a totipotent nuclease (which digests DNA and thus eliminates the detectable signal of DNA in the sample) was applied to the sample and left to incubate. A positive control was always used, and the intensity of the positive control was always reduced by at least half, while the “DNA intensity” of the peptide synthesis sample never decreased significantly. This verifies that “life” (i.e., DNA and RNA) is absent in the peptide synthesis sample.

[0069] Figure 6 shows the results of Marfey reagent derivatization of D and L-amino acids A. The chromatogram shows an overlap between a mixture of four commercially available L-amino acids (black traces) and a mixture of the same four amino acids but D isomers (red traces). The retention time delay of the D-amino acids was observed due to derivatization using Marfey reagent (FDAA, 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide). Figure A shows the chromatogram where the black traces represent a mixture of four L-amino acids (serine, alanine, tyrosine, and methionine) derivatized with Marfey reagent and separated on a C18 column to show their elution times. The light gray traces show four D-amino acids (serine, alanine, tyrosine, and methionine) also derivatized with Marfey reagent to show the difference in elution times—meaning we can distinguish between the D and L isomers in the derivatized amino acid mixture. B shows a non-biological D-amino acid sample obtained on day 28, hydrolyzed into individual amino acids and derivatized with Marfey's reagent (red trace), which overlaps with the chromatograms of four commercially available L-amino acids (black traces). No conversion of D-amino acids to L-amino acids was observed (which could be expected if the biological life form contaminating the sample utilized D-amino acids). C shows a magnified area of ​​the chromatogram, labeled with D- and L-amino acids, indicating that no conversion from D- to L-amino acids occurred in the day 28 D-amino acid sample. This sample was hydrolyzed with 6M HCl (breaking the peptide chain into individual amino acids), derivatized with Marfey's reagent, and then separated on a C18 column. The chromatograms of the derivatized L-amino acids are overlaid in the figure for comparison.

[0070] Figure 7 shows the results of the gamma irradiation experiment (all samples were tested in triplicate). Using *E. coli* (… E. coli The cultures included a positive control for sterilization. The unirradiated sample (left side of A) showed numerous colonies, while the irradiated sample (right side of A) showed no growth after 1000 Gy irradiation, demonstrating effective sterilization. B is a histogram describing the distribution of peptide intensity. The dark green population is the starting material, with low peptide counts (because this is a template-guided experiment—meaning the only product is the peptide itself and a small percentage of miscopying—which occurs with this process) and lower median intensity. The standard incubation sample produced more peptides of higher intensity (i.e., more copies of the peptide), and the irradiated sample showed an increase in peptide production similar to that of the unirradiated sample. The lack of significant differences between the two sample groups suggests that this is a chemical process unaffected by any kind of external contamination. C is a Venn diagram showing the co-occurrence of peptides produced across the conditions. The templated generation of peptides was equivalent between the irradiated and unirradiated samples, demonstrating that the reaction is a chemical reaction independent of bacteria / biocontaminants.

[0071] Figure 8 This is a Venn diagram showing the co-occurrence of detected peptides under various conditions (chloramphenicol, 37°C, standard, sodium azide (NaN3), and D-amino acids). Certain concentrations of chloramphenicol (a broad-spectrum antibiotic), sodium azide (a Gram-negative bactericide), and D-amino acids (most living organisms require L-amino acids, and if living organisms utilize D-amino acids, they convert them to L-amino acids—this does not occur in this case) used in these experiments should eliminate bacterial growth. The peptide synthesis in all experiments is comparable and shows a chemical basis for synthesis that is opposite to the biological origin of the peptide.

[0072] Figure 9 shows the MALDI-TOF mass spectra of commercially available amino acids. These spectra indicate that the starting material was pure and contained no larger molecules that could contribute to the peptides we observed in our experiments. A. MALDI-TOF spectra of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine, and cysteine; B. Scaled MALDI-TOF mass spectra of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine, and cysteine; C. Scaled MALDI-TOF mass spectra of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine, and cysteine; D. MALDI-TOF mass spectra of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine, and alanine; E. Scaled MALDI-TOF mass spectra of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine, and alanine; F. MALDI-TOF scaled mass spectra of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine, and alanine; MALDI-TOF mass spectra of G. glutamate and histidine; and MALDI-TOF scaled mass spectra of H. glutamate and histidine.

[0073] Figure 10 A schematic diagram of the method of the present invention is shown.

[0074] Figure 11 illustrates insulin synthesis using the method of the present invention.

[0075] Figure 12(A) shows the experimental setup used to test the necessity of sunlight and phosphate for the spontaneous polymerization of amino acids into peptides and proteins. All samples were in triplicate. (B) shows the experimental setup for all subsequent experiments with greater sterility. Additional control samples contained sodium azide, chloramphenicol, and D-amino acids to further ensure no biological contamination. (C) shows the experimental setup for the first spontaneous generation experiment, in which two reactions used sterile phosphate buffer as solutions, and the third reaction used pure water, 20 standard amino acids, and were incubated in sunlight (one pure water and one PBS) and in the dark.

[0076] Figure 13 The correlation (Log2 of peptide intensity) of all collection time points in the experiment shown in Figure 12 is illustrated, and the Venn diagram shows that most peptides were shared between samples.

[0077] Figure 14 shows electron micrographs of the structures produced by protein synthesis according to this method. A relative scale can be seen between TEM and SEM (A.), as well as structures that are very similar in appearance to viral capsids (A, right group). Structures were measured and counted using Omero software, and the results are illustrated in B. PBS bright-field samples produced larger but fewer structures, while PBS dark-field and MilliQ bright-field samples produced many but much smaller structures. The reproducibility of biological replication is quite remarkable and can be seen in group C.

[0078] Figure 15 The use of ultrathin sectioning to visualize the interior of the spheres is shown. In earlier samples (A, B, C, approximately day 60), linearly modified protein chains are predominant. In later samples (J, K, L, day 678), spherical structures are the majority that are visible. These range from completely empty spheres (F) to slightly filled spheres and structures with very little dense filling. A significant lack of structure and marked emptiness are evident when compared to conventional cross-sections of cells; however, they remain cellular and may represent prototypical cellular structures.

[0079] Figure 16 shows A: Peptide pair analysis reveals the most and least common amino acid pairings observed in all identified peptides. A preference appears for pairing nonpolar amino acids (I, L, G) with acidic amino acids (E, D). The least favorable pairings are between nonpolar and basic amino acids and predictably between the amino acids with the lowest abundance in the mixture. B describes the utilization of amino acids in the most stable peptides observed in all samples. There appears to be no trend of chemical processes in the amino acids (polar, nonpolar, and acidic amino acids all increase with use). Aspartic acid and asparagine increase with use but are considered to destabilize the helical chain. C: Some examples of the most stable peptides. There is a tendency toward helical structures when random coiling is present, suggesting that hydrogen bonding between the NH group hydrogens of the main chain and the C=O groups of the preceding four residues of the amino acid is sufficiently stable here. Structures predicted by PEP-FOLD (http: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD) Figure 17 shows a comparison between a sample heated to constant temperature in the dark at 37°C and the same sample set using natural light. The Log2 intensity values ​​of the standard set of proteins / peptides, which show an increasing trend over time (indicating stable peptide production and lifetime), are subtracted from the Log2 intensity values ​​of the 37°C sample, and the resulting values ​​are aggregated.

[0080] Figure 18 illustrates the proposed method for the initial polymerization of amino acids, which overcomes the required energy to form amide bonds by utilizing amino acids present in an energy-assisted solution (e.g., powered by sunlight), where the subsequent addition of amino acids to an existing dipeptide is selective in two ways (B). i) The enlarged cross-section increases the likelihood of collisions with amino acids in the correct direction, and ii) the energy required to add an amino acid to the existing dipeptide is 8 times less than that required for the initial amide bond. The additional local chemical environment favors the presence of the peptide / protein, inducing the folding of the peptide structure, excluding water molecules, and forming secondary structures supported by hydrogen and disulfide bonds (C). This produces structural stability, and thus the peptide / protein lifetime results in a reduction in entropy by forming globular, cell-like structures to organize the protein (D). This again protects the formed structures, selecting those that favor them.

[0081] Figure 19 shows the apparatus used to measure temperature fluctuations on a windowsill, characterize differential amino acids in prepared solutions in a controlled heating environment without sunlight (A), the energy consumption of the apparatus (B), and finally, the fully controlled environment used for all subsequent experiments (C). The system was heated and cooled using a Peltier chip with a daylight replication bulb (with a wide range of wavelengths including UV). Temperature was monitored using a medical-grade temperature sensor. The apparatus was heated to 40°C for 12 hours with the bulb lit, then cooled to ambient temperature (approximately 18°C) for 12 hours with the bulb turned off to replicate a controlled circadian rhythm. The data presented in this paper, generated using the controlled environment, are from sterilization experiments (1000 Gy irradiation of the sample). Temperature was measured every 5 minutes with an accuracy of ±0.0625°C. The temperature fluctuation on the windowsill was 10.8125°C (2096.7 J°C). -1 g -1 The temperature fluctuation in the "dark" sample was 0.4444°C (185.8 J°C). -1 g -1 To more accurately identify the differences in energy consumption during the dehydration reaction required to form peptide bonds, we used a controlled temperature environment and a control sample of PBS, along with a PBS + amino acid solution measured at ambient temperature. Measurements were taken every 5 seconds to obtain highly accurate heating and cooling profiles, with the difference in the area under the curve determined to be 28962.97°C. 2The area of ​​the peak in the PBS+amino acid sample was smaller than that in the control at 170°C. The maximum value of the peak in the PBS+amino acid sample was always smaller than that in the control, indicating that energy was always consumed in this system compared to the control. The energy utilized by the PBS+amino acid sample was 1.31 kcal, indicating that a cycle of heating from 24°C to 34°C and cooling for more than 1 mol of dipeptide formation is possible. After 7 days of “growth”, biological replication under each condition was determined to be significantly correlated with each other (up to 0.96 Pearson correlation), although this correlation broadened at about 126 days of growth, but always remained positive. It is hypothesized that the efficiency of peptide replication decreases (through steric hindrance) due to the formation of larger structures (obvious in TEM and SEM), reducing accessibility to the protein surface when hydrophobic regions induce folding.

[0082] Figure 20 The amino acid utilization in the set sample is shown. The percentage of amino acids in TrEMBL%2013 indicates the percentage of amino acids in the starting solution. The difference in amino acids used compared to the starting material may be due to several factors, such as trypsin digestion-induced bias (which we were able to measure in mass spectrometry), the chemical stability of the amino acid sequence combination, and the affinity for the formamide bond.

[0083] Methods and Results Experiment 1: A synthetic experiment was conducted to distinguish the template peptide from the product peptide using amino acids labeled with heavy isotopes. The experiment was performed using chromatographic analysis (industrial standard purity) and mass spectrometry, with an internal standard (benzoic acid), to quantify the peptides (light (template peptide) and heavy (newly synthesized) versions).

[0084] 11 micrograms of the synthetic peptide VR15 (VPDNLQQSLSDEAQR) SEQ ID NO: 1 were added to a 1600 µl amino acid solution containing arginine at a concentration of 0.2 g / ml, which is 6 Daltons heavier than normal arginine. The sample was incubated at 37°C for up to 4 hours using a full-spectrum light source.

[0085] Samples were taken at 2 and 4 hours of synthesis and analyzed in triplicate on a reversed-phase C18 column with UV absorbance measured at 216 nm. Figure 1B and 1C The chromatograms were analyzed, and triplicate were performed on a mass spectrometer to measure the intensity of the heavy and light versions of the peptide. Figure 1D ).

[0086] result In the chromatogram of the synthetic peptide, there was a sustained increase in signal after 2 hours (see...). Figure 1AThe absence of any signal increase (benzoic acid peak) in the internal control and the comparison more clearly indicates the production of more VR15. The signal increase is not due to instrumentation degradation or any other external factor.

[0087] SILAC quantification and labeling of newly synthesized peptides allow us to distinguish between old and new peptides, and this is also evident in the peptide intensities obtained by mass spectrometry.

[0088] Experiment 2: In the initial experiments, it was discovered that proteins and peptides could be synthesized in the absence of cellular organelles and nucleic acids. Then, certain peptides were selected to study the templated amplification process.

[0089] The following peptides were selected due to several factors.

[0090] 1. They do not exist in nature (except for insulin).

[0091] 2. They exhibited high copy numbers in the initial experiments—indicating that they had been amplified. Some peptides were predicted to have unstable structures, which would be detrimental to lifespan and replication. However, over 8000 different sequences were reproducibly produced.

[0092] 3. They have varying lengths to demonstrate the effectiveness of the templated amplification process on a range of different peptides.

[0093] The peptide sequences initially studied are listed in Table 1 below.

[0094]

[0095] A negative control experiment was established in which no template peptide was added to the solution.

[0096] The concentration of amino acids is expressed as the total weight of amino acids per mL of solution. In each case, the relative amount of each amino acid present in the solution is proportional to the amount of amino acids in the template peptide. Taking TR8 as an example, the composition of the amino acid mixture constituting a 0.01 g / mL solution is approximately as follows: 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 other amino acids are present).

[0097] Table 1 shows the number of solutions used in the templated peptide synthesis process.

[0098] 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 in autoclaved Schott flasks, sealed, and then placed in the amplification apparatus.

[0099] The amplification device subjectes the sample to a controlled environment with a constant temperature of 40°C and a constant source of full-spectrum light. (Although the device can be configured in any number of ways).

[0100] Under aseptic conditions, samples (100 µl) were taken daily and analyzed using MS (10 ml injection, 15-minute gradient, 2%–80% acetonitrile, 0.1% formic acid, Thermo Q Exactive Orbitrap) to determine the concentration of peptides over time.

[0101] When a decrease in intensity is observed, the amplification process is stopped by placing the sample in a refrigerator. Absolute cessation of amplification is achieved by chromatographic separation of the peptide from the reagent (amino acid solution).

[0102] Reversed-phase chromatography (using mobile phases from A: 2% acetonitrile, 0.1% formic acid to B: 80% acetonitrile, 0.1% formic acid) was employed, with a high-flow-rate, large-capacity C18 column to achieve chromatographic separation of products from reagents.

[0103] result The amount of peptide produced after purification was measured, and the results are shown in Table 2 below.

[0104]

[0105] WK20 SEQ ID NO: 3 (not shown in Table 1) is a synthetic peptide with the sequence WRWLEHNVVEGNAVNLMFSK.

[0106] Table 2 shows the amount of template peptide before and after the amplification process.

[0107] In each case, the amount of template peptide increases after the amplification process. This is in Figure 2 As shown in the image.

[0108] It was observed that the shorter the peptide, the faster the amplification process. The subsequent decrease in strength was due to the additional amino acids added to the product peptide, resulting in a loss of the desired / template peptide mass.

[0109] Compared to templated synthesis (which is performed in the presence of an MRFA template), Figure 3 Mass spectrometry analyses used for the synthesis of MRFA in the absence of a template (negative control, Table 1) are shown. In contrast to templated synthesis, no perceptible difference in MRFA intensity was observed in peptide synthesis performed in the absence of a template peptide.

[0110] Experiment 3: Excluding other causes of peptide amplification To rule out the possibility of contamination (i.e., peptides present in the starting material that persist during chromatography, bacterial / viral contamination, or any kind of "life"), the following experiments were performed.

[0111] 1. Analyze whether DNA and RNA are present in the sample.

[0112] 2. Use D-amino acids to amplify peptides (because most life forms cannot use D-amino acids, or if they do, they convert D-amino acids into L-amino acids).

[0113] 3. Sterilize the samples using gamma irradiation and verify whether amplification still occurs.

[0114] 4. Conduct experiments in the presence of chloramphenicol or sodium azide.

[0115] 5. Test the purity of the starting components.

[0116] See Figure 4 .

[0117] method Commercially available amino acids were measured as a percentage of total concentrations below 1 g / 100 ml, as shown in Table 3. Amino acids were dissolved in sterile PBS (gibco 1x DPBS 14190-094). The solution was mixed until completely dissolved, then passed through a 0.22 µm sterilicup filter and poured 2 ml into each autoclaved vial in a cell culture fume hood equipped with sterilization equipment. The vials were not opened again until aliquots were needed at the designated time point (all operations were performed under sterile conditions). Samples were either analyzed on the day of the analysis (if prior to incubation on day 14) or immediately reduced and alkylated, followed by digestion in solution (with trypsin). Remaining samples were frozen at -80°C. Aliquots were used for DNA and RNA analysis, and the collection day was also analyzed.

[0118]

[0119] Table 3 shows the protein database data typically found in the UniProtKB / TrEMBL October 2013 edition. http: / / www.ebi.ac.uk / uniprot / TrEMBLstats The percentage of each of the 20 amino acids present in the sample.

[0120] DNA and RNA detection Use Qubit commercially available reagents according to the instructions for use of the Qubit® dsDNA HS Assay Kit, Life Technologies, Q32851 and the Qubit® RNA HS Assay Kit, Life Technologies, Q32855. Use 20 μL of sample for the assay.

[0121] Samples were measured on a Qubit® 2.0 fluorometer Q32866.

[0122] Marfey analysis of D-amino acid composition One sample was prepared for each biological replica of 100 µL of D-amino acid sample (T28 = day 28) and standard sample (T28 = day 28). The samples were rotary evaporated to dryness. Then, 1 mL of 6M HCl was added to the samples, and they were heated at 155°C for 80 min. The samples were then rotary evaporated to dryness. The samples were reconstituted with 300 µL of MilliQ water, and the pH was adjusted to >5 with 3 µL of 10M NaOH. 100 µL of the sample was combined with 200 µL of Marfey's reagent (10 mg / mL acetone solution) and 40 µL of 1M ammonium bicarbonate, and gently shaken at 40°C for 1 h. The reaction was quenched with 20 µL of 2M HCl.

[0123] 100 μL of each sample was injected into an XBridge® BEH 130 C18 column, 130 Å, 3.5 µm, 4.6 mm × 250 mm. Amino acids were separated using a 45-minute gradient from 20% to 65% B (A: 0.1% formic acid, B: 80% acetonitrile, 0.1% formic acid) and UV absorbance was measured at 340 nm.

[0124] Mass acquisition was performed using Thermo Orbitrap QEaxactive, with the first 10 ions selected for ms / ms at a resolution of 140,000 ms and 17,500 ms / ms, scanned from 150 m / z to 2000 m / z.

[0125] Gamma irradiation The sample was prepared as described above (filtration and sterile solubilization of amino acids), but VR15 was used as the template peptide, and the peptide was formed only with essential amino acids. Three copies of the prepared sample, and one containing live *E. coli*, were prepared. E. coli Small vials were used as positive controls for sterilization (irradiated with 1000 Gy in sealed plastic bags). The positive controls were then plated on agar plates to verify that sterilization had occurred, and the peptides obtained in all samples were analyzed by mass spectrometry (as described above).

[0126] result DNA and RNA detection refer to Figure 5 RNA was never detected in the samples where both RNA and DNA were present (detection limit <20 ng / ml). However, samples exposed to full-spectrum light changed color over time and clearly produced positive readings for DNA.

[0127] To determine if it was indeed DNA, a totipotent nuclease (an enzyme known to degrade DNA) was added to the sample, which was then incubated for 1 hour. A positive control (with known bacterial contamination) containing the totipotent nuclease was used. The DNA intensity was approximately halved in the positive control, while no change was observed in the peptide synthesis sample. Therefore, it was inferred that some peptides / proteins formed in this method exhibit autofluorescence at the same wavelengths (485 / 530 nm) as DNA.

[0128] D-amino acid analysis Most amino acids used in nature are in their L-chiral form, and most organisms cannot use them in the production of standard proteins (although they are commonly found in peptidoglycan proteins in bacterial cell walls). The L-type methionine, serine, alanine, and tyrosine were replaced with D-type amino acids from the above-mentioned normal amino acid mixture, but under the same experimental conditions.

[0129] The sample is then tested to determine if the D-amino acids have been converted to L-amino acids by an isomerase (e.g., such isomerases would be expected if contaminating bacteria / life forms are present in the sample). This is done using a reagent called Marfey's reagent (Nα-(2,4-dinitro-5-fluorophenyl)-L-propanamide). When reacting with D-amino acids, Marfey's reagent alters the retention times of those amino acids compared to their L-forms (when separated using chromatography).

[0130] The chromatogram shown in Figure 6 illustrates this change in sample retention time 28 days after synthesis. These data further indicate the absence of bacterial cell contamination in the sample.

[0131] Gamma irradiation The synthetic peptide synthesis experiment was performed in triplicate, but each sample was irradiated with 1000 Gy and then sealed to maintain sterilization achieved by the irradiation. This included *Escherichia coli* (…). E. coli A positive control of the culture was used to verify that sterilization occurred. If peptide synthesis still occurred, it indicates that the process was not a result of contamination by life forms in the sample.

[0132] With Escherichia coli ( E. coliThe cultures included positive controls for sterilization. Unirradiated samples (Fig. 7, left) showed numerous colonies, while irradiated samples (Fig. 7, right) showed no growth at all after 1000 Gy irradiation, demonstrating effective sterilization.

[0133] Figure 7B This is a histogram depicting the distribution of peptide intensity. The dark green population represents the starting material, which has a low peptide count (because not many peptides were produced) and a lower median intensity. The standard incubation sample produced more and higher-intensity peptides (i.e., more copies of the peptide), and the irradiated sample showed an increase in peptide production similar to that of the unirradiated sample.

[0134] Figure 7C This is a Venn diagram showing the co-occurrence of peptides produced under different conditions. The random generation of peptides is equivalent between irradiated and unirradiated samples.

[0135] The lack of significant differences between the two sample groups suggests that peptide synthesis is a chemical process unaffected by existing external contaminants in the samples.

[0136] Samples containing chloramphenicol and sodium azide The chloramphenicol and sodium azide samples were combined with a set of standard samples (amino acids dissolved in sterile vials and PBS) to allow for direct comparison with previously observed non-biological peptide synthesis. Peptides were analyzed in the same manner, and any significant differences were compared with each other.

[0137] Figure 8 The Venn diagram shown indicates that most peptides observed in all samples are identical. Furthermore, there was no significant underproduction of peptides in samples containing bactericides, whereas reduced peptide production would be expected if synthesis were a result of any bacterial or viral contamination. This result further confirms that the peptide synthesis process is chemical.

[0138] Contamination of starting materials for testing All commercially available amino acids, solvents, and digestive enzymes (trypsin) were independently analyzed by mass spectrometry, and the presence of peptides at the start was determined using de novo peptide recognition software.

[0139] In all cases, no undesirable mass greater than that of the reagent was observed.

[0140] Examples related to spontaneous peptide formation Consider the hypothesis that "spontaneous peptide formation requires sunlight and phosphate." Based on these two hypotheses, under three different conditions ( Figure 12ASamples were set up under the following conditions. The first assumption was that, considering most life uses ATP—the transfer of a single phosphate molecule—phosphate might be necessary; therefore, a simple phosphate source in the form of phosphate buffer solution was used. This has the added property of being a loose analogue to seawater (an environment in which life may have begun 4 billion years ago in the Archean Eon). The second assumption was that energy from the sun would be required to input enough kilojoules to overcome the amide bond formation step. Therefore, the samples were as follows: PBS bright field, PBS dark field, and MilliQ bright field. The sample group was considered to be the 'PBS bright field' which was proven positive, the 'MilliQ bright field' which was the negative control, and another 'PBS dark field' which was the negative control.

[0141] However, it has been determined that protein formation is evident under all conditions. Therefore, the inventors have determined that protein synthesis will occur in the presence of the building blocks (amino acids) for life in a solution with spontaneous, even minimal, temperature fluctuations, independent cells and nucleic acids. Figure 13 ).

[0142] Example—The hypothesis that "constant heating (37°C)" will be better for spontaneous peptide production than a day's worth of cyclic heat. Comparing peptide / protein synthesis occurring in two standards (samples provided at room temperature on a windowsill) and a 37°C sample reveals an increasing trend over time (indicating stable peptide production and lifetime). The data show that sunlight (variable wavelength) and cycling temperatures are more favorable than constant temperatures (no sunlight in Figure 17). Since the energy input at 37°C is 24–7, while the energy and light input received on a windowsill is at most half that, the effect is likely greater than the variation observed here.

[0143] in conclusion The above details describe a method for peptide synthesis based on previously unknown properties of proteins and peptides, which, when placed in a solution of amino acids, act as templates to replicate themselves. Accompanying this data is substantial evidence from several different methods (gamma irradiation, D-amino acids, antibacterial agents, and testing for the presence of DNA and RNA): the process is a chemical reaction and is carried out in the absence of nucleic acids, enzymes, coenzymes, other cellular materials, and / or cells. Conversely, the inventors believe that the templated peptide synthesis described herein can be driven by providing small amounts of energy (e.g., in the form of full-spectrum light (including IR and UV) and / or mild, constant heat), the stoichiometry of reagents, and / or stereochemistry.

[0144] exist Figure 9AThe spectrum given in -H is a MALDI-TOF spectrum verifying the purity of the commercially purchased amino acids used as the starting material. The inventors have confirmed that they contain nothing other than the specified amino acids (and have also checked those amino acids to see if they polymerize into larger peptides). This is indeed the case, and the starting material is pure.

Claims

1. A method for synthesizing peptides, the method comprising: A certain amount of template peptide and amino acids capable of forming copies of the template peptide are added to an aqueous solution. A small amount of energy is provided to the solution to synthesize a copy of the template peptide in the solution.

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

3. The method according to claim 1 or 2, wherein the energy comprises infrared, visible, or ultraviolet radiation, or a mixture thereof.

4. The method according to any of the preceding claims, wherein the energy supply is constant, and optionally wherein the energy supply includes exposing the solution to a constant source of full-spectrum light during peptide synthesis.

5. The method according to any of the preceding claims, 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, or at least 1.2 kcal / mol for peptide formation.

6. The method according to any of the preceding claims, wherein the provision 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. The method according to any one of claims 1 to 3, wherein the provision of energy to the solution is periodic.

8. The method of claim 7, wherein the periodic provision of energy comprises: The heat of the solution is periodically increased and / or the solution is periodically exposed to full-spectrum light.

9. The method according to any of the preceding claims, wherein the solvent of the aqueous solution is pure or substantially pure water, or the aqueous solution comprises a phosphate buffer solution.

10. The method according to any of the preceding claims, wherein the aqueous solution comprises or substantially comprises pure or substantially pure water, the template peptide, and the amino acids capable of forming copies of the template peptide.

11. The method according to any of the preceding claims, wherein the aqueous solution is sterile.

12. The method according to any of the preceding claims, wherein the aqueous solution contains only those amino acids present in the template peptide.

13. The method according to any preceding claim, wherein the aqueous solution comprises a stoichiometric amount of the amino acid, the stoichiometric amount being equal to or approximately equal to a stoichiometric amount of each amino acid present in the template peptide.

14. The method according to any of the preceding claims, wherein the total weight of all the amino acids present in the aqueous solution and the weight of the template peptide are provided at a w / w ratio of 20,000:1 to 10:1 or 10,000:1 to 10:

1.

15. The method according to any of the preceding claims, wherein the total weight of the amino acids is provided in an amount of 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. The method according to any of the preceding claims, wherein peptide synthesis is terminated after a period of 3 to 24 hours.

17. The method according to any of the preceding claims, wherein peptide synthesis is terminated after a period of 1 to 5 days.

18. The method according to any of the preceding claims, wherein peptide synthesis is terminated by removing a small amount of energy from the solution sufficient to cause peptide bond formation and / or by separating the synthetic peptide from the aqueous solution.

19. The method according to any of the preceding claims, wherein the method is carried out under atmospheric pressure and / or in the presence of oxygen.

20. The method according to any of the preceding claims, wherein the peptide synthesis occurs in the absence of nucleic acids, enzymes, coenzymes, other cellular materials and / or cells.

21. The use of template peptides for self-amplification in the absence of nucleic acids, enzymes, coenzymes, other cellular materials or cells.

22. The peptide provided by any one or more of the methods according to claims 1 to 20.

23. The structure provided by the peptide of claim 22 or by the method of any one of claims 1 to 19.

24. A kit for synthesizing a copy of a template peptide in solution using the method of any one of claims 1 to 20, said solution comprising said template peptide and at least one amino acid of said template peptide, optionally each amino acid required to form said template peptide.

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