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Analysis of In Vitro Aptamer Selection Parameters

Abstract

Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success.

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References

  1. Alam KK, Chang JL, Burke DH (2015) FASTAptamer: a bioinformatic toolkit for high-throughput sequence analysis of combinatorial selections. Mol Ther Nucleic acids 4:e230. doi:10.1038/mtna.2015.4

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  2. Apweiler R, Bairoch A, Wu CH (2004) Protein sequence databases. Curr Opin Chem Biol 8:76–80. doi:10.1016/j.cbpa.2003.12.004

    CAS  Article  PubMed  Google Scholar 

  3. Baird GS (2010) Where are all the aptamers? Am J Clin Pathol 134:529–531. doi:10.1309/AJCPFU4CG2WGJJKS

    Article  PubMed  Google Scholar 

  4. Barrick JE, Breaker RR (2007) The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol 8:R239. doi:10.1186/gb-2007-8-11-r239

    PubMed Central  Article  PubMed  Google Scholar 

  5. Berezovski M, Musheev M, Drabovich A, Krylov SN (2006) Non-SELEX selection of aptamers. J Am Chem Soc 128:1410–1411. doi:10.1021/ja056943j

    CAS  Article  PubMed  Google Scholar 

  6. Bordeaux J et al (2010) Antibody validation. Biotechniques 48:197–209. doi:10.2144/000113382

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  7. Carothers JM, Oestreich SC, Szostak JW (2006) Aptamers selected for higher-affinity binding are not more specific for the target ligand. J Am Chem Soc 128:7929–7937. doi:10.1021/ja060952q

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  8. Carothers JM, Goler JA, Kapoor Y, Lara L, Keasling JD (2010) Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity. Nucleic Acids Res 38:2736–2747. doi:10.1093/nar/gkq082

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  9. Carter CW Jr, Yin Y (1994) Quantitative analysis in the characterization and optimization of protein crystal growth. Acta Crystallogr Sect D 50:572–590. doi:10.1107/S0907444994001228

    Article  Google Scholar 

  10. Catherine AT, Shishido SN, Robbins-Welty GA, Diegelman-Parente A (2014) Rational design of a structure-switching DNA aptamer for potassium ions. FEBS Open Bio 4:788–795. doi:10.1016/j.fob.2014.08.008

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  11. Cho EJ, Lee JW, Ellington AD (2009) Applications of aptamers as sensors. Annu Rev Anal Chem (Palo Alto, Calif) 2:241–264. doi:10.1146/annurev.anchem.1.031207.112851

    CAS  Article  Google Scholar 

  12. Ciesiolka J, Illangasekare M, Majerfeld I, Nickles T, Welch M, Yarus M, Zinnen S (1996) Affinity selection-amplification from randomized ribooligonucleotide pools. Methods Enzymol 267:315–335

    CAS  Article  PubMed  Google Scholar 

  13. Cruz-Toledo J et al (2012) Aptamer base: a collaborative knowledge base to describe aptamers and SELEX experiments. Database 2012:bas006. doi:10.1093/database/bas006

    PubMed Central  Article  PubMed  Google Scholar 

  14. Deigan KE, Ferre-D’Amare AR (2011) Riboswitches: discovery of drugs that target bacterial gene-regulatory RNAs. Acc Chem Res 44:1329–1338. doi:10.1021/ar200039b

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  15. Deng Q, German I, Buchanan D, Kennedy RT (2001) Retention and separation of adenosine and analogues by affinity chromatography with an aptamer stationary phase. Anal Chem 73:5415–5421

    CAS  Article  PubMed  Google Scholar 

  16. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822. doi:10.1038/346818a0

    CAS  Article  PubMed  Google Scholar 

  17. Ellington AD, Szostak JW (1992) Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. Nature 355:850–852. doi:10.1038/355850a0

    CAS  Article  PubMed  Google Scholar 

  18. Famulok M, Hartig JS, Mayer G (2007) Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem Rev 107:3715–3743. doi:10.1021/cr0306743

    CAS  Article  PubMed  Google Scholar 

  19. Fusco D, Barnum TJ, Bruno AE, Luft JR, Snell EH, Mukherjee S, Charbonneau P (2014) Statistical analysis of crystallization database links protein physico-chemical features with crystallization mechanisms. PLoS One 9:e101123. doi:10.1371/journal.pone.0101123

    PubMed Central  Article  PubMed  Google Scholar 

  20. Gilbert W (1986) Origin of life: the RNA world. Nature 319:618

    Article  Google Scholar 

  21. Gold L et al (2010) Aptamer-based multiplexed proteomic technology for biomarker discovery. PloS one 5:e15004. doi:10.1371/journal.pone.0015004

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  22. Gold L, Janjic N, Jarvis T, Schneider D, Walker JJ, Wilcox SK, Zichi D (2012) Aptamers and the RNA world, past and present. Cold Spring Harb Perspect Biol. doi:10.1101/cshperspect.a003582

    PubMed Central  PubMed  Google Scholar 

  23. Hall B, Micheletti JM, Satya P, Ogle K, Pollard J, Ellington AD (2009) Design, synthesis, and amplification of DNA pools for in vitro selection. In: Ausubel FM et al (eds) Current protocols in molecular biology, Chapter 24, pp 24–22. doi:10.1002/0471142727.mb2402s88

  24. Hianik T, Ostatna V, Sonlajtnerova M, Grman I (2007) Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin. Bioelectrochemistry 70:127–133. doi:10.1016/j.bioelechem.2006.03.012

    CAS  Article  PubMed  Google Scholar 

  25. Hoinka J, Berezhnoy A, Sauna ZE, Gilboa E, Przytycka TM (2014) AptaCluster—a method to cluster HT-SELEX aptamer pools and lessons from its application. Res Comput Mol Biol 8394:115–128. doi:10.1007/978-3-319-05269-4_9

    PubMed Central  Article  PubMed  Google Scholar 

  26. Jagannathan V, Roulet E, Delorenzi M, Bucher P (2006) HTPSELEX–a database of high-throughput SELEX libraries for transcription factor binding sites. Nucleic Acids Res 34:D90–D94. doi:10.1093/nar/gkj049

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  27. Jayasena SD (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 45:1628–1650

    CAS  PubMed  Google Scholar 

  28. Jhaveri S, Ellington A (2002) In vitro selection of RNA aptamers to a small molecule target. In: Beaucage SL et al. (eds) Current protocols in nucleic acid chemistry, Chapter 9, pp 9–5. doi:10.1002/0471142700.nc0905s08

  29. Lee JF, Hesselberth JR, Meyers LA, Ellington AD (2004) Aptamer database. Nucleic Acids Res 32:D95–D100. doi:10.1093/nar/gkh094

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  30. Legiewicz M, Lozupone C, Knight R, Yarus M (2005) Size, constant sequences, and optimal selection. RNA (New York, NY) 11:1701–1709. doi:10.1261/rna.2161305

    CAS  Article  Google Scholar 

  31. Li Y, Geyer CR, Sen D (1996) Recognition of anionic porphyrins by DNA aptamers. Biochemistry 35:6911–6922. doi:10.1021/bi960038h

    CAS  Article  PubMed  Google Scholar 

  32. Lozupone C, Changayil S, Majerfeld I, Yarus M (2003) Selection of the simplest RNA that binds isoleucine. RNA (New York, NY) 9:1315–1322

    CAS  Article  Google Scholar 

  33. Luo X et al (2010) Computational approaches toward the design of pools for the in vitro selection of complex aptamers. RNA (New York, NY) 16:2252–2262. doi:10.1261/rna.2102210

    CAS  Article  Google Scholar 

  34. Mascini M, Palchetti I, Tombelli S (2012) Nucleic acid and peptide aptamers: fundamentals and bioanalytical aspects. Angew Chem (Int Edin English) 51:1316–1332. doi:10.1002/anie.201006630

    CAS  Article  Google Scholar 

  35. Mattice CM, DeRosa MC (2015) Status and prospects of aptamers as drug components. BioDrugs 29:151–165. doi:10.1007/s40259-015-0126-5

    CAS  Article  PubMed  Google Scholar 

  36. McKeague M, DeRosa MC (2012) Challenges and opportunities for small molecule aptamer development. J Nucleic Acids 2012:748913. doi:10.1155/2012/748913

    PubMed Central  Article  PubMed  Google Scholar 

  37. McKeague M, DeRosa MC (2014) Aptamers and SELEX: tools for the development of transformative molecular recognition technology. Aptamers Synth Antib 1:12–16

    Google Scholar 

  38. McKeague M, Giamberardino A, DeRosa MC (2011) Advances in aptamer-based biosensors for food safety. In: Environmental Biosensors. Vernon Somerset, InTech, SBN: 9789533074863, pp 17–18–42

  39. McKeague M et al (2015) Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation. Anal Chem. doi:10.1021/acs.analchem.5b02102

    PubMed  Google Scholar 

  40. McPherson A, Cudney B (2014) Optimization of crystallization conditions for biological macromolecules. Acta Crystallogr Sect F 70:1445–1467. doi:10.1107/S2053230X14019670

    CAS  Article  Google Scholar 

  41. Neves MA, Reinstein O, Saad M, Johnson PE (2010) Defining the secondary structural requirements of a cocaine-binding aptamer by a thermodynamic and mutation study. Biophys Chem 153:9–16. doi:10.1016/j.bpc.2010.09.009

    CAS  Article  PubMed  Google Scholar 

  42. Nimjee SM, Rusconi CP, Sullenger BA (2005) Aptamers: an emerging class of therapeutics. Annu Rev Med 56:555–583. doi:10.1146/annurev.med.56.062904.144915

    CAS  Article  PubMed  Google Scholar 

  43. Ponomarenko JV, Orlova GV, Frolov AS, Gelfand MS, Ponomarenko MP (2002) SELEX_DB: a database on in vitro selected oligomers adapted for recognizing natural sites and for analyzing both SNPs and site-directed mutagenesis data. Nucleic Acids Res 30:195–199

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  44. Price WN 2nd et al (2009) Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data. Nat Biotechnol 27:51–57. doi:10.1038/nbt.1514

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  45. Robertson DL, Joyce GF (1990) Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344:467–468. doi:10.1038/344467a0

    CAS  Article  PubMed  Google Scholar 

  46. Sabeti PC, Unrau PJ, Bartel DP (1997) Accessing rare activities from random RNA sequences: the importance of the length of molecules in the starting pool. Chem Biol 4:767–774. doi:10.1016/S1074-5521(97)90315-X

    CAS  Article  PubMed  Google Scholar 

  47. Sefah K, Shangguan D, Xiong X, O’Donoghue MB, Tan W (2010) Development of DNA aptamers using Cell-SELEX. Nat Protoc 5:1169–1185. doi:10.1038/nprot.2010.66

    CAS  Article  PubMed  Google Scholar 

  48. Serganov A, Patel DJ (2007) Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat Rev Genet 8:776–790. doi:10.1038/nrg2172

    CAS  Article  PubMed  Google Scholar 

  49. Silverman SK (2009) Artificial functional nucleic acids: aptamers ribozymes, and deoxyribozymes identified by in vitro selection. Funct Nucleic Acids Anal Appl 1:47–108. doi:10.1007/978-0-387-73711-9_3

    Article  Google Scholar 

  50. Stoltenburg R, Reinemann C, Strehlitz B (2005) FluMag-SELEX as an advantageous method for DNA aptamer selection. Anal Bioanal Chem 383:83–91. doi:10.1007/s00216-005-3388-9

    CAS  Article  PubMed  Google Scholar 

  51. Thodima V, Pirooznia M, Deng Y (2006) RiboaptDB: a comprehensive database of ribozymes and aptamers. BMC Bioinform 7(Suppl 2):S6. doi:10.1186/1471-2105-7-S2-S6

    Article  Google Scholar 

  52. Tolle F, Brandle GM, Matzner D, Mayer G (2015) A versatile approach towards nucleobase-modified aptamers. Angew Chem. doi:10.1002/anie.201503652

    Google Scholar 

  53. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510

    CAS  Article  PubMed  Google Scholar 

  54. Velez TE et al (2012) Systematic evaluation of the dependence of deoxyribozyme catalysis on random region length. ACS Comb Sci 14:680–687. doi:10.1021/co300111f

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  55. Waldrop MM (1989) Did life really start out in an RNA world? Science 246:1248–1249

    CAS  Article  PubMed  Google Scholar 

  56. Walsh R, DeRosa MC (2009) Retention of function in the DNA homolog of the RNA dopamine aptamer. Biochem Biophys Res Commun 388:732–735. doi:10.1016/j.bbrc.2009.08.084

    CAS  Article  PubMed  Google Scholar 

  57. Zhu G, Ye M, Donovan MJ, Song E, Zhao Z, Tan W (2012) Nucleic acid aptamers: an emerging frontier in cancer therapy. Chem Commun 48:10472–10480. doi:10.1039/c2cc35042d

    CAS  Article  Google Scholar 

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Acknowledgments

We thank Dr. Alex Wahba, Dr. Heather Shanks-McElroy, Ben Dorion, and Amal Awad for useful discussions in designing this research and analyzing the data. This work was supported by funds from the Natural Sciences and Engineering Research Council (NSERC) (Grant to M.C.D.) and Health Canada (Grant to R.A-R.).

Author Contributions

The manuscript was written through contributions of all authors. M.M., E.M.M., M.D., and M.C.D. wrote the article.

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Correspondence to Michel Dumontier or Maria C. DeRosa.

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Authors Maureen McKeague and Erin M. McConnell contributed equally.

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McKeague, M., McConnell, E.M., Cruz-Toledo, J. et al. Analysis of In Vitro Aptamer Selection Parameters. J Mol Evol 81, 150–161 (2015). https://doi.org/10.1007/s00239-015-9708-6

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Keywords

  • Aptamer
  • In vitro selection
  • SELEX
  • Aptamer target
  • Database