Phi29 DNA polymerase mutant lacking exonuclease activity, preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biology and provides a phi29 DNA polymerase mutant lacking exonuclease activity, a preparation method therefor and a use thereof. By modifying at least one of amino acid residues at positions 54, 55, 90, 96, 102, 140, 163, 203, 212, 277, 284, 327, 439, 502, 505, 534, and 552 in an amino acid sequence of a phi29 DNA polymerase, a protein having reduced exonuclease activity and retained polymerase activity is generated, thereby solving problems possibly caused by excessive exonuclease activity during the actual application of phi29 DNA polymerases, and improving the stability and reliability of phi29 DNA polymerases in DNA amplification, sequencing, and other molecular biology experiments.

Description

A phi29 DNA polymerase mutant lacking exonuclease activity, its preparation method, and its applications. Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a phi29 DNA polymerase mutant that has lost its exonuclease activity, its preparation method, and its application. Background Technology

[0002] Phi29 DNA polymerase is a carefully studied and optimized biopolymerase extracted from bacteriophage phi 29 of Bacillus subtilis. Its superior biological properties make it an important tool in the field of in vitro DNA amplification. This enzyme is unique in its exceptional continuous synthesis capacity and strong strand displacement activity, meaning it can continuously synthesize extremely long DNA strands, up to 70 kilobase pairs, in a single catalytic event. This characteristic greatly improves the efficiency and product length of DNA amplification, making it particularly suitable for DNA amplification techniques that require isothermal amplification under constant temperature conditions, such as rolling circle amplification (RCA) and multiple displacement amplification (MDA). These techniques have demonstrated outstanding performance in various scientific and clinical applications, such as the efficient amplification of whole or metagenomic genomes of pathogenic microorganisms for subsequent analysis, and the sensitive detection of low-abundance nucleic acid molecules such as viral genomes and microRNAs (miRNAs).

[0003] However, it is worth noting that while Phi29 DNA polymerase performs excellently in many applications, its use in third-generation sequencing technologies requires special consideration. Third-generation sequencing technologies, such as single-molecule real-time sequencing (SMRT) and nanopore sequencing, are based on the principle of real-time monitoring of DNA synthesis. This necessitates that the polymerase used should not unnecessarily degrade the newly synthesized DNA strand while adding individual nucleotides. Although Phi29 DNA polymerase possesses ideal 5'-3' polymerase activity, its simultaneous 3'-5' exonuclease activity may negatively impact the newly synthesized sequence during sequencing, leading to incomplete sequence recording.

[0004] Therefore, when designing systems for third-generation sequencing, it is necessary to significantly reduce exonuclease activity while retaining excellent polymerization activity. This ensures the acquisition of high-quality long-read sequence data during sequencing, thereby promoting the development of high-precision, long-fragment DNA sequence analysis. This is of great significance for improving the accuracy and efficiency of the entire sequencing process. Technical issues

[0005] The purpose of this invention is to address the aforementioned problems in the prior art by proposing a phi29 DNA polymerase mutant that retains good DNA polymerase activity but loses exonuclease activity. Technical solutions

[0006] The objective of this invention can be achieved through the following technical solution: a phi29 DNA polymerase mutant that loses exonuclease activity, wherein the phi29 DNA polymerase mutant includes any one of A1) to A2):

[0007] A1) Modify at least one amino acid residue from the amino acid sequence of phi29 DNA polymerase, specifically from the sequence numbered 54, 55, 90, 96, 102, 140, 163, 203, 212, 277, 284, 327, 439, 502, 505, 534, and 552.

[0008] A2) An amino acid sequence obtained by attaching a tag to the middle and / or N-terminus and / or C-terminus of the amino acid sequence shown in A1).

[0009] The tags described in this invention include at least one of the tags that facilitate the dissolution, expression, purification, fixation, and detection of phi29 DNA polymerase mutants.

[0010] It is understood that the phi29 DNA polymerase mutant of the present invention may contain one or more tags; multiple tags may include combinations of multiple identical tags, or combinations of multiple different tags. For example: tags that facilitate the dissolution of the phi29 DNA polymerase mutant include, but are not limited to, MBP tags, SUMO tags, NusA tags, thioredoxin, etc.; tags that facilitate the expression and purification of the phi29 DNA polymerase mutant include, but are not limited to, strep tags, His tags, GST tags, FLAG tags, dihydrofolate reductase (DHFR), pelB signal sequences, or ompA signal sequences; tags that facilitate the immobilization of the phi29 DNA polymerase mutant include, but are not limited to, streptavidin, biotin; tags that facilitate the detection of the phi29 DNA polymerase mutant include, but are not limited to, horseradish peroxidase (HRP), β-galactosidase, luciferase, hemagglutinin A (HA), green fluorescent protein (GFP), orange fluorescent protein (OFP), red fluorescent protein (RFP), and yellow fluorescent protein (YFP), etc.

[0011] Preferably, the label can be a His label.

[0012] In the aforementioned phi29 DNA polymerase mutant with low exonuclease activity, the phi29 DNA polymerase mutant has an amino acid sequence that is at least 90% identical to SEQ ID NO.26-48.

[0013] Preferably, the phi29 DNA polymerase mutant is an amino acid residue sequence of a protein derived from Bacillus subtilis that has more than 90% identity with the protein in SEQ ID NO.26-48.

[0014] Further preferred, the phi29 DNA polymerase mutant is an amino acid residue sequence of a protein derived from Bacillus subtilis that has more than 95% identity with the protein in SEQ ID NO.26-48.

[0015] As a preferred embodiment, the amino acid sequence of the phi29 DNA polymerase mutant is shown in SEQ ID NO.26-48.

[0016] Biological materials related to the aforementioned phi29 DNA polymerase mutant are also within the scope of protection of this invention, and the biological materials are any one of B1) to B4):

[0017] B1) A nucleic acid molecule encoding the phi29 DNA polymerase mutant as described in claim 1 or 2;

[0018] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0019] B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0020] B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0021] The recombinant vector contains the nucleic acid molecule described in B1) or the expression cassette described in B2), and the recombinant biological cell contains the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0022] In the above-mentioned biological materials, the nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NO.3 to SEQ ID NO.25.

[0023] Preferably, the expression cassette refers to DNA capable of expressing the phi29 DNA polymerase mutant in host cells. This DNA may include not only a promoter to initiate transcription of the phi29 DNA polymerase mutant gene, but also a terminator to terminate transcription of the protein gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0024] Preferably, the vector can be a plasmid, granule, bacteriophage, or viral vector. Specifically, it can be the PET-28a vector.

[0025] Preferably, the recombinant vector can be a recombinant vector obtained by inserting a nucleic acid molecule encoding the phi29 DNA polymerase mutant into the multiple cloning site of the vector.

[0026] Preferably, biological cells include prokaryotic cells and eukaryotic cells.

[0027] The prokaryotic cells include at least one of bacteria and algae.

[0028] The eukaryotic cells include at least one of fungi, mammalian cells, and insect cells. The bacteria may be *Escherichia coli*, such as *Escherichia coli* BL21(DE3).

[0029] The recombinant organism does not contain reproductive material.

[0030] The recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into a biological cell.

[0031] Specifically, it can be recombinant Escherichia coli obtained by introducing a recombinant vector into Escherichia coli BL21(DE3).

[0032] The present invention also provides an enzyme preparation comprising the above-described phi29 DNA polymerase mutant.

[0033] The present invention also provides a method for preparing the above-mentioned phi29 DNA polymerase mutant, the method comprising the following steps: introducing the coding gene of the above-mentioned phi29 DNA polymerase mutant into a biological cell, so that the coding gene is expressed, thereby obtaining the phi29 DNA polymerase mutant.

[0034] The present invention also provides a method for purifying the above-mentioned phi29 DNA polymerase mutant, the method comprising the following steps: high-throughput purification of the above-mentioned phi29 DNA polymerase mutant using His tag protein purification magnetic beads.

[0035] The present invention also provides a method for amplifying or sequencing template DNA, the method comprising the following steps: amplifying or sequencing template DNA using the above-mentioned phi29 DNA polymerase mutant.

[0036] The present invention also provides the application of any one of C1) to C3) in any one of D1) to D4), wherein C1) to C3) and D1) to D4) are as follows:

[0037] C1) The above-mentioned phi29 DNA polymerase mutant;

[0038] C2), the aforementioned biological materials;

[0039] C3), the enzyme preparations mentioned above;

[0040] D1) Nucleic acid amplification;

[0041] D2) Preparation of nucleic acid amplification-related products;

[0042] D3), ​​sequencing;

[0043] D4) Prepare sequencing-related products.

[0044] The present invention also provides a nucleic acid amplification or sequencing related product, including the above-mentioned phi29 DNA polymerase mutant or the above-mentioned enzyme preparation.

[0045] In one of the aforementioned nucleic acid amplification or sequencing-related products, the product also includes non-natural substrates. Beneficial effects

[0046] Compared with the prior art, the present invention has the following beneficial effects: The present invention designs and provides a mutant of phi29 DNA polymerase. By modifying at least one amino acid residue from the amino acid sequence of phi29 DNA polymerase at positions 54, 55, 90, 96, 102, 140, 163, 203, 212, 277, 284, 327, 439, 502, 505, 534, and 552, a novel protein with lower exonuclease activity but maintaining polymerase activity is generated. This successfully solves the problem that phi29 DNA polymerase may cause due to excessive exonuclease activity in practical applications, and improves its stability and reliability in DNA amplification, sequencing, and other molecular biology experiments. Attached Figure Description

[0047] Figure 1 is a schematic diagram of the pET28a carrier structure of Example 1.

[0048] Figure 2 shows the DNA polymerase activity detection results of the phi29 DNA polymerase mutant in Example 1.

[0049] Figure 3 shows the results of 3'-5' exonuclease activity detection in the phi29 DNA polymerase mutant in Example 1. Embodiments of the present invention

[0050] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the scope of protection of the present invention is not limited thereto.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0053] Unless otherwise specified, the solvent for all solutions or buffer solutions in the following examples is water.

[0054] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0055] The lysis buffer in the following examples is: 300 mmol / L NaCl, 50 mmol / L NaH2PO4, 10 mmol / L imidazole, pH=8.0.

[0056] The washing buffer in the following examples consists of 300 mmol / L NaCl, 50 mmol / L NaH2PO4, 20 mmol / L imidazole, and pH=8.0.

[0057] The elution buffer in the following examples is: 300 mmol / L NaCl, 50 mmol / L NaH2PO4, 250 mmol / L imidazole, pH=8.0.

[0058] The protein storage buffer used in the following examples is: 10 mmol / L Tris-HCl, 100 mmol / L KCl, 1 mmol / L DTT, 0.1 mmol / L EDTA, 0.5% Tween® 20, 0.5% IGEPAL® CA-630, 50% glycerol, pH=7.4.

[0059] The 10X phi29 reaction buffer in the following examples consists of: 500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl2, 100 mM (NH4)2SO4, and 40 mM DTT.

[0060] OligoA in the following examples: Oligo A is a 19nt single-stranded DNA with the sequence 5'-GsAsCTGACCGACCAGCCTTG-3'; "s" represents phosphate thioester.

[0061] The pET28-phi29 plasmid in the following examples: Based on the wild-type amino acid sequence SEQ ID NO.1 of Phi29 DNA polymerase and the codon preference of E. coli, the wild-type nucleic acid sequence SEQ ID NO.1 was synthesized and inserted between the NcoI and XhoI sites of the pET28a vector shown in Figure 1, without retaining the C-terminal His tag, to obtain the recombinant plasmid. Example 1

[0062] S1. Using pET28-phi29 plasmid as a template, the phi29 nucleic acid sequence shown in SEQ ID NO.1 was amplified with high-fidelity polymerase. Then, the pET28-phi29 plasmid and the phi29 nucleic acid sequence shown in SEQ ID NO.1 were digested with restriction endonucleases NcoI and XhoI. The digested plasmid fragments and the sequence were spliced ​​together using T4 DNA ligase to obtain the nucleic acid sequence shown in SEQ ID NO.2. The splicing product was transformed into E. coilDH5α competent cells for verification to obtain the original expression plasmid of Phi29 DNA polymerase.

[0063] S2. Using the partially overlapping primers (containing mutation sites) shown in Table 1, the phi29 nucleic acid sequence shown in SEQ ID NO. 3-25 was sequentially amplified using the original expression plasmid of phi29 DNA polymerase as a template. The amplification reaction system (50 μL) consisted of: 10 μL 5x Pfu Reaction Buffer, 4 μL dNTP Mix (2.5 mM), 30 ng pET28-Phi29 plasmid, 1 μL Pfu DNA Polymerase, 1 μL forward primer (10 μM), and 1 μL reverse primer (10 μM). The amplification conditions were: 95℃ for 2 min, 30 cycles for [95℃ 20 s, 55℃ 20 s, 72℃ 7 min], 72℃ for 5 min, and 16℃ infinity. After being digested with Dpnl, the amplified product was transformed into E. coil DH5α competent cells, plated on LB plates containing kanamycin, and cultured at 37°C. Single colonies were then picked, plasmids were extracted, and sequenced to verify whether the phi29 DNA polymerase mutant plasmid was successfully constructed. The phi29 DNA polymerase mutant plasmid was obtained.

[0064] Table 1: Partially Overlapping Primers

[0065]

[0066] S3. The verified original plasmid and mutant plasmid of phi29 DNA polymerase were transformed into the expression host bacterium E. coil BL21(DE3) for further induction and purification. Due to the large number of mutant proteins, His-tagged protein purification beads were used for high-throughput purification of the mutant proteins.

[0067] Inducible expression of protein:

[0068] (1) Pick a single colony and inoculate it into LB liquid medium containing 100ug / mL Kana resistance, and incubate overnight at 37°C to activate it;

[0069] (2) Take the activated bacterial solution and inoculate 1% into 100ml of LB liquid medium containing 100ug / mL Kana resistance. Incubate at 37℃ and 200 r / min for about 2h until the logarithmic growth phase. The bacterial cell OD 600 It is approximately 0.6;

[0070] (3) Add IPTG to the culture medium to a final concentration of 0.2 mmol / L, and incubate overnight at 16℃ and 200 r / min for 8 h;

[0071] (4) Centrifuge at 4000 r / min and 4℃ for 5 min, and collect the bacterial cells;

[0072] (5) Discard the supernatant and resuspend the bacterial cells in 20 mL of Lysis buffer;

[0073] (6) Add PMSF to each tube of bacterial cells at a ratio of 1 wt%, and sonicate under ice bath conditions. Sonicate for 5 seconds with a 7-second interval, for a total of 99 times. The bacterial concentration difference after each collection is considered, and the bacterial solution becomes clear as the termination criterion.

[0074] (7) Add PEI to a final concentration of 0.3wt%, stir slowly for 10 min, and remove DNA from the extract;

[0075] (8) Centrifuge at 18000 r / min for 40 min and collect the supernatant;

[0076] (9) SDS-PAGE gel electrophoresis was used to analyze protein expression. If the target protein was distributed in the supernatant, subsequent purification operations were performed.

[0077] Purification of the target protein:

[0078] (1) Vibrate the Ni-Charged MagBeads thoroughly to mix them;

[0079] (2) Take an appropriate amount of Ni-Charged MagBeads into a centrifuge tube and place the centrifuge tube on a magnetic separator to collect the magnetic beads;

[0080] (3) Add 1 mL of Lysis buffer to (2) and invert the tube several times to mix. Collect the magnetic beads using a magnetic separator and discard the supernatant. Repeat this step twice;

[0081] (4) Add the cell lysate containing the multihistidine-labeled protein to the test tube and gently invert the test tube to mix;

[0082] (5) Oscillate at a lower temperature for 60 minutes;

[0083] (6) Collect the magnetic beads using a magnetic separator and discard the supernatant;

[0084] (7) Add 1 mL of washing buffer to (6), stir well, collect the magnetic beads with a magnetic separator, discard the supernatant, and repeat twice;

[0085] (8) Add 500uL of Elution buffer to (7), mix well, and incubate in an ice bath for 5 min. You can wash repeatedly to increase the yield of the target protein.

[0086] (9) Collect the magnetic beads using a magnetic separator and transfer the supernatant containing the eluted protein into a clean test tube;

[0087] (10) Inject the supernatant containing the eluted protein from (9) into an ultrafiltration tube, centrifuge at 3000xg, replace and concentrate the protein storage buffer, and store at -20℃;

[0088] (11) SDS-PAGE gel electrophoresis was used to analyze the protein purification status. If there was a single target band with the correct molecular weight, the purification was successful and the amino acid sequence of phi29 DNA polymerase as shown in SEQ ID NO 26-48 was obtained. Example 2

[0089] The phi29 DNA polymerase and wild-type phi29 DNA polymerase prepared in Example 1 were used as the test enzyme solutions. Rolling circle amplification was performed using the single-stranded M13mp18 genome as a template. A mixture of 0.1 μM M13mp18 and 1 μM M13-II specific primers was incubated at 95 °C for 3 min, followed by annealing on ice for 5 min. The template / primer complex, dNTPs, 10X phi29 reaction buffer, and each phi29 DNA polymerase (200 nM) were incubated at 30 °C for 30 min. The reaction was terminated by adding 0.5 M EDTA; the negative control was the absence of phi29 DNA polymerase. The samples were mixed with alkaline buffer and alkaline loading buffer, and electrophoresed slowly at 50 V on an alkaline agarose gel for 6 h. The agarose gel was then equilibrated in neutralization buffer for 20 min and stained in 1X TAE buffer containing SYBR Gold (10000x) for 40 min. As shown in Figure 2, gel imaging revealed that the above reaction system and conditions resulted in the formation of numerous long-fragment nucleic acid products, with amplified fragments ranging from approximately 48.5 kb. The markers ranged in size from 10171 bp to 48502 bp. Lane 1 contained the product obtained by adding wild-type phi29 DNA polymerase, while lanes 2-23 contained products obtained by adding various mutant phi29 DNA polymerases. Example 3

[0090] The phi29 DNA polymerase and wild-type phi29 DNA polymerase prepared in Example 1 were used as the test enzyme solutions. Exonuclease activity was determined by incubating 0.1 μM phi29 DNA polymerase with 1 μM Oligo A, with the negative control being the absence of phi29 DNA polymerase. After incubation with 10X phi29 DNA reaction buffer at 30°C for 10 minutes, the sample (10 μL) was mixed with an equal volume of 2X TBE-urea DNA loading, and the products were analyzed using a 20% TBE-urea gel. As shown in Figure 3, the marker ranges from 20 nt to 75 nt. Lane 1 represents the product obtained with the addition of wild-type phi29 DNA polymerase, and lanes 2-23 represent the products obtained with the addition of each mutant phi29 DNA polymerase. Gel imaging revealed that the wild-type phi29 DNA polymerase exhibited strong exonuclease activity, almost completely degrading the single-stranded template. The phi29 DNA polymerase mutants, however, did not show significant exonuclease activity.

[0091] In summary, this invention designs and provides a mutant of phi29 DNA polymerase. By modifying specific amino acid residues, particularly at least one of the amino acid residues at positions 54, 55, 90, 96, 102, 140, 163, 203, 212, 277, 284, 327, 439, 502, 505, 534, and 552 in the amino acid sequence of phi29 DNA polymerase, a novel protein with lower exonuclease activity but maintaining polymerase activity is generated. This successfully solves the problem that phi29 DNA polymerase may cause due to excessive exonuclease activity in practical applications, and improves its stability and reliability in DNA amplification, sequencing, and other molecular biology experiments.

[0092] The embodiments herein cover any points not exhaustively within the scope of the technical claims of this invention, as well as new technical solutions formed by equivalent substitutions of one or more technical features in the embodiments. These are all within the scope of the claims of this invention. Furthermore, in all listed or unlisted embodiments of this invention, each parameter in the same embodiment merely represents an instance (i.e., a feasible solution) of its technical solution, and there is no strict coordination or limitation relationship between the parameters. The parameters can be substituted for each other without violating axioms and the claims of this invention, unless otherwise stated.

[0093] The technical means disclosed in this invention are not limited to those described above, but also include technical solutions composed of any combination of the above technical features. The above descriptions are specific embodiments of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

[0094] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A phi29 DNA polymerase mutant lacking exonuclease activity, characterized in that, The phi29 DNA polymerase mutant includes any one of A1) to A2): A1) Modify at least one amino acid residue from the amino acid sequence of phi29 DNA polymerase, specifically from the sequence numbered 54, 55, 62, 90, 96, 102, 140, 163, 203, 212, 277, 284, 327, 439, 502, 505, 534, and 552. A2) An amino acid sequence obtained by attaching a tag to the middle and / or N-terminus and / or C-terminus of the amino acid sequence shown in A1).

2. The phi29 DNA polymerase mutant with lost exonuclease activity according to claim 1, characterized in that, The tags include at least one of those that facilitate the dissolution, expression, purification, fixation, and detection of the phi29 DNA polymerase mutant.

3. A phi29 DNA polymerase mutant lacking exonuclease activity according to claim 1 or 2, characterized in that, The tag is His.

4. The phi29 DNA polymerase mutant with lost exonuclease activity according to claim 1, characterized in that, The phi29 DNA polymerase mutant has an amino acid sequence that is at least 90% identical to SEQ ID NO. 26-48.

5. The phi29 DNA polymerase mutant with lost exonuclease activity according to claim 1, characterized in that, The amino acid sequence of the phi29 DNA polymerase mutant is shown in SEQ ID NO.26-48.

6. A biomaterial related to the phi29 DNA polymerase mutant of claim 1, characterized in that, The biomaterial is any one of B1) to B4): B1) A nucleic acid molecule encoding the phi29 DNA polymerase mutant of claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

7. The biomaterial according to claim 6, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NO.3 to SEQ ID NO.

25.

8. An enzyme preparation, characterized in that, Includes the phi29 DNA polymerase mutant as described in claim 1.

9. A method for preparing the phi29 DNA polymerase mutant according to claim 1, characterized in that, The method includes the following steps: introducing the coding gene of the phi29 DNA polymerase mutant according to claim 1 into a biological cell, so that the coding gene is expressed, and obtaining the phi29 DNA polymerase mutant.

10. A method for amplifying or sequencing template DNA, characterized in that, The method includes the following steps: amplifying or sequencing template DNA using the phi29 DNA polymerase mutant as described in claim 1.

11. The application of any one of C1) to C3) in any one of D1) to D4), characterized in that, C1) to C3) and D1) to D4) are shown below: C1) The phi29 DNA polymerase mutant according to claim 1; C2), the biomaterial as described in claim 6 or 7; C3), the enzyme preparation according to claim 8; D1) Nucleic acid amplification; D2) Preparation of nucleic acid amplification-related products; D3), ​​sequencing; D4) Prepare sequencing-related products.

12. A nucleic acid amplification or sequencing related product, characterized in that, Includes the phi29 DNA polymerase mutant of claim 1 or the enzyme preparation of claim 8.

13. The product according to claim 12, characterized in that, The product also includes non-natural substrates.

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