Synthetic trinucleotides

Another important technique was based on the observation that synthetic trinucleotides induced the binding to ribosomes of tRNA molecules that were "charged" with their specific amino acids 38/39 For example, the trinucleotides UpUpU and ApApA stimulated the binding to ribosomes of 14C-labeled phenylalanyl-tRNA and lysyl-tRNA, respectively. The corresponding dinucleotides had no effect, an observation that not only verified the two codons but also provided direct evidence for the triplet nature of the genetic code. Another powerful approach was the use of artificial RNA polymers, synthesized by combined chemical and enzymatic approaches.40 For example, the polynucleotide CUCUCUCUCU led to the synthesis by ribosomes of a regular alternating polypeptide of leucine and serine. 

The use of synthetic trinucleotides as building blocks would obviate the problems of stop codons or other unwanted amino acids turning up randomly in hypervariable sequences, and would indeed allow the predetermined definition of amino acid codon frequency bias. This has been demonstrated by Glaser et al. [97] for phage-display library construction see also Ref. 98 for a review of current DNA synthesis technology. 

In the binding assay technique, various tRNA molecules, one of which is radioactively labeled with are mixed with ribosomes and synthetic trinucleotides bound to a filter. If the radioactive label is detected on the filter, then it is known that the particular tRNA bound to that triplet. The binding experiments can be repeated until all the triplets are assigned. 

Di- and trinucleotides may be used as units instead of the monomers. This convergent synthetic strategy simplifies the purification of products, since they are differentiated by a much higher jump in molecular mass and functionality from the educls than in monomer additions, and it raises the yield. We can illustrate the latter effect with an imaginary sequence of seven synthetic steps, c.g. nucleotide condensations, where the yield is 80% in each step. In a converging seven-step synthesis an octanucleotide would be obtained in 0.8 x 100 = 51% yield, compared with a 0.8 x 100 = 21% yield in a linear synthesis. 

Figure 4.3 The synthesis of an oligonucleotide from an activated mononucleotide, (a) Adenonine triphosphate (ATP), the substrate of enzymatic nucleic-acid synthesis. (b) An imidazolide of a nucleotide of the kind used in many non-enzymatic template-directed reactions, (c) The synthetic reaction leading to the formation of a trinucleotide. (Modified from Orgel, 2002.)...

Work is well under way on the high resolution structure of the uninhibited nuclease as are attempts to prepare crystals with other mono-, di-, and trinucleotides of various types so that more insight may be gained into the actual workings of the nuclease. As described elsewhere in this volume (25), some of the most exciting prospects for the future are the correlations to he made between the crystallographic studies of the three-dimensional structure and the properties of the fragmented and synthetic versions of this nuclease. 

Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., et al. (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus framework and CDRs randomized with trinucleotides. J. Mol. Biol. 296,57-86. 

In an effort to uncover the structures of DNA adducts of cisplatin, many reactions with the drug and synthetic oligonucleotides have also been carried out. Recently it has been shown that (1) and (2) can react in a cross-linking manner with the guanine residues in the trinucleotide d(GpTpG) [26]. In both cases, NMR measurements showed that the donor atoms bound to the platinum ions are two ammonia ligands and N-7 of guanine in positions 1 and 3 of the trinucleotide. 

As the structural features of V regions become further elucidated [28], it is possible that rules describing the amino acids, or amino acid combinations, permitted or frequently present at different CDR positions may be derived. For such rules to be useful, they are likely to be more complicated than a statistical analysis of amino acid frequency at different CDR positions may provide, since it is possible that the presence of particular amino acids at particular positions may preclude the presence of other amino acids at other sites. Neural networks are capable of understanding relationships of this kind. Such rules, in association with trinucleotide chemistry and the use of well-expressed V genes, may lead to the creation of synthetic libraries which are better than those presently derived from natural sources. 

A variety of techniques were used to determine the genetic code. The initial experiments used synthetic mRNA with only a single nucleotide, such as AAAAAAAA. These were then translated to see what protein homopolymer was produced. In this way the translation for AAA, UUU, GGG, and GCG, were determined. The most comprehensive experiment was the filter-binding experiment of Nirenberg, which used specific trinucleotides bound to a filter to see which tRNA molecules would bind. 

In the initiation complex, location of the charged initiator tRNA in the P site of the ribosome allows transfer of the methionine residue to the amino group of another aminoacyl-tRNA in the A site (Fig. 7b) by pep-tidyl transferase to form dipeptidyl-tRNA (see Fig. 7c). Functional insertion of Met-tRNAf directly into the P site can be demonstrated using the trinucleotide AUG as a synthetic mRNA and another trinucleotide, for example UUU, to bind an acceptor aminoacyl-tRNA (in this case Phe-tRNA). 

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