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“Look, this is something”: self-replication of artificial DNA



One of the main distinguishing features of any living organism is the ability to save and reproduce the necessary information to create their own kind. This primarily manifests itself in DNA replication, when a pair of daughter ones appear from the same parent DNA, which are an exact copy of their progenitor. In nature, this process is observed everywhere, but it is extremely difficult to recreate it under laboratory conditions from scratch, however, it is quite realistic.

Scientists from the Institute of Biochemistry. Max Planck (Germany) successfully created a biological system that has the ability to replicate its own DNA. What techniques were used to create synthetic replicating DNA, how effective is the resulting system, and what does this discovery mean for modern synthetic biology? We will find answers to these questions in the report of scientists. Go.

Study basis


In the field of creating artificial biological systems, the ability to replicate is a key aspect of the usefulness of the created system. According to scientists, this can be achieved through the implementation of the basic components: replication * , transcription * and DNA translation * .
* — .
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In other words, in order to create a full-fledged artificial life, it is necessary to implement self-encoded reproduction, i.e. self-replication. This is an extremely complicated and confusing process.

The authors of the study note that in systems based on existing biochemistry, for example, MPC ( minimal protein-based cells) , self-replication requires a complete rethinking of molecular biology, including replication, transcription and translation of DNA, taking into account a completely cell-free environment.

The synthesis of protein from DNA can be achieved in certain recombinant systems based on phage * RNA polymerases - the main parts of the translation mechanism of Escherichia coli and the system of minimal energy regeneration, i.e. PURE - Protein synthesis Using Recombinant Elements (protein synthesis using recombinant elements).
Bacteriophages or phages * are viruses that selectively infect bacterial and archaeal cells. In biology, it is used as a vector (DNA for transferring genetic material into the cell).
Meanwhile, it remains extremely difficult to recreate the process of DNA replication of the genome based on transcription and translation (TTcDR - transcription – translation-coupled DNA replication ), which encodes all macromolecular components of the PURE system using self-encoded replicoma * .
Replisoma * is a multi-protein complex that replicates bacterial DNA.
DNA replication based on DNA polymerases (DNAP) from phages (e.g., Phi29) can help implement the TTcDR technique.

However, in the TTcDR method, the full-sized Phi29 genome could be achieved only by blocking some of the replicating factors.

All the above methods have their theoretical advantages, but in practice they did not give a full result. In the work we are considering today, scientists describe a translation system that provides self-encoded replication and expression of large DNA genomes under well-defined cell-free conditions. In particular, self-replication of the genome of over 116 kb was achieved. (thousand pairs of nucleotides), covering the full range of translation factors Escherichia coli(Escherichia coli), all three ribosomal RNAs, the energy regeneration system, as well as RNA and DNA polymerases * .
Polymerase * is an enzyme that performs the synthesis of polymers of nucleic acids. DNA polymerase synthesizes DNA, and RNA polymerase synthesizes RNA. This process proceeds through complementary copying of parental DNA or RNA.
In addition, the created system demonstrated the ability to synthesize more than 30 translation factors, half of which expresses in amounts equal to or greater than the introductory ones.

Research results


At the first stage, scientists tested the self-encoded Phi29-DNAP-dependent TTcDR using the standard protocol of the PURExpress system .

The Phi29-DNAP coding region flanked by the * T7 promoter was first cloned into the pCR-Blunt TOPO vector (pREP, 1a ).
Promoter * is a DNA nucleotide sequence used by RNA polymerase as a region for transcription initiation.


Image No. 1

In theory, this architecture should provide spontaneous RNA replication as a rolling ring * using self-encoded DNAP without additional replication proteins or externally provided DNA primers.
Rolling Ring Type Replication * is a unidirectional nucleic acid replication process during which a rapid synthesis of multiple copies of ring DNA or RNA molecules occurs.
However, using the standard PURExpress reaction with dNTP (nucleotide containing C 5 H 10 O 4 deoxyribose ) and 4 nM pREP (LB medium supplemented with zeocin C 55 H 86 N 20 O 21 S 2 ), DNA synthesis was not detected using agarose gel electrophoresis * , nor by real-time polymerase chain reaction * ( 1b and 1c ).
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To improve DNA replication without the involvement of specialized PURE systems, it was decided to optimize the standard PURExpress reaction protocol. For this, the relative number of translation factors, ribosomes, and a reducing agent was increased while lowering the levels of tRNA (transport RNA) and rNTP (ribonucleoside triphosphate) ( 1d ).

Using this optimized PURE composition (PURErep), it was possible to achieve ∼5–12-fold replication of pREP monomeric units in the TTcDR reactions ( 1b and 1e ). Full size pREP synthesis was confirmed by MluI digestion * of the replication product ( 1c ).
MluI * is a commercially available restriction endonuclease, i.e. an enzyme that catalyzes the hydrolysis of nucleic acids.
The expression of green fluorescent protein (sfGFP) was used as an evaluation factor of translation activity. It was found that a change in the composition of PURE led to a decrease in protein synthesis by 20-40% compared with PURE without TTcDR. However, this decrease in protein expression remains acceptable given the improved compatibility of the PURErep system with DNA replication.

Analysis of DNA replication based on qPCR revealed a stable doubling time (1-2 hours) for various initial matrix concentrations, while DNA replication continued even after 24 hours at 30 ° C ( 1e ).

TTcDR was also stable for more than five generations of sequential dilution when only 4% of the finished PURErep / pREP reaction product was directly transferred to the new PURErep mixture ( 1f) The main conclusion of this observation is that TTcDR products can serve as templates for DNA self-replication over several generations.

Scientists note that a significant amount of product with low electrophoretic mobility is typical for replication as a rolling ring, which was observed during the experiments. These reaction products can be represented by high molecular weight concatemers * and / or DNA / MgPPi clusters.
Concatemer * - multiple copies of DNA sequences assembled in a sequential cluster.
The formation of products with a size of ~ 5 kb was also detected. in untreated samples. It follows that TTcDR reactions can produce significant amounts of pREP monomeric copies.

In addition, scientists were able to transform E. coli products after removal of the parent plasma. The resulting purified reaction products were identical in size to the monomeric pREPs.

At the next stage, the scientists decided to create a set of genes encoding the important components of the PURE reaction: 31, an essential translation factor (FT) of E. coli bacteria .

For this, TTcDR from pREP (4.6 kb) was studied in conjunction with each of the three large plasmids *: pLD1 (30 bp, 13 FT), pLD2 (20 bp, 8 FT) and pLD3 (23 bp, 9 FT). All of them were cloned to provide recombinant expression of 30 of the 31 translation factors of E. coli bacteria .
Plasmids * are small DNA molecules capable of independent replication.
TTcDR products of all four plasmids (including pREP) showed identical MluI restriction features: clonal plasmids, typically propagated in E. coli ( 2a ).


Image No. 2

It is also worth noting that pLD TTcDR products can be directly transformed into E. coli , where they are stored as monomeric plasmids.

The modified PURErep mixture also provided complete replication of all three pLD plasmids together with PURErep during the reaction in a common medium ( 2b ).

Scientists did not stop there and already at the next stage of the study they tried to expand the genetic load of the TTcDR system by co-replicating plasmids encoding additional components of the PURE system: EF-Tu (pEFTu), which is absent in the pLD system, as well as the ribosomal RNA operon * rrnB ( prRNA is a precursor rRNA) that encodes 23S rRNA (ribosomal RNA), 16S rRNA, 6S rRNA and tRNA (transport RNA, in this case Glu2) ( 2c ).
Operon * is a functional unit of the unicellular genome, which includes genes encoding proteins.
QPCR experiments targeting plasmid-specific amplicons * confirmed that the monomeric units of all six plasmids (total DNA length was 93 kb) replicated about 2-8 times compared to the original number ( 2c ).
Amplicon * is an extrachromosomal unit of amplification ( copying of DNA sections).
Additional confirmation of the success of joint replication of plasmids was the formation of colonies resistant to either zeocin (pREP), or to kanamycin (plasmid pLD and prRNA), or to carbenicillin (pEFTu). These colonies were the result of the transformation of PURErep reaction products treated with DpnI ( 2d ).

The selected 26 clonal colonies followed by restriction analysis once again confirmed the success of the TTcDR of all six plasmids ( 2e ).

Using the same method as previously described, scientists carried out another procedure for joint replication of five additional plasmids: genes for the minimal regeneration system of nucleoside triphosphate based on creatine kinase (pCKM), adenylate kinase (pAK1) and nucleoside diphosphate kinase (pNDK); as well as T7 RNA polymerase (T7RNAP) and pyrophosphatase (pIPP).


Image No. 3

With a total size of 116.3 kb this set of 11 plasmids reaches> 100% of the estimated genome length proposed for a minimal, self-replicating system that depends only on low molecular weight nutrients ( 3a ).

The scientists then decided to check whether PURErep can provide parallel gene expression during replication. For this, the multicistronic expression of FT encoded on three pLD plasmids: pLD1, pLD2 and plD3 (not including pEFTu) was examined.

In order to investigate whether PURErep is able to generally support multicistronic expression from these plasmids, cell-free expression was performed from each individual plasmid in the presence of BODIPY-Lys-tRNALys, which provides fluorescence labeling of translation products.

In order to improve detection sensitivity and enable the quantification of newly synthesized proteins, a quantitative analysis of protein expression was further performed based on mass spectrometry using stable isotopic labels.


Image No. 4

This analysis provided convincing evidence for the synthesis of all FT subunits of proteins encoded by pLD ( 4a ). Partial or complete regeneration of proteins encoded in both pLD2 and pLD3 was also observed.

Analysis of expression showed that even in unmodified PURErep in combination with pREP, there is a complete replication of 32 FT cidrons encoded by pLD and expression of about half of the encoded FT peptide chains, the number of which corresponds to or exceeds the initial one.

For a more detailed acquaintance with the nuances of the study, I recommend that you look into the report of scientists and additional materials to it.

Epilogue


In this work, scientists managed to achieve what many of their colleagues previously could only dream of - creating an artificial system that is capable of self-replicating.

Exaggerated saying this means that they were able to take the first steps towards creating a system that will self-reproduce, thereby simulating biological processes as much as possible.

The authors of the study are extremely pleased with the results and plan to expand the artificial genome with additional DNA segments in the future. They want to create a system with a shell that can maintain viability by absorbing nutrients and disposing of waste. Such an artificial structure can be used as a specialized production bio-device for natural substances or as a basis for creating even more complex systems.

Cloning is a complex process, of which there is no doubt, but the creation of a complete, viable synthetic system is a completely different level. Someone will say that such studies are dangerous, because no one has the right to assume the responsibilities of mother nature. However, the curiosity of a person is almost impossible to calm down. Our desire to understand everything and repeat everything is one of the driving factors in the progress of science. For better or worse, this is a question for which there is no single answer. We can only admire the achievements of bright minds and cautiously look to the future, while hoping for a utopia.

Thank you for your attention, stay curious and have a great weekend everyone, guys! :)

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