PEPTIDE SYNTHESIS

Abstract

There is provided a method to synthesise peptides, a peptide being synthesised based on the amino acid sequence of a template peptide, peptides provided by the method and use of the peptides.

Claims

1.-24 (canceled)

25. A method of synthesising a peptide, the method comprising: adding a quantity of a template peptide and amino acids capable of forming copies of the template peptide into an aqueous solution, and providing at least 1.2 kcal/mol and light of wavelengths 350 to 700 nm and light intensity from 0 to 100% (in the form of Thermal IR, UV or full spectrum or mixture of any of these) to the solution in order to synthesise copies of the template peptide in solution.

26. The method according to claim 25, wherein the provision of energy comprises maintaining the solution at a constant temperature between about 10° C. and 100° C.

27. The method according to claim 25, wherein the provision of energy to the solution is cyclical.

28. The method according to claim 27, wherein the cyclical provision of energy comprises periodically increasing the heat of the solution and/or periodically exposing the solution to full spectrum light.

29. The method according to claim 25, wherein the solvent of the aqueous solution is pure or substantially pure water, or the aqueous solution comprises a phosphate buffered saline solution, acids or bases (such as HCl, Formic Acid, NaOH etc.).

30. The method according to claim 25, wherein the aqueous solution consists or consists essentially of pure or substantially pure water, the template peptide and the amino acids capable of forming copies of the template peptide.

31. The method according to claim 25, wherein the aqueous solution is sterile.

32. The method according to claim 25, wherein the aqueous solution comprises only those amino acids present in the template peptide.

33. The method according to claim 25, wherein the aqueous solution comprises the amino acids in a stoichiometric amount which equates to or is about equal to the stoichiometric amount of each amino acid found in the template peptide.

34. The method according to claim 25, wherein the total weight of all of the amino acids and the weight of the template peptide present in the aqueous solution are provided in a w/w (weight by weight) ratio of between 20,000 to 1 and 1 to 1, or between 10,000 to 1 and 10 to 1.

35. The method according to claim 25, wherein the total weight of the amino acids is provided in such an amount to provide a solution having a concentration between about 0.001 g/mL and 10 g/mL, or between about 0.005 g/mL and 5 g/mL, or between about 0.01 g/mL and 1 g/mL.

36. The method according to claim 25, wherein peptide synthesis is terminated after a period of between 10 minutes and 5 days.

37. The method according to claim 25, wherein peptide synthesis is terminated by removing the energy from the solution and/or by separation of the synthesised peptide from the aqueous solution.

38. The method according to claim 25, wherein the method is carried out at atmospheric pressure and/or in the presence of oxygen, or any other gas such as Nitrogen, hydrogen, CO.sub.2 for example.

39. The method according to claim 25, wherein the peptide synthesis using a template peptide for amplification of itself takes place in the absence of nucleic acids, enzymes, co-enzymes, other cellular material and/or cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention will now be further described by way of example and with reference to the following figures which show:

[0066] FIG. 1A shows the quantitative analysis of a tem plated peptide synthesis using mass spectrometry (Thermo Q Exactive Orbitrap) for a synthesised peptide VR15 (SEQ ID NO: 1—VPDNLQQSLSDEAQR—this peptide does not occur in nature). The newly synthesised peptides are differentiated from the template peptide by the incorporation of heavy Arginine (R, denoted SILAC here), which means this peptide can be measured distinctly from its templating peptide in Mass Spectrometric analysis. B shows the UV absorbance of the samples over time, with the significant increase in signal of the new peptide compared to time point 0. C shows the complete chromatogram. All samples were amplified with an internal standard (benzoic acid) which does not change throughout the process—this is seen in B and C. D shows the intensity as measured in the Mass Spectrometer (intensity represents ions detected within the mass spectrometer and is a direct indication of quantity).

[0067] FIG. 2 shows the amount by weight of the template peptide before and after the templated peptide synthesis, of four different peptides; Insulin (110 amino acids long), MRFA (4 amino acids long), VR9 (synthetic peptide not occurring in nature VMDSSYLSR, 9 amino acids long SEQ ID NO: 2), and WK20 SEQ ID NO: 3 (synthetic peptide not occurring in nature, WRWLEHNVVEGNAVNLMFSK). This shows that peptides of any length can be amplified, with structure and disulphide bonds not presenting an issue in the amplification process.

[0068] FIG. 3 shows the quantitative analysis of MRFA peptide synthesis using mass spectrometry (Thermo Q Exactive Orbitrap) in the presence of a MRFA template peptide (“amplification”) and in the absence of a template peptide (“control”). This shows that the addition of the template does indeed catalyse production of the peptide.

[0069] FIG. 4 shows the experimental designs for the subsequent experiments in order to demonstrate the lack of contamination present in the exemplified peptide synthesis process. The inclusion of additional control samples with sodium azide (NaN.sub.3), chloramphenicol (Chlor) and D amino acids (Daa's) were used to further guarantee the absence of biological contamination.

[0070] FIG. 5 shows the results of the testing for presence of DNA and RNA. The samples were tested for the presence of DNA and RNA using the high sensitivity Qubit® kits and representations of the observed intensities are shown. No RNA was ever detected and DNA was detected after the samples changed colour. To verify whether this was in fact DNA, benzonase (which digests DNA, and would therefore eliminate the detectable signal of the DNA in the samples) was applied to the samples and left to incubate. A positive control was always used and the intensity of the positive control always reduced by at least half, while the peptide synthesis samples “DNA intensity” was never reduced significantly. This verifies that there is not “life” present in the peptide synthesis samples (i.e. DNA and RNA).

[0071] FIG. 6 shows the results of the Marfey's Reagent derivatisation of D and L amino acids A. This chromatogram shows the overlay of a mixture of 4 commercial L amino acids (the black trace) and a mixture of the same 4 amino acids but the D isomers (the red trace). The delay in retention time can be seen in the D amino acids due to their derivatisation with Marfey's Reagent (FDAA, 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide). B shows the abio D amino acids sample taken at day 28, hydrolysed into single amino acids and derivatised with Marfey's (red trace) overlaid with the 4 commercial L amino acid chromatogram (black trace). It was observed that there was no conversion of D amino acids into L amino acids (which may be expected to occur if a biological life form contaminating the sample were utilising the D amino acids).

[0072] FIG. 7 shows the results of the gamma irradiation experiment (all samples run in triplicate). A positive control for sterilization was included in the form of an E. coli culture. The non-irradiated samples (A left side) show numerous colonies, and the irradiated samples (A right side) show after 1000 Gy irradiation no growth at all, demonstrating effective sterilisation. B is a histogram describing the peptide intensity distributions. The dark green population is the starting material, which has a low peptide count (as this was a template guided experiment—meaning the only products were the peptide itself and a small percentage of miscopies—which occur with this process) as well as lower median intensity. The standard incubated samples generated more peptides of higher intensity (i.e. more copies of peptides), with the irradiated samples showing a similar increase in peptide generation to that of the non-irradiated sample. The lack of distinct difference between these two sample sets suggests this is a chemical process not influenced by external living contamination of any sort. C is a Venn diagram showing the co-occurrence of peptides generated between conditions. The templated generation of peptides between the irradiated and not irradiated samples are equivalent, demonstrating the reaction is a chemical one, independent of bacterial/biological contamination.

[0073] FIG. 8 shows a Venn diagram showing the co-occurrence of detected peptides between various conditions (chloramphenicol, 37° C., standard, sodium azide (NaN.sub.3), and D Amino Acids). Chloramphenicol (broad spectrum antibiotic), sodium azide (gram negative bacterioside) and D amino acids (most living organisms require L-amino acids—and if D-amino acids are utilised by a living organism they will be converted into L-amino acids—which didn't occur in this case (see Marfey's reagent experiment) in the concentrations used in these experiments should eliminate bacterial growth. Peptide synthesis in all experiments is comparable and shows the chemistry basis for the synthesis as opposed to biological origin of the peptides.

[0074] FIG. 9 shows MALDI-TOF Mass Spectrometry of Commercial Amino Acids. These spectra show the starting materials are pure and free from any larger molecules which could contribute to the peptides we see in the experiments. A. MALDI-TOF spectra of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine and cysteine; B. MALDI-TOF zoomed mass spectra of leucine, glutamine, arginine, proline, serine, tryptophan, valine, methionine and cysteine; C. MALDI-TOF zoomed 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. MALDI-TOF zoomed mass spectra of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine, and alanine; F. MALDI-TOF zoomed mass spectra of tyrosine, asparagine, threonine, phenylalanine, aspartic acid, glycine, isoleucine, lysine, and alanine; G. MALDI-TOF mass spectra of glutamic acid and histidine; and H. MALDI-TOF zoomed mass spectra of glutamic acid and histidine.

[0075] FIG. 10 illustrates a schematic of the method of the present invention

[0076] FIG. 11 illustrates insulin synthesis using the method of the present invention.

[0077] FIG. 12 (A) illustrates an experimental set up to test the necessity of sun light and phosphate to the spontaneous polymerization of amino acids into peptides and proteins. All samples were run in triplicate (B) illustrates the experimental set up of all subsequent experiments with greater sterility. The inclusion of additional control samples with sodium azide, chloramphenicol and D amino acids were used to further guarantee the absence of biological contamination

[0078] FIG. 13 illustrates correlations (Log2 of peptide intensities) of all collected time points in the experiment of FIG. 12 and the venn diagram shows the majority of peptides seen were shared between samples.

[0079] FIG. 14 illustrates electron microscopy images of the structures generated by protein synthesis according to the method. The relative scales can be seen (A.) between TEM and SEM, and the structures that are very similar in appearance to viral capsids (A right side panels). The structures were measured and counted using Omero software and the results are graphed in B. The PBS sun samples generate larger and less numerous structures, whilst the PBS Dark and MilliQ Sun generate numerous and much smaller structures. The reproducibility of the bio-replicates is quite remarkable and can be seen in panel C.

[0080] FIG. 15 illustrates ultra-microtomy was used to visualise the interior of the spheres. In earlier samples (A, B, C, ˜day 60) there is a prevalence of linear decorated strands of protein. In later samples (J, K, L, 678 days) the spherical structures are the majority of what can be seen. These vary from completely empty spheres (F) to slightly filled spheres, and very rarely densely filled structures. When compared to traditional cross sections of cells there is a distinct lack of structure and notable emptiness, none-the-less these are cell-like and could possibly represent prototype cell structures.

[0081] FIG. 16 illustrates A: Peptide pair analysis revealed the most common and least common amino acid pairings seen in all identified peptides. There appears to be a preference for non-polar amino acids (I, L, G) to pair with acidic amino acids (E, D). The least favourable pairings are between non polar and basic amino acids—and predictably the lowest abundance amino acids in the mix and B describes the utilisation of amino acids in the most stable peptides seen in all samples. It does not appear that there is a trend in the chemistry of amino acids (polar, non-polar and acidic amino acids all increased in usage). Aspartic acid and Asparagine are increased in usage but are thought to destabilize helices. C: Some examples of the most stable peptides. While random coil is present there is a tendency towards helical structures, suggesting the hydrogen bond formation between a backbone N—H group hydrogen to the backbone C═O group of the amino acid four residues earlier is sufficiently stabilizing in this context. Structures predicted by PEP-FOLD (http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD)

[0082] FIG. 17 illustrates comparison between constant heating of samples in the dark at 37° C. and the same sample set up but with a natural light source. The Log2 Intensity values of the standard set up proteins/peptides which showed increasing trends over the time course (indicating stable peptide generation and longevity) were subtracted from that of the 37° C. samples and the resulting values clustered.

[0083] FIG. 18 illustrates a proposed method for the initial polymerisation of amino acids by the presence of amino acids in solution aided by energy (for example provided by sunlight energy) to overcome the required energy to form an amide bond wherein subsequent additions of amino acids to existing di-peptides are selected for in 2 ways (B). i) an enlarged cross-section increasing the likelihood of collisions with amino acids in the correct orientation, and ii) the energy required to add amino acids to existing di-peptides is 8× less than the initial amide bond. Additional local chemical environments favour of peptide/protein existence, inducing the folding of the peptide structures, excluding water molecules and forming secondary structures supported with hydrogen bonds and di-sulphide bonds (C). This generates structural stability and therefore longevity of peptides/proteins formed Reduction of entropy by forming spherical cell-like structures (D) to organize the proteins occurs. This again protects the structures formed, selecting in favour of these structures.

[0084] FIG. 19 illustrates apparatus to measure temperature fluctuations on the windowsill (A), then in a controlled heating environment without sunlight, to characterize the difference amino acids in solution made, and the amount of energy consumed in the apparatus (B) and finally the completely controlled environment used for all subsequent experiments (C). The system could be heated and cooled using a Peltier chip, with the addition of a sunlight replicating bulb (with a large range of wavelengths, including UV). Temperatures were monitored with medical grade temperature sensors. The apparatus was heated to 40° C. constantly for 12 hours with the bulb illuminated, and cooled to ambient temperature (˜18° C.) with the bulb off for 12 hours to replicate a controlled circadian cycle. Data presented here which was generated using the controlled environment was the sterilization experiment (1000 Gy irradiation samples). Temperatures were measured every 5 minutes with an accuracy of ±0.0625° C. Fluctuations in temperature on the window sills were 10.8125° C. (2096.7 J ° C..sup.−1g.sup.−1), whilst the temperature fluctuations in the “dark” sample were 0.4444° C. (185.8 J ° C..sup.−1g.sup.−1). To more accurately discern a difference between energy that may be consumed in the dehydration reactions required to make peptide bonds we used a controlled temperature environment and a control sample of PBS, and the PBS+amino acids solutions along with an ambient temperature measurement. Measurements were taken every 5 seconds for highly accurate heating and cooling curve description with the difference in area under the curve determined as 28962.97° C..sup.2s, or 170° C.s, with the area being less in the PBS amino acids sample. The maximum of the peak in the PBS+amino acids was always less that the control, suggesting energy was always consumed in this system compared to the control. The resulting energy utilised by the PBS+amino acids sample was 1.31 kcal, suggesting that in one cycle of heating from 24° C. to 34° C. and cooling more than 1 mol of di-peptide formation was possible. It was determined that biological replicates of each condition had remarkable correlation to each other (Pearson correlations of up to 0.96) after 7 days of “growth” but this correlation broadens at ˜126 days growth, but always remains positive. It is hypothesized to be due to larger structures forming (evident in the TEM and SEM, causing the efficiency of peptide replication to tail off (via steric hindrance), when hydrophobic areas induce folding, reducing accessibility to protein surfaces.

[0085] FIG. 20 illustrates Amino acid utilitisation in sample set up. The percentage of amino acids in TrEMBL % 2013 represents the percentages of amino acids in the starting solution. The differences in amino acids utilized compared to starting material are likely due to several factors such as trypsin digestion introducing bias (and therefore what we can measure in the Mass Spectrometer), chemical stability of amino acid sequence combinations and affinity to form amide bonds.

Methods and Results

Experiment 1

[0086] A synthesis experiment utilising heavy isotope labelled amino acids to distinguish the template peptide from the product peptide was carried out. This experiment was done with an internal standard (Benzoic acid) with chromatographic analysis (the industry standard for purity) and Mass Spectrometry to quantify the peptides (both light (template peptide) and heavy (newly synthesised) versions.

[0087] Eleven micrograms of synthetic peptide VR15 (VPDNLQQSLSDEAQR) SEQ ID NO: 1 was added to 1600 μl of amino acids solution at 0.2 g/ml concentration including Arginine which was 6 Daltons heavier than normal Arginine. Samples were incubated at 37° C., with a full spectrum light source, constantly for up to 4 hours.

[0088] 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 FIGS. 1B and 1C), and on the Mass Spectrometer in triplicate to measure the intensity of the heavy and light versions of the peptides (FIG. 1D).

Results

[0089] The consistent increase in signal after 2 hours in the chromatogram of the synthesised peptide (see FIG. 1A) and the lack of any signal increase in the internal control (benzoic acid peak) shows clearly more VR15 was generated. The increase in signal was not due to instrument variation or any other external factor.

[0090] The SILAC quantification and labelling of newly synthesised peptides allows us to differentiate between the old and the new peptide, and this is also clear in the resulting peptide intensity measured with Mass Spectrometry.

Experiment 2

[0091] During initial experiments it was found that proteins and peptides could be synthesised in the absence of cellular organelles and nucleic acids. Certain peptides were then selected to investigate a templated amplification process.

[0092] The following peptides were selected due to several factors [0093] 1. They do not occur in nature (with the exception of insulin). [0094] 2. They exhibited high copy numbers in initial experiments—suggesting they had been amplified. It was predicted that some peptides have unstable structure which is not conducive to longevity and replication. However, over 8000 different sequences were capable of being made reproducibly. [0095] 3. They had varying lengths to demonstrate the utility of the templated amplification process to a range of different peptides.

[0096] The peptide sequences initially investigated are set out in Table 1 below.

TABLE-US-00003 Template Concentration of Concentration Peptide Sequence MW template peptide Volume of Amino acids MRFA MRFA 523.65 0.001 g/ml 10 ml 0.01 g/ml SEQ ID NO: 4 MRFA* MRFA 523.65 0 10 ml 0.01 g/ml TR8 SEQ TGASLNSR 805.4163 0.00031 g/ml 10 ml 0.01 g/ml ID NO: 5 VR9 VMDSSYLSR 1057.4983 0.00023 g/ml 10 ml 0.063 g/ml SEQ ID NO: 2 Insulin MALWMRLLP 11974.026 0.00002 g/ml 100 ml  0.2 g/ml SEQ ID LLALLALWG NO: 6 PDPAAAFVN QHLCGSHLV EALYLVCGE RGFFYTPKT RREAEDLQV GQVELGGGP GAGSLQPLA LEGSLQKRG IVEQCCTSI CSLYQLENY CN VR15 RQAEDSLSQ 1699.8246 0.00044 g/ml 10 ml 0.01 g/ml QLNDPV *A negative control experiment was set up wherein no template peptide was added to the solution.

[0097] The concentration of amino acids represents 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 the amino acid in the template peptide. By way of example, taking TR8, the composition of the amino acid mixture making up the 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 (with no additional amino acids being present).

[0098] Table 1 shows a number of solutions used in the templated peptide synthesis process.

[0099] 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 bottles, sealed and then placed in an amplification apparatus.

[0100] The amplification apparatus subjected the samples to a controlled environment with a consistent temperature of 40° C. and constant source of full spectrum light. (Although it is possible to configure this apparatus in any number of ways).

[0101] Samples were taken (100 μl) daily under sterile conditions and analysed using MS (10 ml injected, 15 minute gradient, 2%-80% Acetonitrile 0.1% formic acid, Thermo Q Exactive Orbitrap) to determine the concentration of the peptides over time.

[0102] When a decrease in intensity was observed the amplification process was halted by placing the samples in a refrigerator. Absolute cessation of amplification is achieved by chromatographic separation of the peptides from the reagents (amino acids in solution).

[0103] The chromatographic separation of the product from reagents was achieved with a high flow rate large capacity C18 column, using reversed phase chromatography (the mobile phase used was A: 2% Acetonitrile, 0.1% Formic acid to B: 80% Acetonitrile, 0.1% Formic acid).

Results

[0104] The amount of peptide produced was measured following purification and the results are indicated in Table 2 below.

TABLE-US-00004 Peptide Before (g) After (g) Amount made (g) Increase (%) Insulin 0.0000342 0.000075959 0.000041759 122 MRFA 0.0085 0.010811 0.002310764 27 VR9 0.0027 0.005286 0.00258607 96 WK20* 0.0003 0.000906 0.000605756 201 *WK20 SEQ ID NO: 3 (not shown in Table 1) is a synthetic peptide having the sequence WRWVLEHNVVEGNAVNLMFSK.

[0105] Table 2 shows the amount of template peptide before and after the amplification process.

[0106] In each case, the amount of template peptide increased following the amplification process. This is illustrated in FIG. 2.

[0107] It was observed that the shorter the peptide, the more rapid the amplification process. The drop in intensity thereafter is due to additional amino acids being added onto the product peptide, resulting in the loss of the mass of the desired/template peptide.

[0108] The mass spectral analysis for the synthesis of MRFA in the absence of a template (negative control, Table 1) is shown in FIG. 3, in comparison to the templated synthesis (carried out in the presence of an MRFA template). In contrast to the templated synthesis, no appreciable difference in the intensity of MRFA is observed in the peptide synthesis carried out in the absence of a template peptide.

Experiment 3

Excluding Other Causes of Peptide Amplification

[0109] To exclude the possibility of contamination (i.e. peptides present in starting material, carry over in the chromatography process, bacterial/viral contamination, or “life” of any sort) being present, the following experiments were carried out. [0110] 1. Analysis of samples for presence of DNA and RNA. [0111] 2. Use of D amino acids to amplify peptides (as most life forms cannot use these, or if they do they convert them into L amino acids). [0112] 3. Use of gamma irradiation to sterilise the samples and verify if amplification still occurs. [0113] 4. Perform the experiments in the presence of Chloramphenicol or Sodium Azide. [0114] 5. Testing the starting components for purity.

[0115] See FIG. 4.

Methods

[0116] Commercial amino acids were measured out according to the percentages in Table 3 below to a total concentration of 1 g/100 ml. The amino acids were solubilised in sterile PBS (gibco 1× DPBS 14190-094). Solutions were mixed until complete solubilisation had occurred then passed through 0.22 μm stericup filters, and decanted 2 ml per autoclaved vial, in a cell culture fume hood with sterilised equipment. Vials were not opened again until an aliquot was required at the specified time point (again all done in a sterile environment). The samples were either analysed that day (if prior to day 14 incubation) or immediately reduced and alkylated, followed by in-solution digestion (with trypsin). The remainder of the sample was frozen at −80° C. An aliquot was taken for DNA and RNA analysis and analysed the day of collection also.

TABLE-US-00005 Amino Acid % Amino Acid % Ala (A) 8.73 Gln (Q) 4.00 Arg (R) 5.38 Glu (E) 6.20 Asn (N) 4.11 Gly (G) 7.10 Asp (D) 5.34 His (H) 2.18 Cys (C) 1.19 Ile (I) 6.09 Leu (L) 10.0 Ser (S) 6.52 Lys (K) 5.29 Thr (T) 5.52 Met (M) 2.50 Trp (W) 1.29 Phe (F) 4.03 Tyr (Y) 3.06 Pro (P) 4.55 Val (V) 6.81

[0117] 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).

DNA and RNA Detection

[0118] The 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. Twenty microliters of sample were used for the assays. The samples were measured on the Qubit® 2.0 Fluorometer, Q32866.

Marfey's Analysis of D Amino Acids Composition

[0119] One hundred microliters of each bio replicate of the D amino acids samples (T28=day 28) and the standard samples (T28=day 28) were pooled together resulting in 1 sample for each condition. The samples were rotary evaporated to dryness. 1 mL of 6M HCl was then added to the samples and they were heated at 155° C. for 80 minutes. The samples were then rotary evaporated to dryness. Samples were re-constituted with 300 μl of MilliQ water, and pH adjusted (to >5) with 3 μl of 10M NaOH. One hundred microliters were combined with 200 μl of Marfey's reagent (10 mg/ml in acetone) and 40 μl of 1M ammonium bicarbonate and gently shaken at 40° C. for 1 hour. The reaction was quenched with 20 μl of 2M HCl.

[0120] One hundred microliters of each sample were injected onto an XBridge® BEH 130 C18 Column, 130Å, 3.5 μm, 4.6 mm×250 mm. A gradient from 20% to 65% B over 45 minutes (A: 0.1% Formic acid, B: 80% Acetonitrile 0.1% formic acid) was used to separate the amino acids and the UV absorbance measured at 340 nm.

[0121] Mass Acquisition was performed using a Thermo Orbitrap QEaxactive, top 10 ions selected for ms/ms with an ms resolution of 140,000, ms/ms resolution 17,500, scanning from 150 m/z-2000 m/z.

Gamma Irradiation

[0122] Samples were prepared as described above (solubilisation of amino acids, filtered and sterile) but with VR15 as a template peptide, and only with the required amino acids to constitute that peptide. Samples were prepared in triplicate, as well as vials containing live E. coli, as a positive control for sterilisation (irradiation with 1000 Gy, in sealed plastic bags). The positive controls were then spread on agar plates to verify sterilisation had occurred, and the resulting peptides in all samples analysed by mass spectrometry (as described above).

Results

DNA and RNA Detection

[0123] With reference to FIG. 5, in the process of testing for RNA and DNA presence in the samples, RNA was never detected (lower limit of detection <20 ng/ml). Samples subjected to full spectrum light source however, changed colour over time, and apparently generated a positive reading for DNA.

[0124] To determine whether this was truly DNA, benzonase (an enzyme known to degrade DNA) was added to the samples, which were then incubated for an hour. A positive control with benzonase (with known bacterial contamination) was used. The DNA intensity was roughly halved in the positive control while in the peptide synthesis samples the DNA intensity did not change. Therefore it was concluded that certain of peptides/proteins formed in the method, autofluoresce at the same wavelength as DNA (485/530 nm).

D amino acid analysis

[0125] Most amino acids used in nature are the L chiral forms of amino acids, and most organisms cannot use them in standard protein production (they are however found commonly in peptidoglycan proteins in bacterial cell walls). The L forms of methionine, serine, alanine and tyrosine were substituted with the D versions in the normal amino acid mixture described above but otherwise the same experimental conditions were used.

[0126] The samples were then tested to test if the D amino acids had been converted into L amino acids by isomerases (e.g. such isomerases would be expected to be present if contaminant bacteria/life were present in the sample). A reagent known as Marfey's reagent (Nα-(2,4-Dinitro-5-fluorophenyl)-L-alaninamide) was utilised. On reaction with D amino acids, Marfey's reagent changes the retention time of those amino acids (when separated using chromatography) when compared to the L versions of the same amino acids.

[0127] The chromatograms illustrated in FIG. 6 exhibit this shift in retention time, from samples after 28 days of synthesis. These data further indicate there was no bacterial cellular contamination present in the samples.

Gamma Irradiation

[0128] Synthetic peptide synthesis experiments were run in triplicate, but with 1000 Gy of radiation applied to the samples, which were then sealed to maintain the sterilisation achieved by the radiation. Positive controls of E. coli culture were included to verify the sterilisation occurred. If the peptide synthesis still occurred, then it was indicative that the process is not a result of contamination in the form of life in the samples.

[0129] A positive control for sterilization was included in the form of an E. coli culture. The non-irradiated samples (FIG. 7, A left side) show numerous colonies, and the irradiated samples (FIG. 7, A right side) show after 1000 Gy irradiation no growth at all, demonstrating effective sterilisation.

[0130] FIG. 7B is a histogram describing the peptide intensity distributions. The dark green population is the starting material, which has a low peptide count (as not many peptides had been generated) as well as lower median intensity. The standard incubated samples generated more peptides of higher intensity (i.e. more copies of peptides), with the irradiated samples showing a similar increase in peptide generation to that of the non-irradiated sample.

[0131] FIG. 7C is a Venn diagram showing the co-occurrence of peptides generated between conditions. The stochastic generation of peptides between the irradiated and not irradiated samples are equivalent.

[0132] The lack of distinct difference between these two sample sets suggests that the peptide synthesis is a chemical process not influenced by external living contamination in the samples.

Chloramphenicol and Sodium Azide Containing Samples

[0133] The chloramphenicol and sodium azide samples were run in conjunction with the standard samples set up (sterile vial and amino acids solubilised in PBS) to allow a direct comparison to the abiotic peptide synthesis previously observed. The peptides were analysed in the same way, and compared to each other for any significant differences.

[0134] The Venn diagram shown in FIG. 8 illustrates the majority of peptides seen are identical in all samples. Additionally, there was no significant deficit in peptide production in the samples containing bacteriosides, which if the synthesis was a result of any bacterial or viral contamination, would have been expected to be reduced. This result further demonstrates that the described peptide synthesis process is a chemical one.

Testing Starting Materials for Contamination

[0135] All commercial amino acids, solvents and digestive enzymes (trypsin) were analysed independently by mass spectrometry, and put through the de novo peptide identification software to determine if the peptides were present to begin with.

[0136] In all cases, there were no unexpected masses observed that were greater than the stated reagent.

Example in Relation to Spontaneous Peptide Formation

[0137] Considering the hypothesis “Spontaneous peptide formation requires Sunlight and Phosphate”. Samples were set up in 3 different conditions (FIG. 12A) based on 2 assumptions. The first assumption was that Phosphate may be required given the energy unit used by most life is ATP—and the transfer of single phosphate molecules; therefore a simple source of phosphate in the form of Phosphate Buffered Saline was used. This had the additional properties of being a loose analogue to sea water (the environment where life may have begun 4 billion years ago in the Archaean). The second assumption was that energy from the sun was required to input sufficient kilojoules to overcome the amide bond formation step. Thus the samples were as follows: PBS Sun, PBS Dark, MilliQ Sun. The sample groups were intended as ‘PBS Sun’ being the proven positive, the ‘MilliQ Sun’ being a negative control and ‘PBS Dark’ another negative control.

[0138] However, it was determined that protein formation was apparent in all conditions. Thus, the inventor has determined that where there are building blocks for life (amino acids), in solution, with even minimal temperature fluctuations, spontaneous, cell and nucleic acid independent, protein synthesis will occur (FIG. 13).

Example—Test of the Hypothesis “Constant Heating (37° C.) Will be Better for Spontaneous Peptide Generation than the Cycling Heat of a Day

[0139] Peptides/protein synthesis was compared which occurred in both standard (samples provided at room temperature on a window sill) and 37° C. samples, which showed increasing trends over the time course (indicating stable peptide generation and longevity). The data showed it is favourable to have sun light (variable wavelengths) and cycling temperature over constant temperature (and no sun light FIG. 17). The effect is likely greater than the change seen here due to the fact that the energy input of 37° C. was 24-7, whereas the energy and light input received on the window sill was at best half this.

Conclusions

[0140] Detailed above is a method for peptide synthesis, based upon a previously unknown property of proteins and peptides acting as a template to replicate themselves, when placed in a solution of amino acids. Accompanying this data is extensive proof by several different means (gamma irradiation, D amino acids, bacteriocidal agents, as well as testing for presence of DNA and RNA) that this process is a chemical reaction and proceeds in the absence of nucleic acids, enzymes, co-enzymes, other cellular material and/or cells. Rather, the inventors believe that the templated peptide synthesis described herein may be driven by the provision of a low amount of energy (e.g. in the form of full spectrum light (including IR and UV) and/or gentle constant heat), the stoichiometry of the reagents and/or stereochemistry.

[0141] The spectra covered in FIGS. 9A-H are MALDI-TOF spectra verifying the purity of the starting commercially bought amino acids. The inventor has verified they were not containing anything aside from the specified amino acid (checking also for polymerisation of those amino acids into bigger peptides). This was indeed the case and the starting material was pure.