Synthetic Biology
Course for Master Students
Cover image from: https://www.technologynetworks.com/drug-discovery/blog/how-is-synthetic-biology-shaping-the-future-of-drug-discovery-340290
part 2
Cover image from: https://www.technologynetworks.com/drug-discovery/blog/how-is-synthetic-biology-shaping-the-future-of-drug-discovery-340290
xenobiology
Xenobiology is a branch of synthetic biology with the goal of developing life forms with different biochemistry or genetic code.
The term "xenobiology" comes from the ancient Greek ξενος, meaning "foreign" or "guest."
xenobiology
01
origin of life
02
exobiology
03
system chemistry
04
Synthetic biology
"Generatio spontanea" refers to the idea that life could suddenly arise from non-living matter - Aristotel.
The spontaneous generation of life, however, was rejected by scientific experiments conducted by Pasteur, whose results demonstrated that modern organisms do not arise spontaneously in nature from non-living matter.
However, on sterile Earth about 4 billion years ago, at least once, abiogenesis must have occurred to ultimately lead to the emergence of the Last Universal Common Ancestor (LUCA). The genetic code of LUCA was expected to be based on DNA with four nitrogenous bases forming trinucleotides, codons encoding twenty amino acids.
Orthogonal life
refers to the concept of modularity in contemporary industry. Modularity involves constructing complex products from smaller subsystems that can be created independently but function together as a unified whole. One of the fundamental requirements of modularity is orthogonality. The term orthogonality comes from the Greek orthos-, meaning "straight," and gonia-, meaning "angle" or "corner."
�In the natural world, there is no existence of the principle of orthogonality in living organisms, meaning that a change in one component or mechanism leads to changes in the whole. Research on orthogonality is conducted in two directions: creating metabolic orthogonality and biochemical building blocks.
Metabolic orthogonality
Metabolic orthogonality is one of the approaches in synthetic biology aimed at creating modular platforms for the highly efficient synthesis of complex chemical compounds. The goal of this approach is to extract individual synthetic modules from the cell's metabolic system that will not interact with each other. For example, an energy module and a sugar synthesis module can be designed without enzymatic interaction. This allows the exclusion of feedback principles. Thus, separating two metabolic modules will enable adjusting the performance of one module by reconfiguring its key enzymes without affecting the other.
Biochemical orthogonality
Biochemical orthogonality in synthetic biology involves modifying certain natural biochemical blocks. This enables the use of alternative biomolecules to support natural processes. Amino acids, nucleotides, proteins, and DNA have been modified, allowing the creation of proteins that do not exist in nature. Additionally, research focused on creating "mirror life," living molecules with chirality opposite to existing natural molecules, presents significant possibilities.
The modification in the translation process from mRNA to protein using tRNA has allowed the incorporation of certain modified amino acids (p-methoxyphenylalanine-pMpa, p-acetylphenylalanine-pAra, p-benzoylphenylalanine-pBra, p-iodophenylalanine-pIra, p-azidophenylalanine-pAzra, and p-propargyloxycarbonylphenylalanine-pPra).
The incorporation of phenylalanine derivatives into the structure of proteins in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells has allowed triplets to code for 64 amino acids. It has also been demonstrated that amino acids can be encoded by an additional four nucleotides, theoretically providing 256 amino acids with new assignments. The ability to include more than the 20 natural amino acids will lead to the creation of new proteins that do not exist in nature, increasing the diversity in the interpretation of the genetic code.
To introduce amino acids that are not naturally occurring, it is necessary to have clean codons and adapt mRNA to use quadruplets instead of triplets. To achieve this, a library of 16S r-RNA codes was created using site-directed mutagenesis. It is noteworthy that a synthetic, orthogonal ribosome named Ribo-X was successfully created through these efforts.
�Azido-phenylalanine was introduced using the glutathione-S-transferase-maltose creating protein. An alkyl amino acid was also introduced. Calmodulin protein was synthesized with two non-natural amino acids, azido-phenylalanine, and N6-[(2-propinyl)carbonyl]-L-lysine, which linked together. Thus, one-third of the protein structure became more stable.
To build a xenobiological system, it is necessary to create a synthetic orthogonal chromosome. This is only possible with the presence of xeno-nucleotides.
Analogs of nucleotides can be diverse, with changes possible in three parts of the molecule. Experiments on creating artificial DNA began in 1989 when Steven Benner and colleagues synthesized DNA with six nucleotide sequences. Two of these were artificially created and did not occur in nature.
Xeno-DNA was introduced into bacterial cells using a plasmid.
�Synthetic pair of nucleotides: Z-purine nucleotide (6-amino-5-nitro-3-(1'-β-2'-deoxyribofuranosyl)-2(1H)-pyridone); P-pyrimidine nucleotide (2-amino-8-(1-β-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)). Three intermolecular hydrogen bonds between the purine base amine/carbonyl and the pyrimidine base carbonyl/amine.
IsoG----------IsoC
Synthetic pair of nucleotides IsoG-IsoC: This is a mirror reflection of the natural nucleotides Cytosine and Guanine: the purine amine/ketone of isoguanine forms three intermolecular hydrogen bonds with the pyrimidine isocytosine ketone/amine.
(DAP-T) synthetic nucleotide pair: The purine amine/amine/amine forms three intermolecular hydrogen bonds with the pyrimidine ketone/amine/ketone.
� In xenonucleic acids, the nucleotides have replacements for deoxyribose and ribose with molecules containing four carbon compounds instead of the standard five-carbon carbohydrates. Others may contain up to seven carbon atoms. These substitutions make XNA functionally and structurally analogous to natural DNA and RNA, but they are unnatural and synthetic.
In 2014, two complementary synthetic nucleotides, X and Y, which do not naturally occur anywhere, were inserted into bacterial DNA. Moreover, they were present in a cultural environment. This allowed the bacteria, E. coli, to be passaged 24 times. The DNA with these additional nucleotide bases replicated normally during cell division and was passed on to other bacteria, but these new nucleotides were not utilized in protein synthesis.
XNA demonstrates a diversity of structural chemical changes compared to its natural analogs. Types of synthetic XNA that have already been created include 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), triose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA). Morpholino-peptide xenonucleic acid forms a triple helix structure.
https://futuristech.info/posts/dna-has-been-created-with-a-6-letter-code-x-and-y-base-pairs-have-been-added-to-the-genetic-alphabet
The genetic information is stored using the natural four bases; therefore, natural DNA polymerase cannot replicate information in XNA. In other words, the genetic information stored in XNA is 'invisible' and, therefore, useless for natural organisms whose life is based on DNA.
Xenonucleotides with different sugar-phosphate backbones:
More attention is given to the search for alternatives to the sugar-phosphate backbone of RNA, as ribose is the weak link in RNA. It is less stable than other sugars (glucose, erythrose, glyceraldehyde) and breaks down more quickly. The hydroxyl group on the second carbon atom in each ribose in the RNA chain facilitates the chain breakage.
Low-xeno nucleic acids were synthesized, where ribose is replaced by a four-carbon sugar (treose) or a three-carbon alcohol (glycerol) - treose nucleic acid (TNA) and glycol nucleic acid (GNA).
The molecules of threose nucleic acid form double helices with each other and with RNA through complementary pairs of nitrogenous bases. The strength of the bond between the two helices in such complexes is approximately similar to that in double-stranded DNA. Mutant forms of RNA polymerase have been obtained, capable of building TNA on a DNA template. Using these forms, the technology of systematic evolution of ligands by exponential enrichment (SELEX) was successfully adapted for TNA. This technology is used to obtain new ribozymes. With TNA, it was possible to obtain aptamers - molecules that selectively bind to a specific substance, in this case, the thrombin protein.
Glycol nucleic acid (GNA), like TNA, also forms a double helix with each other and with RNA. The structure of GNA helices makes the nucleotide pairs more stable. A high temperature is required to unwind this double helix for subsequent copying. TNA and GNA, due to their catalytic activity, are inferior to RNA. This is because they lack free -OH groups in the second position. These groups, as described above, make RNA more stable, but free -OH groups in the second position are necessary for catalytic activity.
Peptide nucleic acids (PNA) consist of a peptide chain to which nitrogenous bases are attached. The peptide chain can consist of various amino acids, not only the 20 amino acids found in proteins. This backbone differs from the sugar-phosphate backbone in greater stability. The most studied variant has a backbone composed of N-aminoethylglycine (aeg-PNA). This PNA forms a double helix with itself and stable heteroduplexes with DNA and RNA. Aeg-PNA does not contain chiral centers, unlike natural nucleic acids and xenonucleic acids, and cannot have left or right isomers.
The most promising approach to altering the genetic code is the reassignment of rarely used or non-essential codons. Ideally, the genetic code is expanded by one codon, thereby freeing itself from its previous function and switching to the encoding of a non-natural amino acid (nnAA) ('code expansion').
As these methods are complex to implement, there is the possibility of employing shorter paths ('code development'), such as in autotrophic bacteria, which in experiments receive isostuctural analogs instead of natural amino acids. In this scenario, residues of natural amino acids in native proteins are replaced with xenogenic amino acids. Moreover, it is possible to introduce several diverse xenogenic amino acids. In addition, the set of 20 natural amino acids can not only be expanded but also reduced to 19.
The codon specificity can be altered by reassigning nucleotide pairs of transfer RNA (tRNA) / aminoacyl-tRNA synthetase. Cells possessing such aminoacyl-tRNA synthetases are capable of reading mRNA sequences that are unreadable for the existing gene expression system. Changing the codon-tRNA synthetase pairs can facilitate the incorporation of unnatural amino acids into proteins in vivo.
In 2013, Farren Isaacs and George Church from Harvard University made synonymous changes to all 314 TAG stop codons in the E. coli genome, replacing them with TAA codons. This demonstrated that extensive substitutions can be made in strains while maintaining their viability. Subsequently, they further reprogrammed 13 codons across the entire genome, directly affecting 42 essential genes.
The replacement of DNA with XNA can also be achieved through another method, namely by altering the surrounding environment rather than genetic modules. Marlière and Muytjens created an E. coli strain whose DNA consists of standard nucleotides A, C, and G, with its synthetic analog of thymine, 5-chlorouracil, inserted instead of T. The growth of these cells depends on whether 5-chlorouracil is supplied in the culture medium, but otherwise, the cells behave like a typical E. coli strain.
Additionally, a strain of E. coli has been engineered to import unnatural triphosphates from Phaeodactylum tricornutum using nucleoside triphosphate. Subsequently, these imported phosphates are utilized for the replication of a plasmid into which unnatural complementary nucleotides dNaM-dTPT3 have been inserted.
To utilize this new information, in vivo transcription of this unnatural base pair into mRNA, tRNA, aminoacylated tRNA with unnatural amino acid, and unnatural nucleotide pairs in the decoding on the ribosome is required. Through these manipulations, it was possible to obtain a recombinant green fluorescent protein containing azidophenylalanine, which is encoded by an additional pair of synthetic nucleotides. The inclusion of the unnatural synthetic amino acid did not lead to a disruption of the protein's properties.
Ribozymes and Xenozymes
Ribozyme (shortened from 'ribonucleic acid' and 'enzyme') is also called catalytic RNA or enzymatic RNA. This RNA molecule possesses catalytic activity. Many naturally occurring ribozymes catalyze the cleavage of themselves or other RNA molecules.
In 1967, Carl Woese, Francis Crick, and Leslie Orgel first proposed the idea that RNA could be a catalyst. This hypothesis was based on the fact that RNA can form complex secondary structures. It is now known that ribozymes and many other RNA molecules have not only complex secondary structures but also intricate tertiary structures.
The catalytic activity of RNA was first discovered in the 1980s by Thomas Cech, who studied the splicing of a new mRNA in the ciliate Tetrahymena thermophila, and by Sidney Altman, who worked with the bacterial ribonuclease P. The ribozyme turned out to be a region of the pre-rRNA molecule in Tetrahymena thermophila, which is encoded by an intron outside the chromosomal rDNA gene. This activity could be provided by the ribozyme without the involvement of proteins. In 1982, the term ribozyme was introduced by Cech and Kruger. Despite the fact that most ribozymes are relatively rare in cells, sometimes they are crucial for the cell's existence. For example, the active part of the ribosome, the molecular machine that carries out the translation of proteins from mRNA, is a ribozyme.
The fact that RNA can contain genetic information led Walter Gilbert to propose the hypothesis that in ancient times, RNA served both as genetic material and as catalysts and structural components of the cell. Later, these roles were redistributed between DNA and proteins. This hypothesis is now known as the RNA world hypothesis.
Recent studies on prion folding have shown that RNA can catalyze protein folding, leading to its pathological behavior. Thus, ribozymes can perform functions similar to proteins (enzymes) acting as chaperones.
As mentioned earlier, there are not many ribozymes in cells. Here are some examples:
Group I and II introns (these are ribozymes capable of auto-splicing)
Leadzyme (a ribozyme that cleaves RNA in the presence of lead) - several natural examples have been found, though the first one was created in the laboratory. There is a hypothesis that this ribozyme is a key pathological factor in lead poisoning.
A ribozyme containing a hammerhead motif refers to RNA molecules that exhibit enzyme-like properties. They are found in plant viruses and participate in the self-cleavage reaction of the viral RNA replication products. These ribozymes are independent of metal ions. The hammerhead ribozyme, for example, is a small self-cleaving RNA with a conservative sequence found in certain viroids and associated with satellite RNAs of some plant viruses. They catalyze specific cleavage of phosphodiester bonds, and their secondary structure resembles a hammerhead.
Here are some examples of ribozymes in various contexts:
The ribozyme of the delta-hepatitis virus is involved in auto-splicing.
The Tetrahymena thermophila ribozyme is involved in mRNA splicing.
VS ribozymes are found in satellite RNAs of Neurospora.
Ribozymes of glucosamine-6-phosphate synthase are non-coding RNAs present in gram-positive bacteria. They interact with glucosamine-6-phosphate (GlcN6P), and upon binding to mRNAglmS, they cleave it.
These ribozymes play diverse roles in cellular processes, showcasing the versatility of RNA molecules in catalytic functions.
Secondary structure of ribozymes of different types: a - "hammerhead," b - hairpin, c - hepatitis virus ribozyme, d - Neurospora VS ribozyme.
Within the studies dedicated to the origins of life, it has been possible to create artificial ribozymes similar to RNA polymerases that, under certain conditions, can catalyze their own assembly. However, laboratory samples have shown a low catalytic efficiency: they are capable of assembling a chain of no more than 14 nucleotides within 24 hours, after which they break down through the hydrolysis of phosphodiester bonds.
Xenoenzymes are molecules of certain types of xenonucleic acids (with specific structures and nucleotide sequences) capable of specifically catalyzing certain biochemical reactions. In these xenoenzymes, deoxyribose/ribose has been replaced with arabinose, 2-fluoroarabinose, hexitol, and cyclohexene. Thus, nucleic acids with four different chemical structures were formed (arabinonucleic acid, 2-fluoroarabinonucleic acid, hexitol nucleic acid, cyclohexene nucleic acid). The artificial enzymes resembled ribozymes and exhibited various enzymatic activities, including nuclease and ligase activities.
xenoenzymes
The enzyme was obtained based on fluorinated arabinose which contained a metal ion (characteristic of natural enzymes). It stitched together two molecules of XeNA-XeNA, forming oligo-xeno-nucleotides. These artificial xenozymes obtained are highly resistant to degradation. This is due to the fact that natural RNases can cleave them.
Xenoamino acids
In addition to the synthesis of xeno-nucleotides, there are many non-natural analogs of amino acids that can be inserted into proteins under certain conditions. To achieve this, modifications need to be made from mRNA to protein using tRNA. Here are some non-natural analogs of amino acids: p-methoxyphenylalanine (pMpa), p-acetylphenylalanine (pAra), p-benzoylphenylalanine (pVra), p-iodophenylalanine (pIra), p-azidophenylalanine (pAzra), and p-propargyloxiphenylalanine (pPra); 2,2'-bipyridin-5-yl-alanine (Bpy-ala) and 2-amino-3-(8-hydroxyquinolin-3-yl)alanine (HQ-ala).
Xenoamino acids
Tryptophan (1) and its derivatives (4-amino tryptophan-1a, 4-fluorotryptophan-1b, 7-azatryptophan-1c); Proline (2) and its derivatives (cis-4-fluoroproline-2a, trans-4-fluoroproline-2b, cis-4-hydroxyproline-2c, trans-4-hydroxyproline-2d); Tyrosine (3) and its analogs (o-fluorotyrosine-3a, m-fluorotyrosine-3b); Methionine (4); Norleucine (4a); Azidohomoalanine (4b); Phenylalanine (5) and its analogs (m-fluorophenylalanine-5a, o-fluorophenylalanine-5b).
�The incorporation of these non-natural amino acids into the lipase enzyme from Thermoanaerobacter thermohydrosulfuricus enhances its activity in processing organic solvents and surfactants. Additionally, the enzyme's resistance to denaturation significantly increases. Introducing these non-natural analogs of amino acids into proteins can alter the properties of proteins, enzyme proteins, and contribute to the development of advanced biotechnologies.
The most important obstacles that need to be overcome before a safe XNA organism is created include:
Environmental Safety: It is crucial to ensure that the introduction of synthetic XNA does not cause negative ecological consequences or harm to natural biodiversity.
Biocompatibility: XNA organisms should interact with other living organisms and natural processes without adverse effects.
Ethical Considerations: The use of XNA raises ethical questions, and defining ethical standards for genome editing and the creation of modified organisms is essential.
Health and Safety: Achieving a high level of safety for consumers and other organisms interacting with XNA organisms.
Public Opinion: Considering and engaging with public opinion to ensure acceptance and support for synthetic XNA within society.
Control and Regulation: Developing effective methods for controlling and regulating the introduction of XNA organisms into natural environments to avoid unforeseen consequences.
Education and Research: Conducting additional research and education to understand the potential risks and benefits of using XNA organisms.
End of part 2