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. 1998 Feb 1;26(3):697–702. doi: 10.1093/nar/26.3.697

Scoring functions for computational algorithms applicable to the design of spiked oligonucleotides.

L J Jensen 1, K V Andersen 1, A Svendsen 1, T Kretzschmar 1
PMCID: PMC147326  PMID: 9443959

Abstract

Protein engineering by inserting stretches of random DNA sequences into target genes in combination with adequate screening or selection methods is a versatile technique to elucidate and improve protein functions. Established compounds for generating semi-random DNA sequences are spiked oligonucleotides which are synthesised by interspersing wild type (wt) nucleotides of the target sequence with certain amounts of other nucleotides. Directed spiking strategies reduce the complexity of a library to a manageable format compared with completely random libraries. Computational algorithms render feasible the calculation of appropriate nucleotide mixtures to encode specified amino acid subpopulations. The crucial element in the ranking of spiked codons generated during an iterative algorithm is the scoring function. In this report three scoring functions are analysed: the sum-of-square-differences function s, a modified cubic function c, and a scoring function m derived from maximum likelihood considerations. The impact of these scoring functions on calculated amino acid distributions is demonstrated by an example of mutagenising a domain surrounding the active site serine of subtilisin-like proteases. At default weight settings of one for each amino acid, the new scoring function m is superior to functions s and c in finding matches to a given amino acid population.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arkin A. P., Youvan D. C. Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis. Biotechnology (N Y) 1992 Mar;10(3):297–300. doi: 10.1038/nbt0392-297. [DOI] [PubMed] [Google Scholar]
  2. Balint R. F., Larrick J. W. Antibody engineering by parsimonious mutagenesis. Gene. 1993 Dec 27;137(1):109–118. doi: 10.1016/0378-1119(93)90258-5. [DOI] [PubMed] [Google Scholar]
  3. Botstein D., Shortle D. Strategies and applications of in vitro mutagenesis. Science. 1985 Sep 20;229(4719):1193–1201. doi: 10.1126/science.2994214. [DOI] [PubMed] [Google Scholar]
  4. Bryan P. N. Site-directed mutagenesis to study protein folding and stability. Methods Mol Biol. 1995;40:271–289. doi: 10.1385/0-89603-301-5:271. [DOI] [PubMed] [Google Scholar]
  5. Chattopadhyaya J. B., Reese C. B. Chemical synthesis of tridecanucleoside dodecaphosphate sequence of SV40 DNA. Nucleic Acids Res. 1980 May 10;8(9):2039–2053. doi: 10.1093/nar/8.9.2039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Delagrave S., Youvan D. C. Searching sequence space to engineer proteins: exponential ensemble mutagenesis. Biotechnology (N Y) 1993 Dec;11(13):1548–1552. doi: 10.1038/nbt1293-1548. [DOI] [PubMed] [Google Scholar]
  7. Derbyshire K. M., Salvo J. J., Grindley N. D. A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. Gene. 1986;46(2-3):145–152. doi: 10.1016/0378-1119(86)90398-7. [DOI] [PubMed] [Google Scholar]
  8. Dreher T. W., Bujarski J. J., Hall T. C. Mutant viral RNAs synthesized in vitro show altered aminoacylation and replicase template activities. Nature. 1984 Sep 13;311(5982):171–175. doi: 10.1038/311171a0. [DOI] [PubMed] [Google Scholar]
  9. Glaser S. M., Yelton D. E., Huse W. D. Antibody engineering by codon-based mutagenesis in a filamentous phage vector system. J Immunol. 1992 Dec 15;149(12):3903–3913. [PubMed] [Google Scholar]
  10. Hermes J. D., Parekh S. M., Blacklow S. C., Köster H., Knowles J. R. A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers. Gene. 1989 Dec 7;84(1):143–151. doi: 10.1016/0378-1119(89)90148-0. [DOI] [PubMed] [Google Scholar]
  11. Hui A., Hayflick J., Dinkelspiel K., de Boer H. A. Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli. EMBO J. 1984 Mar;3(3):623–629. doi: 10.1002/j.1460-2075.1984.tb01858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hutchison C. A., 3rd, Nordeen S. K., Vogt K., Edgell M. H. A complete library of point substitution mutations in the glucocorticoid response element of mouse mammary tumor virus. Proc Natl Acad Sci U S A. 1986 Feb;83(3):710–714. doi: 10.1073/pnas.83.3.710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jarnagin A. S., Ferrari E. Extracellular enzymes: gene regulation and structure function relationship studies. Biotechnology. 1992;22:189–217. [PubMed] [Google Scholar]
  14. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  15. Little J. W. Saturation mutagenesis of specific codons: elimination of molecules with stop codons from mixed pools of DNA. Gene. 1990 Mar 30;88(1):113–115. doi: 10.1016/0378-1119(90)90067-2. [DOI] [PubMed] [Google Scholar]
  16. Lyttle M. H., Napolitano E. W., Calio B. L., Kauvar L. M. Mutagenesis using trinucleotide beta-cyanoethyl phosphoramidites. Biotechniques. 1995 Aug;19(2):274–281. [PubMed] [Google Scholar]
  17. Matteucci M. D., Heyneker H. L. Targeted random mutagenesis: the use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5' of an ATG initiation codon. Nucleic Acids Res. 1983 May 25;11(10):3113–3121. doi: 10.1093/nar/11.10.3113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McNeil J. B., Smith M. Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs. Mol Cell Biol. 1985 Dec;5(12):3545–3551. doi: 10.1128/mcb.5.12.3545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Oliphant A. R., Nussbaum A. L., Struhl K. Cloning of random-sequence oligodeoxynucleotides. Gene. 1986;44(2-3):177–183. doi: 10.1016/0378-1119(86)90180-0. [DOI] [PubMed] [Google Scholar]
  20. Ono A., Matsuda A., Zhao J., Santi D. V. The synthesis of blocked triplet-phosphoramidites and their use in mutagenesis. Nucleic Acids Res. 1995 Nov 25;23(22):4677–4682. doi: 10.1093/nar/23.22.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ophir R., Gershoni J. M. Biased random mutagenesis of peptides: determination of mutation frequency by computer simulation. Protein Eng. 1995 Feb;8(2):143–146. doi: 10.1093/protein/8.2.143. [DOI] [PubMed] [Google Scholar]
  22. Reidhaar-Olson J. F., Sauer R. T. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science. 1988 Jul 1;241(4861):53–57. doi: 10.1126/science.3388019. [DOI] [PubMed] [Google Scholar]
  23. Schier R., Balint R. F., McCall A., Apell G., Larrick J. W., Marks J. D. Identification of functional and structural amino-acid residues by parsimonious mutagenesis. Gene. 1996 Mar 9;169(2):147–155. doi: 10.1016/0378-1119(95)00821-7. [DOI] [PubMed] [Google Scholar]
  24. Siderovski D. P., Mak T. W. RAMHA: a PC-based Monte-Carlo simulation of random saturation mutagenesis. Comput Biol Med. 1993 Nov;23(6):463–474. doi: 10.1016/0010-4825(93)90094-h. [DOI] [PubMed] [Google Scholar]
  25. Siezen R. J., Leunissen J. A. Subtilases: the superfamily of subtilisin-like serine proteases. Protein Sci. 1997 Mar;6(3):501–523. doi: 10.1002/pro.5560060301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Siezen R. J., de Vos W. M., Leunissen J. A., Dijkstra B. W. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. Protein Eng. 1991 Oct;4(7):719–737. doi: 10.1093/protein/4.7.719. [DOI] [PubMed] [Google Scholar]
  27. Smith M. In vitro mutagenesis. Annu Rev Genet. 1985;19:423–462. doi: 10.1146/annurev.ge.19.120185.002231. [DOI] [PubMed] [Google Scholar]
  28. Sondek J., Shortle D. A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3581–3585. doi: 10.1073/pnas.89.8.3581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Suzuki M., Christians F. C., Kim B., Skandalis A., Black M. E., Loeb L. A. Tolerance of different proteins for amino acid diversity. Mol Divers. 1996 Oct;2(1-2):111–118. doi: 10.1007/BF01718708. [DOI] [PubMed] [Google Scholar]
  30. Tomandl D., Schober A., Schwienhorst A. Optimizing doped libraries by using genetic algorithms. J Comput Aided Mol Des. 1997 Jan;11(1):29–38. doi: 10.1023/a:1008071310472. [DOI] [PubMed] [Google Scholar]
  31. Virnekäs B., Ge L., Plückthun A., Schneider K. C., Wellnhofer G., Moroney S. E. Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 1994 Dec 25;22(25):5600–5607. doi: 10.1093/nar/22.25.5600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wells J. A., Estell D. A. Subtilisin--an enzyme designed to be engineered. Trends Biochem Sci. 1988 Aug;13(8):291–297. doi: 10.1016/0968-0004(88)90121-1. [DOI] [PubMed] [Google Scholar]
  33. Wells J. A., Vasser M., Powers D. B. Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. Gene. 1985;34(2-3):315–323. doi: 10.1016/0378-1119(85)90140-4. [DOI] [PubMed] [Google Scholar]

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