Computational Tools: RNA Interference in Fungal Therapeutics

  • Chakresh Kumar Jain
  • Gulshan Wadhwa


There is steady rise in the number of immunocompromised population due to increased use of potent immunosuppression therapies. This is associated with increased risk of acquiring fungal opportunistic infections in immunocompromised patients which account for high morbidity and mortality rates, if left untreated. The conventional antifungal drugs to treat fungal diseases (mycoses) are increasingly becoming inadequate due to observed varied susceptibility of fungi and their recurrent resistance. RNA interference (RNAi), sequence-specific gene silencing, is emerging as a promising new therapeutic approach. This chapter discusses various aspects of RNAi, viz., the fundamental RNAi machinery present in fungi, in silico siRNA features, designing guidelines and tools, siRNA delivery, and validation of gene knockdown for therapeutics against mycoses. Target gene identification is a crucial step in designing of gene-specific siRNA in addition to efficient delivery strategies to bring about effective inhibition of fungi. Subsequently, designed siRNA can be delivered effectively in vitro either by soaking fungi with siRNA or by transforming inverted repeat transgene containing plasmid into fungi, which ultimately generates siRNA(s). Finally, fungal inhibition can be verified at the RNA and protein levels by blotting techniques, fluorescence imaging, and biochemical assays. Despite challenges, several such in vitro studies have spawned optimism around RNAi as a revolutionary new class of therapeutics against mycoses. But, pharmacokinetic parameters need to be evaluated from in vivo studies and clinical trials to recognize RNAi as a novel treatment approach for mycoses.


RNA interference Short interfering RNA Mycoses Immunosuppression Opportunistic infection siRNA design Target gene Soaking Inverted repeat transgene Therapeutics 


  1. Amarzguioui M, Prydz H (2004) An algorithm for selection of functional siRNA sequences. Biochem Biophys Res Commun 316(4):1050–1058CrossRefPubMedGoogle Scholar
  2. Barton L, Prade R (2008) Inducible RNA interference of brlAβ in Aspergillus nidulans. Eukaryot Cell 7:2004–2007CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bhanderi B et al (2009) Antifungal drug resistance – concerns for veterinarians. Veterinary World 2:204–207CrossRefGoogle Scholar
  4. Chamilos G, Kontoyiannis D (2005) Update on antifungal drug resistance mechanisms of Aspergillus fumigatus. Drug Resist Updat 8:344–358CrossRefPubMedGoogle Scholar
  5. Cogoni C, Macino G (1997) Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc Natl Acad Sci U S A 94:10233–10238CrossRefPubMedPubMedCentralGoogle Scholar
  6. Drinnenberg I et al (2009) RNAi in budding yeast. Science 326:544–550CrossRefPubMedPubMedCentralGoogle Scholar
  7. Dujon B et al (2004) Genome evolution in yeasts. Nature 430:35–44CrossRefPubMedGoogle Scholar
  8. Erental A et al (2007) Type 2A phosphoprotein phosphatase is required for asexual development and pathogenesis of Sclerotinia sclerotiorum. MPMI 20:944–954CrossRefPubMedGoogle Scholar
  9. Fire A et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefGoogle Scholar
  10. Fulci V, Macino G (2007) Quelling: post-transcriptional gene silencing guided by small RNAs in Neurospora crassa. Curr Opin Microbiol 10:199–203CrossRefPubMedGoogle Scholar
  11. Hammond T, Keller N (2005) RNA silencing in Aspergillus nidulans is independent of RNA-dependent RNA polymerases. Genetics 169:607–617CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hammond T et al (2007) Aspergillus Mycoviruses are targets and suppressors of RNA silencing. Eukaryot Cell 7:350–357CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hammond T et al (2008) RNA silencing gene truncation in the filamentous fungus Aspergillus nidulans. Eukaryot Cell 7:339–349CrossRefPubMedGoogle Scholar
  14. Holen T (2006) Efficient prediction of siRNAs with siRNArules 1.0: an open-source JAVA approach to siRNA algorithms. RNA 12(9):1620–1625CrossRefPubMedPubMedCentralGoogle Scholar
  15. Ibrahim A et al (2008a) Comparison of lipid amphotericin B preparations in treating murine zygomycosis. Antimicrob Agents Chemother 52:1573–1576CrossRefPubMedPubMedCentralGoogle Scholar
  16. Ibrahim A et al (2008b) Combination echinocandin-polyene treatment of murine mucormycosis. Antimicrob Agents Chemother 52:1556–1558CrossRefPubMedPubMedCentralGoogle Scholar
  17. Ibrahim ML et al (2009) Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals whole-genome duplication. PLoS Genet 5:e1000549CrossRefPubMedPubMedCentralGoogle Scholar
  18. Janus D et al (2007) An efficient fungal RNA-silencing system using the DsRed reporter gene. Appl Environ Microbiol 73:962–970CrossRefPubMedGoogle Scholar
  19. Jinek M, Doudna J (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457:405–412CrossRefPubMedGoogle Scholar
  20. Jong J et al (2006) RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune. Appl Environ Microbiol 72:1267–1269CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kadotani N et al (2003) RNA silencing in the phytopathogenic fungus Magnaporthe oryzae. MPMI 16:769–776CrossRefPubMedGoogle Scholar
  22. Kalidas S, Smith DP (2002) Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila. Neuron 33(2):177–184CrossRefPubMedGoogle Scholar
  23. Kawasaki K et al (2003) Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 3. Bioorg Med Chem Lett 13:87–91CrossRefPubMedGoogle Scholar
  24. Khatri M, Rajam M (2007) Targeting polyamines of Aspergillus nidulans by siRNA specific to fungal ornithine decarboxylase gene. Med Mycol 45:211–220CrossRefPubMedGoogle Scholar
  25. Krcmery V (1996) Emerging fungal infections in cancer patients. J Hosp Infect 33:109–117CrossRefPubMedGoogle Scholar
  26. Leuschner P et al (2006) Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep 7:314–320CrossRefPubMedPubMedCentralGoogle Scholar
  27. Li L et al (2010) RNA interference pathways in filamentous fungi. Cell Mol Life Sci 67:3849–3863CrossRefPubMedGoogle Scholar
  28. Liu H et al (2002) RNA interference in the pathogenic fungus Cryptococcus neoformans. Genetics 160:463–470PubMedPubMedCentralGoogle Scholar
  29. Matveeva O et al (2007) Comparison of approaches for rational siRNA design leading to a new efficient and transparent method. Nucleic Acids Res 35:e63CrossRefPubMedPubMedCentralGoogle Scholar
  30. McDonald T et al (2005) RNA silencing of mycotoxin production in Aspergillus and Fusarium species. MPMI 18:539–545CrossRefPubMedGoogle Scholar
  31. Moriwaki A et al (2007) RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae. FEMS Microbiol Lett 269:85–89CrossRefPubMedGoogle Scholar
  32. Moussian B (2008) The role of GlcNAc in formation and function of extracellular matrices. Comp Biochem Physiol 149:215–226CrossRefGoogle Scholar
  33. Mouyna I et al (2004) Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol Lett 237:317–324PubMedGoogle Scholar
  34. Mukherjee P et al (2005) Combination treatment of invasive fungal infections. Clin Microbiol Rev 18:163–194CrossRefPubMedPubMedCentralGoogle Scholar
  35. Münsterkötter M, Mannhaupt G (2008) The posttranscriptional machinery of Ustilago maydis. Fungal Genet Biol 45:S40–S46CrossRefPubMedGoogle Scholar
  36. Naito Y et al (2006) siVirus: web-based antiviral siRNA design software for highly divergent viral sequences. Nucleic Acids Res 34((Web Server issue)):W448–W450CrossRefPubMedPubMedCentralGoogle Scholar
  37. Nakayashiki H (2005) RNA silencing in fungi: mechanisms and applications. FEBS Lett 579:5950–5957CrossRefPubMedGoogle Scholar
  38. Nakayashiki H et al (2005) RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol 42:275–283CrossRefPubMedGoogle Scholar
  39. Namekawa S et al (2005) Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing. Microbiology 151:3669–3678CrossRefPubMedGoogle Scholar
  40. Ndegwa D et al (2007) Protocols for gene silencing in schistosomes. Exp Parasitol 117:284–291CrossRefPubMedPubMedCentralGoogle Scholar
  41. Nicolás F et al (2003) Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes. EMBO J 22:3983–3991CrossRefPubMedPubMedCentralGoogle Scholar
  42. Oliveira J et al (2008) Efficient cloning system for construction of gene silencing vectors in Aspergillus niger. Appl Microbiol Biotechnol 80:917–924CrossRefPubMedGoogle Scholar
  43. Paddison PJ, Vogt PK (2008) RNA interference, Current topics in microbiology and immunology, vol 320. Springer-Verlag, BerlinGoogle Scholar
  44. Pauw B, Picazo J (2008) Present situation in the treatment of invasive fungal infection. Int J Antimicrob Agents 32:S75–S79Google Scholar
  45. Rappleye C et al (2004) RNA interference in Histoplasma capsulatum demonstrates a role for α-(1,3)-glucan in virulence. Mol Microbiol 53:153–165CrossRefPubMedGoogle Scholar
  46. Reynolds A et al (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330CrossRefPubMedGoogle Scholar
  47. Ribes J et al (2000) Zygomycetes in human disease. Clin Microbiol Rev 13:236–301CrossRefPubMedPubMedCentralGoogle Scholar
  48. Richardson M (1991) Opportunistic and pathogenic fungi. J Antimicrob Chemother 28:1–11CrossRefPubMedGoogle Scholar
  49. Rogers T (2008) Treatment of zygomycosis: current and new options. J Antimicrob Chemother 61:i35–i39CrossRefPubMedGoogle Scholar
  50. Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343–3353CrossRefPubMedGoogle Scholar
  51. Ruddon R (2007) Cancer biology, 4th edn. Oxford University Press, New YorkGoogle Scholar
  52. Schepers U (2005) RNA interference in practice: principles, basics, and methods for gene silencing in C. elegans, drosophila, and mammals, 1st edn. Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimGoogle Scholar
  53. Segers G et al (2006) Hypovirus papain-like protease p29 suppresses RNA silencing in the natural fungal host and in a heterologous plant system. Eukaryot Cell 5:896–904CrossRefPubMedPubMedCentralGoogle Scholar
  54. Sigova A et al (2004) A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. Genes Dev 18:2359–2367CrossRefPubMedPubMedCentralGoogle Scholar
  55. Singh S et al (2005) New fungal metabolite geranylgeranyltransferase inhibitors with antifungal activity. Nat Prod Res 19:739–747CrossRefPubMedGoogle Scholar
  56. Siomi H, Siomi M (2009) On the road to reading the RNA-interference code. Nature 457:396–404CrossRefGoogle Scholar
  57. Solis C, Guillén N (2008) Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica. In: Barik S (ed) RNAi: design and application. Humana Press, Totowa, pp 113–128CrossRefGoogle Scholar
  58. Song J et al (2004a) The Candida albicans lanosterol 14-α Demethylase (ERG11) gene promoter is maximally induced after prolonged growth with antifungal drugs. Antimicrob Agents Chemother 48:1136–1144CrossRefPubMedPubMedCentralGoogle Scholar
  59. Song J et al (2004b) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305:1434–1437CrossRefPubMedGoogle Scholar
  60. Takeno S et al (2005) Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with Δ12-desaturase gene expression. Appl Environ Microbiol 71:5124–5128CrossRefPubMedPubMedCentralGoogle Scholar
  61. Tinoco M et al (2010) In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA. BMC Biol 8:27CrossRefPubMedPubMedCentralGoogle Scholar
  62. Tuschl T et al (1999) Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13:3191–3197CrossRefPubMedPubMedCentralGoogle Scholar
  63. Ui-Tei K et al (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res 32(3):936–948CrossRefPubMedPubMedCentralGoogle Scholar
  64. Vert JP et al (2006) An accurate and interpretable model for siRNA efficacy prediction. BMC Bioinformatics 30(7):520CrossRefGoogle Scholar
  65. Wapinski I et al (2007) Natural history and evolutionary principles of gene duplication in fungi. Nature 449:54–61CrossRefPubMedGoogle Scholar
  66. Warnock D, Richardson M (1991) Fungal infection in the compromised patient, 2nd edn. Wiley, ChichesterGoogle Scholar
  67. Wesley SV et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27(6):581–590CrossRefPubMedGoogle Scholar
  68. Yamada O et al (2007) Gene silencing by RNA interference in the Koji Mold Aspergillus oryzae. Biosci Biotechnol Biochem 71:138–144CrossRefPubMedGoogle Scholar
  69. Yiu S et al (2005) Filtering of ineffective siRNAs and improved siRNA design tool. Bioinformatics 21:144–151CrossRefPubMedGoogle Scholar
  70. Yu J et al (2004) Transgenic RNAi-mediated reduction of MSY2 in mouse oocytes results in reduced fertility. Dev Biol 268(1):195–206CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Chakresh Kumar Jain
    • 1
  • Gulshan Wadhwa
    • 2
  1. 1.Department of BiotechnologyJaypee Institute of Information TechnologyNoidaIndia
  2. 2.Department of Biotechnology, Apex Bioinformatics CentreMinistry of Science & TechnologyNew DelhiIndia

Personalised recommendations