TelN Protelomerase: A New Force in Enzymatic DNA Synthesis
When it comes to telomeres and Protelomerase in biology, people's minds often first think of aging, an inevitable natural phenomenon. Telomeres is repetitive DNA sequences at the ends of eukaryotic chromosomes, which bears the responsibility of maintaining chromosome integrity and regulating the cell division cycle.
Protelomerase is a reverse transcriptase DNA synthesis enzyme that extends telomeres. It is a nucleoprotein composed of RNA and proteins. The protein component can catalyze the synthesis of telomere repeat sequences with the RNA component.
Figure 1. Mechanism of Protelomerase-mediated Telomere Extension[1]
Protelomerase can repair and lengthen telomeres, make up for defects in DNA replication, prevent telomere loss during cell division, and increase the number of cell divisions. In 2009, the Nobel Prize in Physiology or Medicine was awarded to Elizabeth H Blackburn, Carol W Greider, and Jack W Szostak for their outstanding contributions in Revealing the crucial role of telomeres and Protelomerase in cellular function and aging.
Today, I want to introduce to you a unique Protelomerase: TelN proProtelomerase. TelN Protelomerase comes from bacteriophage N15 and is a component of the N15 replication system, participating in the generation of linear prophage DNA. Unlike Protelomerase in eukaryotes, TelN is a pure protein enzyme without any RNA components. It has cleavage-ligation activity and leaves a covalently closed end at the cleavage site after cutting double-stranded DNA (dsDNA).
Figure 2. TelN Protelomerase Cleavage Site
TelN Protelomerase Mechanism of Action
The recognition site of TelN Protelomerase is a palindrome sequence that is 56 bp long, with telR and telL on both sides. The central position of the target sequence is also a palindrome sequence telO. TelN Protelomerase cleaves within the telO sequence and forms a covalently closed hairpin structure at the two cleavage ends. The end of the DNA after digestion is still composed of telR and telL, which is known as doggybone DNA (dbDNA).
Figure 3. Protelomerase cleavage mechanism at the TelN site[2]
Due to the special enzyme cleavage-ligation activity of TelN Protelomerase, circular plasmid DNA can be converted into linear covalently closed dumbbell-shaped molecules through a one-step enzymatic reaction. Compared with linear open DNA molecules, linear closed DNA has higher protein expression level in cells, which is very suitable for constructing linear closed end mini DNA with high stability and minimal foreign sequence. Plasmid DNA plays a vital role as the core element of mRNA vaccine, DNA vaccine and cell gene therapy. The traditional plasmid production method involves fermentation with E. coli and multi-stage amplification, and the uncontrollable risk in the fermentation process limits the yield of high-quality plasmid, which becomes the key to limit the production capacity of vaccines. Touchlight company in the UK has launched an innovative dbDNA technology. This technology disrupts the traditional method of bacterial fermentation for plasmid DNA preparation and instead of using in vitro enzymatic synthesis of DNA. The special enzyme cleavage-ligation activity of TelN Protelomerase is also employed to generate linear closed-end mini DNA in the above method.
Application of TelN Protelomerase in DNA Enzymatic Synthesis
DNA in vitro enzymatic method based on phi29 DNA polymerase and TelN Protelomerase can avoid many uncontrollable risks in the biological fermentation process, and the in vitro enzymatic method can synthesize DNA with fast speed and high yield. The synthesized DNA can be used in many emerging technologies, such as mRNA vaccine, DNA vaccine, gene therapy vector and gene editing.
Figure 4. Flowchart of DNA enzymatic synthesis[3]
Enzymatic DNA Synthesis Process
- Template denaturation
The circular plasmid DNA template is converted into two single-stranded circular DNAs by a denaturation process.
- Rolling circle amplification
Rolling circle amplification was carried out by using Phi29 DNA polymerase and single-stranded circular DNA to generate long linear double-stranded concatemeric DNA with Protelomerase recognition sequence intervals.
- Cutting and Covalent Closure
TelN Protelomerase recognizes the telRL on the DNA of telomeric complexes, and performs cleavage-ligation activities to generate linear, covalently connected DNA monomers.
- Removal of bacterial backbone DNA
Restriction endonucleases or exonucleases degrade the bacteria skeleton DNA with an open structure at the end to obtain DNA containing only the target gene expression elements.Enzymatic synthesis of DNA technology bypasses biological fermentation and can quickly achieve GMP-level plasmid DNA synthesis, solving the production capacity limitations in the fields of gene therapy and mRNA vaccines. It holds enormous potential for industrialization. To promote the development of DNA enzymatic synthesis technology, YEASEN Biology can provide core enzyme materials for DNA enzymatic synthesis, such as phi29 DNA polymerase (14404ES) and TelN Protelomerase (14540ES), to assist in the research and production of DNA enzymatic synthesis technology.
Performance presentation of YEASEN's TelN Protelomerase
- Cleavage-ligationperformance is excellent, which is equivalent to imported brands.
Using a supercoiled plasmid containing the TelN Protelomerase recognition site as a template, The gradient addition (0.078-5 U) enzyme from YEASEN and the brand A was added. 0.5 μg of supercoiled plasmid was converted to closed linear double stranded DNA (dsDNA). Gel electrophoresis was used to detect the conversion efficiency, and the results showed that the cleavage and ligation activity of YEASEN's TelN Protelomerase was equivalent to that of Brand A.
Figure 5. Protelomerase cleavage activity detection of TelN M:Marker, C: supercoiled plasmid control
- The closure integrityof linear dsDNA > 90%
Using the supercoiled plasmid containing TelN Protelomerase recognition site as template, adding TelN Protelomerase of YEASEN and brand A, converting 0.5 μg of supercoiled plasmid into closed linear dsDNA, and adding T5 exonuclease to detect its end integrity through band degradation degree. The results showed that the dsDNA end closure integrity generated by TleN Protelomerase of YEASEN was > 90%, which was equivalent to that of brand A.
Figure 6. TelN Protelomerase end closure integrity test. C1: plasmid containing TelN recognition site, without TelN Protelomerase; C2: plasmid containing TelN recognition site, TelN Protelomerase, without T5 exonuclease; Plasmid: plasmid containing TelN recognition site.
Related Products of DNA Enzymatic Synthesis
|
Product Classification |
Product Name |
Catalog No. |
|
Protelomerase |
14540ES |
|
|
Phi29 DNA Polymerase |
14404ES |
|
|
Exonuclease |
Exonuclease III |
14525ES |
|
T5 Exonuclease (10 U/µL) |
14538ES |
|
|
dNTP |
10125ES |
References
[1] Giardini MA, Segatto M, da Silva MS, Nunes VS, Cano MI. Telomere and Protelomerase biology. Prog Mol Biol Transl Sci. 2014;125:1-40.
[2] Heinrich J, Schultz J, Bosse M, Ziegelin G, Lanka E, Moelling K. Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells. J Mol Med (Berl). 2002;80(10):648-654.
[3] Heinrich J, Schultz J, Bosse M, Ziegelin G, Lanka E, Moelling K. Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells. J Mol Med (Berl). 2002;80(10):648-654. doi:10.1007/s00109-002-0362-2.