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Oligonucleotides generated by using Trimer Phosphoramidites

Metkinen Chemistry offers randomized oligonucleotide libraries synthesized by using synthetic Trinucleotide (codon and anticodon) phosphoramidites.

1. Synthetic Trinucleotide (codon & anticodon) phosphoramidites for Protein mutagenesis.

Oligonucleotide-directed mutagenesis is probably the most widely used approach for the production of proteins with variations at specific sites. 1-6

Randomization of oligonucleotide sequence for generation of protein libraries is best accomplished by using trimer phosphoramidites. Out of the 64 possible triplets, just 20 are sufficient to code for the 20 amino acids commonly used in protein sequences. Trimer phosphoramidites offer an elegant solution that circumvents problems of codon bias, frameshift mutations and stop-codon introduction, when compared to usage of monomers to generate codon mixtures.

Several reports describing 7-9 the synthesis of trimer phosphoramidites have been published. General structure of commercially available set of Trinucleotide phosphoramidites is shown in Figure 1. Once a mutagenic oligonucleotide library has been synthesized using Trimer Phosphoramidites, it can be introduced into a plasmid for construction of a gene library. 10,11

2. "Codon" and "Anticodon" Trimers for protein mutagenesis. Why do we need "codon" and "anticodon" Trimers for randomization of Genes?

Nowadays full chemical synthesis of genes has become very common and widely commercially available. It is usually done through PCR extension of overlapping oligonucleotides, partially representing coding and noncoding strands of the required sequence.



This is also a convenient way of assembling gene libraries where variable part (blue bar) of the oligo library is positioned in the gap of the opposite strand to be filled with polymerase.



With earlier commercially available set of trimers (Table 1, Column 2) it was only possible to introduce variations through the coding strand. As a result adjacent stretches of the sequences were sometimes difficult to randomize.

Now, when the “anticodon” trimers, which can be used as part of complementary (noncoding) strand are available, the following scheme could be implemented:



Similar scheme could be implemented in template-dependent overlapping PCR that will also allow an easy placement of variable regions in close proximity.



In order to develop an approach for Protein Mutagenesis, where oligonucleotides with randomizations could be designed as forward or reverse primers, the set of Trimer phosphoramidites had to be expanded to 25 (Table 1).

The set of 20 trimers contains optimal E.coli codons for all 20 amino acids, that can be placed in the sense strand (Table 1, column 2). Out of these 20 trimers, 15 can be employed for antisense strand synthesis (Table 1, column 3, Trimers given in black), that would give rise to optimal E.coli codons upon replication (Table 1, column 3, Trimers given in brackets) and subsequently translated into 15 different amino acids (Table 1, column 1). For the remaining 5 amino acids, there are five trimers (Table 1, column 4, Trimers given in red) that can be used specifically for antisense strand sequence. These "anticodons", in turn will give rise to optimal "codons" upon replication Table 1, column 4, Trimers given in brackets). Thus, the available set of 25 unique trimers presented in Tables 1 is sufficient to produce oligonucleotides with randomizations in forward and/or reverse orientation.

Table 1. "Codon" Trimers and "Antcodon" Trimers of Metkinen Chemistry

1 2 3 4
Amino acid encoded Trimers for placing into
coding (sense) strand
Same Trimers placed
in the antisense strand
Additional trimers for
introducing into
antisense strand
Tyr TAC - GTA (TAC)
Ser TCT - AGA (TCT)
Gly GGT ACC (GGT)  
Arg CGC GCG (CGC)  
Ala GCA TGC (GCA)  
Asp GAT ATC (GAT)  
Asn AAC GTT (AAC)  
Gln CAG CTG (CAG)  
Glu GAA TTC (GAA)  
His CAT ATG (CAT)  
Ile ATC GAT (ATC)  
Leu CTG CAG (CTG)  
Lys AAA - TTT (AAA)
Met ATG CAT (ATG)  
Phe TTC GAA (TTC)  
Pro CCA TGG (CCA)  
Thr ACC GGT (ACC)  
Trp TGG - CCA (TGG)
Val GTT AAC (GTT)  
Cys TGC - GCA (TGC)


3. 25 standard trimeric building blocks of Metkinen Chemistry

25 standard trimeric building blocks are normally used by our company for the synthesis of randomized oligonucleotide libraries: TAC, TCT, GGT, CGT, GCG, GAT, AAC, CAG, GAA , CAT, ATC, CTG, AAA, ATG, TTC, CCA, ACC, TGG, GTT, TGC, GTA, AGA, TTT, CCA, GCA.

4. References

1. J. Sondek and D. Shortle, Proc Natl Acad Sci U S A, 1992, 89, 3581-3585.
2. P. Gaytan, J. Yanez, F. Sanchez, H. Mackie, and X. Soberon, Chem Biol, 1998, 5, 519-527.
3. P. Gaytan, J. Yanez, F. Sanchez, and X. Soberon, Nucleic Acids Res, 2001, 29, E9.
4. F.A. Fellouse, et al., J Mol Biol, 2007, 373, 924-40.
5. E-C. Brockmann, S. Akter, T. Savukoski, A. Lehmusvuori, J. Leivo, O. Saavalainen, A. Azhayev, T. Lövgren, J. Hellman, U. Lamminmäki, Protein Eng. Des.Sel. 2011, 1-10.
6. T. Huovinen, M. Syrjänpää, H. Sanmark, E-C. Brockmann, A. Azhayev, Q. Wang, M. Vehniäinen, U. Lamminmäki "Two ScFv antibody libraries derived from identical VL-VH framework with different bindingsite designs display distinct binding profiles" Protein Eng. Des.Sel. 2013, 683-693
7. A.L. Kayushin, M.D. Korosteleva, A.I. Miroshnikov, W. Kosch, D. Zubov, and N. Piel, Nucleic Acids Research, 1996, 24, 3748-3755
8. T. Mauriala, S. Auriola, A. Azhayev, A. Kayushin, M. Korosteleva, and A. Miroshnikov, J.Pharmaceutical and Biomedical Analysis, J Pharm Biomed Anal, 2004, 34, 199-206.
9. A. Yagodkin, A. Azhayev, J. Roivainen, M. Antopolsky, A. Kayushin, A., M. Korosteleva, A. Miroshnikov, A., J. Randolph, H. Mackie, Nucleosides, Nucleotides and Nucleic Acids 2007, 473-497 and references cited therein.
10. J.Randolph, A.Yagodkin, M.Lamaitre, A.Azhayev, H.Mackie, Nucleic Acids Symposium Series 02/2008; DOI:10.1093/nass/nrn243
11. S.S. Sidhu, H.B. Lowman, B.C. Cunningham, and J.A. Wells, Methods Enzymol, 2000, 328, 333-63


5. Advantages of using Trinucleotide phosphoramidites for generation of randomized oligonucleotide libraries:
  • Absence of synonymous, rare and stop codons
  • Absence of frameshift mutations
  • Library variability can be more that an order higher than the ones obtained by common methods
  • Occurrence of each amino acid at a certain position can be easily controlled.
6. Additional information:
  • Purification of randomized oligo libraries: preparative PAGE
  • QC: PAGE
  • Length and amount: up to 80-mer; 20 - 30 nmol (10-20 OD)
  • Time of order execution: up to 1 month
  • Storage: 1 year at -20 ˚C
7. Price for oligonucleotide synthesis:
  • The price is €195 per randomization with standard MC Trimer phosphoramidites and their mixes
  • Price will depend on amount of randomizations (trimer couplings; 10 couplings max within each randomized oligonucleotide) and will not depend on amount of trimer mixes used for randomization
  • No charge for regular monomer insertion (dA, dG, dC or T); the charge for 5'-phosphorylation of any oligo is €39
  • Maximum length of each oligo should be 80 bases
8. Contacts. How to order: E-mail: info@metkinenchemistry.com