Chemical Synthesis of a Gene: Phosphodiester Approach

Chemical Synthesis of a Gene: Phosphodiester Approach Chemical synthesis of a gene is the process of synthesizing an artificially designed gene into a physical DNA sequence by chemical methods. The amino acid sequence of the protein encoded by a gene enables the deduction of base sequence of the concerned gene. From the amino acid sequence of the protein and using a set of optimal codons, the nucleotide sequence of the gene can be back translated. However, the degeneracy of genetic code may present some problems, but a functional sequence of the gene can nonetheless be worked out and can be optimized for codon usage as well as for base composition. In principle, a DNA synthesizer can be used to synthesize the DNA sequence chemically and this can be cloned in the usual manner. But this is not so simple. A synthesizer will add bases sequentially one at a time to the growing oligonucleotide chain through a series of chemical reactions and washing steps. Synthesis of oligonucleotides 30-50 bases long is very reliable, longer sequences can be synthesized but the practical limit is not more than 100 bases. One way to solve this is to synthesize short fragments and join them chemically or enzymatically to create the longer fragment. However, the synthesizer makes single-stranded DNA, so the complementary strand has to be synthesized again to create a double-stranded DNA. It involves a lot of work but is achievable. Early studies. The synthesis of nucleic acids in the laboratory started about thirty years ago. Early synthetic efforts used phosphodiester approach which enabled the synthesis of short oligonucleotides of 10-20 nucleotides. This approach was based on the selection of the proper condensing agents for phosphodiester bond formation and at the same time suitable protective groups were employed for the bases and the ribose moiety. These oligonucleotides were then assembled into longer DNA fragments with the help of kinase and DNA ligase. From the known primary structure of a ribonucleic acid, tyrosine tRNA, Dr H Khorana and his colleagues deduced the DNA sequence and synthesized successfully a DNA segment containing 200 bp coding for the structural gene for tyrosine tRNA. However, the low yields in the condensation step, the long reaction times, and especially the time-consuming purification of intermediates led to believe that chemical gene synthesis is unlikely to become a standard lab oratory method. Since then, the procedure for oligonucleotide synthesis has been improved by several workers and they provide different approaches for synthesis as well as protection of bases and sugar moieties. There are three distinct methods: (1) phosphodiester approach, (2) phosphotriester or phosphate triester approach and (3) phosphite triester or phosphoramidite approach. Phosphodiester approach This method involves the formation of an ester linkage between an activated phosphate group of one nucleotide with the hydroxyl group of another nucleoside, thus forming the natural phosphodiester bridge between the 5-OH of one nucleoside unit and the 3-OH of the next. Here, 3-O-acetylnucleoside-5-O-phosphate (a) is activated by N,N-dicyclo- hexylcarbodiimide (DCC) or p-toluenesulphonylchloride(PTS/PTsCl) and subjected to react with a 5-O-protected nucleoside (b) to give a protected dinucleoside monophosphate or phosphodiester (c). Activation of phosphate moiety is essential for easier formation of the phosphodiester linkage and this is mediated by DCC or PTsCl. Now, to increase the chain length, one has to remove the 3-O-acetyl group by base catalysed hydrolysis. Further chain elongation is carried out by repeating the process. The major drawback of the phosphodiester method is the formation of pyrophosphate oligomers and oligonucleotides branched at the internucleosidic phosphate. Phosphotriester approach In this method, oligonucleotide branch formation is avoided by protecting the phosphate group with an ethylcyano group. A nucleotide containing 5-OH protected and phosphate protected by MMT and 2-cyanoethyl group respectively (compound a) is activated with 2,4,6-Triisopropylbenzenesulfonyl chloride (TPSCl) and subjected to reaction with a 3-O-protected nucleoside (b). This generates a dinucleoside monophosphate or phosphotriester (c) in which phosphate group is protected by 2-cyanoethyl group. The basic difference between phosphodiester and phosphotriester method is that, in phosphodiester method, the phosphate group is protected by two phosphoester linkage but in phosphotriester method the phosphate group is protected by one extra phosphoester linkage with 2-cyanoethyl group. In phosphotriester method, the formation of oligonucleotide branch at the internucleosidic phosphate is avoided. Phosphite triester or phosphoramidite approach The phosphite triester or phosphoramidite approach for oligonucleotide synthesis was based upon the use of phosphoramidite monomers and the use of tetrazole catalysis. In phosphite triester method, the starting compound is N-6-benzoyldeoxyadenosinephosphoramidite (if adenine is the first base) where the phosphorous atom is in the +3 oxidation state. So unlike the other methods, the formation of oligonucleotides branch is not possible in this process. In this approach, the oligonucleotide is synthesized by a series of reactions described below. Protection of base and sugar In this step, the free -NH2 group of the bases are protected by benzoylation or acylation depending upon the nature of bases. The 5-hydroxyl group is also protected by dimethoxytrityl group (DMT), which protects only primary hydroxyl group but not secondary. The reactions are illustrated in CSG_Fig 3., the blocked bases are shown in the inset. Formation of phosphite triester or phosphoramidite In this step phosphite triester is synthesized by a series of reactions. First, 2-cyanoethanol on reaction with phosphorus trichloride produces an intermediate compound which on further reaction with di-isopropylamine (two-equivalent) and 5-OH protected nucleoside (one-equivalent) produces phosphite triester (CSG_Fig 4). This phosphoramidite will be repeatedly used during the oligonucleotide synthesis process described below. The synthesis procedure The synthesis is carried out in several steps described below: Step 1: The deblocking step The first base, which is attached to the solid support, is at first inactive because all the active sites have been blocked or protected. The free -NH2 groups in the bases remains protected by benzoylation or acylation depending upon the bases and the -OH group is protected by dimethoxytrityl group (DMT). To add the next base, the DMT group protecting the 5-hydroxyl group must be removed (deblocking). This step is also called detritylation. This is done by adding either dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM), to the reaction column. The 5-hydroxyl group is now the only reactive group on the base monomer. This ensures that the addition of the next base will only bind to that site. The reaction column is then washed to remove any extra acid and by-products. Step 2: Base condensation The step2 is basically a condensation step. Now prior to addition of the well protected nucleotide to the column, it is essential to activate the phosphate group, so that the nucleophilic attack on phosphorous atom takes place easily. This is best done by adding tetrazole to the nucleotide in dichloromethane medium. In presence of tetrazole, diisopropylamine group of the nucleotide becomes positively charged and hence its departure would be easier after nucleophilic attack of 5-hydroxyl group of the previous nucleotide which is attached with resin column. After the reaction, the column was washed to remove extra tetrazole, unbound nucleotide and byproduct (diisopropylamine). Step 3: Capping In case of unreacted nucleoside attached with resin, the 5-hydroxyl group is unprotected this may react later with the addition of different nucleotides. If left unprotected, it will lead to the formation of a mixture of oligonucleotides. The 5-hydroxyl group is therefore blocked by adding acetic anhydride and N-methylimidazole (capping). After capping, the reaction column is thoroughly washed to remove extra acetic anhydride and N-methylimidazole. Step 4: Oxidation This step is basically an oxidation step. In this step, the phosphite linkage is oxidized to give more stable phosphate linkage. The oxidation is best done by adding a mixture of dilute aqueous iodine solution, pyridine (Py) and tetrahydorfuran (THF) to the reaction column. The steps one through four, i.e., deblocking, base condensation, capping and oxidation, are repeated until all desired bases have been added to the column. This cycle is completed once for each additional base. Step 5 Detachment of oligonucleotide from solid support After all bases have been added the oligonucletide must be cleaved from the solid support and deprotected before it can be effectively used. For detachment of oligonucleotides form resin, the column is treated with 28% ammonium hydroxide solution (NH4OH), and at the same time the ethylcyano group on the phosphate group is removed. Step 6: Purification and isolation of oligonucleotide In this step, NH4OH is evaporated from the ammonium hydroxide solution of oligonucleotides to get crude product. The crude product is a mixture of oligonucleotide, cleaved protective groups and oligonucleotides with internal deletions. Now this crude product is subjected to boiling in a sealed tube with NH4OH at 55 °C. The main purpose of this reaction is to remove the base protecting group. After evaporation of NH4OH, the crude product is subjected to desalting followed by Polyacrylamide Gel Electrophoresis, to purify the oligonucleotides. Desalting is used mainly to remove the ammonium ion. This is done by ethanol precipitation, size-exclusion chromatography, or reverse-phase chromatography. Oligonucleotides are synthesized by the stepwise addition of nucleoside-3à ¢Ã¢â€š ¬Ã‚ ²-phosphoramidite monomers to solid-phase supports in an automated DNA synthesizer. In solid-phase synthesis, 3-terminal hydroxy group of the first added nucleoside is attached to the solid surface by covalent interaction. The solid support is contained in columns whose dimensions depend on the scale of synthesis. The two most frequently used solid phase materials are Control Pore Glass (CPG) and macroporous polystyrene (MPPS). CPG is commonly defined by its pore size, for example pore sizes of 500Ã… are used to allow the oligonucleotides preparation of about 50 -mer. To improve the performance of native CPG some modification is required. This is done by treating the material with (3-aminopropyl)triethoxysilane) to give Aminopropyl CPG. The amino group then serves as the anchoring point for the first added oligonucleoside. MPPS is synthesized by polymerization of divinylbenzene, styrene, and 4-chloromethylstyrene in the presence of a porogeneous agent. It is a low-swellable, highly cross-linked polystyrene and suitable for oligonucleotide synthesis. The macroporous chloromethyl MPPS obtained is often converted to aminomethyl MPPS to improve the efficiency of the support. Annealing of oligonucleotides For chemically synthesize a gene, the next step will be to assemble the oligonucleotides to form a complete gene. This is achieved by enzymatic methods which include polymerase cycling and ligase reactions. Some of the strategies are discussed below. Assembling oligonucleotides by single-step PCR. For synthesis of a gene, the oligonucleotides (about 30-60 nt long) are synthesized chemically so that each oligonucleotide has a 6-9 nt overlap with its neighboring oligonucleotide. These are then assembled in a single-step PCR. In this method, oligonucleotides are first ligated and then the product, the entire gene, is PCR amplified using the outmost oligonucleotides as primers. This method was first used to synthesize a 924-bp gene coding for an isozyme of horseradish peroxidase. Another method was developed by WPC Stemmer which did not use any ligase for joining the oligonucleotide products. It however, relied on Taq DNA polymerase (PCR cycling) for joining the individual oligonucleotides. Assembling oligonucleotides by two-step PCR. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Several modifications of the above procedure have been presented. One such method called PAS (PCR-based accurate synthesis) involves (i) synthesis of oligonucleotides to cover the entire DNA sequence (ii) PCR to synthesize DNA fragments (iii) second PCR for assembly of the products of the first PCR and (iv) cloning of the synthetic DNA and then verification by DNA sequencing. Besides, other methods in use for gene synthesis are successive extension PCR, simplified gene synthesis (PCR based), synthons and ligation by selection, to name a few. Review questions and problems What is the advantage of phosphatetriester method over phosphatediester method? What is the advantage of phosphitetriester method over phosphatetriester and  phosphatediester method? What is the main advantage to use DMTCl for protecting the 5-hydroxyl group? How could you attach the first nucleoside to the solid support? What is the utility of capping step in the oligonucleotides synthesis? Why capping is done by aceticanhydride? What is the function of iodine in the oxidation step of oligonucleotides synthesis? How could you protect only the free -NH2 group of the bases of a nucleoside? What is the reagent used for the removal of 2-cyanoethyl group from the  synthesized oligonucleotides? What is the byproduct produced from the base-condensation step of oligonucleotides  synthesis in phosphite triester method? How could you deprotect the bases of oligonucleotides? What is the function of tetrazole in the base condensation step of oligonucleotide synthesis? What is the basic principle for synthesizing a gene from the corresponding oligonucleotides by (a) PCR-based one-step DNA synthesis, (b) PCR-based two-step DNA synthesis?