WO2011079356A1 - Carbon nanotube conjugate for inhibiting pathogenic infection structures in plants - Google Patents

Carbon nanotube conjugate for inhibiting pathogenic infection structures in plants Download PDF

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Publication number
WO2011079356A1
WO2011079356A1 PCT/BR2010/000411 BR2010000411W WO2011079356A1 WO 2011079356 A1 WO2011079356 A1 WO 2011079356A1 BR 2010000411 W BR2010000411 W BR 2010000411W WO 2011079356 A1 WO2011079356 A1 WO 2011079356A1
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carbon nanotube
pathogen infection
structures
carbon nanotubes
conjugate
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PCT/BR2010/000411
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French (fr)
Portuguese (pt)
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Luiz Orlando Ladeira
Leonardo Rodrigues
Ary CORRÊA JUNIOR
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Universidade Federal De Minas Gerais - Ufmg
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Publication of WO2011079356A1 publication Critical patent/WO2011079356A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/34—Shaped forms, e.g. sheets, not provided for in any other sub-group of this main group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism

Definitions

  • the present invention describes the construction and use of a hybrid system involving the conjugation of carbon nanotubes and oligonucleotides.
  • process and methodology for the inhibition or control of pests and pathogen infections in vegetables, especially in crops of important commercial interest such as: beans, soy, coffee and eucalyptus.
  • the oligonucleotide-carbon nanotube conjugate is used as a cell internalizing agent carrying a specific nucleic acid sequence, also called an oligonucleotide, from outside into the microorganism cell cytoplasm.
  • This internalization is to allow the oligonucleotide to pass into the microorganism's cytoplasm and interfere with the protein synthesis mechanisms regulated by the microorganism's messenger RNA, which as a result leads to inhibition of infection structures, causing the microorganism to die or to reduce the harmful effects of the microorganism. aggressor to the host.
  • Carbon nanotubes are quasi one-dimensional structures formed by carbon-carbon bonds in sp 2 hybridization in the form of tubes whose diameter can range from 1 nm (10 "9 M) at 100 nm and typical length of approximately 10 4 times its diameter (HERBST , MH; MACEDO, MIF & ROCCO, AM Carbon Nanotube Technology: Trends and Perspectives in a Multidisciplinary Area Chem.Nova. 27 (6): 986-992. 2004; IIJIMA, S & ICHIHASHI, T. Single-shell carbon nanotubes of 1-nm dimeter Nature 363: 603-605 1993; IIJIMA, S. Helical microtubules of graphical carbon Nature 354: 56-58 1991) Carbon nanotubes can be produced.
  • SWNT single-walled carbon nanotubes
  • MWNT concentric multiple-walled carbon nanotubes
  • Carbon nanotubes have high structural rigidity, high biocompatibility, low cytotoxicity, and their diameter in the range of a few nanometers are small enough to passively pass through the cell wall and cytoplasmic membrane of cells, thus carrying bio-molecules attached to their middle surface.
  • telomerase transcriptase small interfering RNA in complex with positively charged single- walled carbon nanotubes suppresses tumor growth Cancer Clin Res 12 (16): 4933-4939 2006; KAM, NWS & DAI, H. Carbon nanotubes as intrace llular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, NWS; JESSOP, TC; WENDER, PA & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004). The attachment or immobilization of biomolecules to the outer wall of carbon nanotubes can be accomplished by two processes:
  • Covalent immobilization in this case a bridge molecule has one end covalently attached to the carbon nanotube wall and the biomolecule covalently linked to the other end of the bridge molecule (CHEN, S .; SHEN, W .; WU, G .; CHEN , D. & JIANG, M.
  • CHEN, S .; SHEN, W .; WU, G .; CHEN , D. & JIANG, M A new approach to the functionalization of single-walled carbon nanotubes with both alkyl and carboxyl groups Chem.Phys.Letters 402: 312-317 2005; HE, P. & URBAN, MW Controlled phospholipids functionalization of single-walled carbon nanotubes Biomacromolecules 6: 2455-2457 2005; WANG, Y; IQBAL, Z.
  • Non-covalent immobilization in this case the biomolecule non-covalently adsorbs to the carbon nanotube wall and is weakly adhered to its wall by Van der Walls forces (BECKER, ML; FAGAN, JA; GALLANT, ND; BAUER , BJ; BAJPAI, V.; HOBBIE, EK; LACERDA, SH; MIGLER, KB & JAKUPCIAK, JP Length-dependent uptake of DNA-wrapped single-walled carbon nanotubes Adv.Mater. 19: 939-945.
  • BECKER ML
  • FAGAN JA
  • GALLANT ND
  • BAUER BJ
  • BAJPAI V.
  • HOBBIE EK
  • LACERDA SH
  • the common bean is grown in all units of the federation, being the fourth agricultural product in planted area and the sixth in value of the country's grain production (IBGE - Brazilian Institute of Geography and Statistics Foundation. Systematic survey of agricultural production: men- salt forecasting and monitoring of the crop in the calendar year Rio de Janeiro, v.21, n.1, 79p, 2009; ZIMMERMANN, MJ DE; ROCHA, M .; YAMADA, T. Bean culture. Piracicaba : Brazilian Association for Research Potash and Phosphate (POTAFOS), 689p. 1988).
  • U. appendiculatus is a obligate parasitic fungus characterized by high pathogenic variability.
  • HALEY SD; MIKLAS, PN; AFANADOR, L; KELLY, JD
  • RAPD Random amplified polymorphic DNA
  • a family of genes expressed between the period coincident with the formation of the appressorium was characterized (XUEI, X .; BHAIRI, S .; STAPLES, RC & YODER, OC. Characterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus. Gene 110: 49-55 1992; BHAIRI, SM; STAPLES, RC; FREVE, P. & YODER, OC Characterization of a structure-specific gene infection from the rust fungus, Uromyces appendiculatus. Gene. 81: 237-243. 1989).
  • the antisense oligonucleotide technique is extensively used as a tool to inhibit expression of target mRNAs and to enable elucidation of gene function both in vitro and in vivo (OEKELEN, DV; LUYTEN, WHML AND LEYSEN, JE).
  • OEKELEN, DV LUYTEN, WHML AND LEYSEN, JE.
  • Ten years of antisense inhibition of brain G-protein-coupled receptor function (Brain Res. Rev. 42: 123-142. 2003). It has been previously demonstrated by a member of the team filing this patent that microinjection of antisense sequences of the INF24 gene results in inhibition of U. appendiculatus appressorium formation in vitro (BARJA, F .; CORR ⁇ A JR.
  • SWNT single wall
  • MWNT multiple wall carbon nanotubes
  • Biological molecules such as protein and nucleic acids, interact easily with the surface of NTC and have been successfully used as surfactants of these nanostructures (ZHAO, X. & JOHNSON, JK. Simulation of DNA adsorption on carbon nanotubes. J.Am 129: 10438-10445 2007; MALIK, S.; VOGEL, S.; ROSNER, H.; ARNOLD, K.; HENNRICH, R.; KOHLER, A.; RICHERT, C. & KAPPES, MM Physical Chemical characterization of DNA-SWNT suspensions and associated composites Composites Science and Technology 67: 916-921 2007.
  • Nanotube molecular transporters Internationalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004; CHEN, R.J .; BANGSARUNTIP, S .; DROUVALAKIS, KA; KAM, N.W.S .; SHIM, M .; LI, Y; KIM, W .; UTZ, PJ. & DAI, H. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).
  • Patent application WO2005104179 describes the use of carbon nanotubes for analysis of chemical and biological samples, but does not report the use of nanotubes to introduce substances into the cytoplasm of cells.
  • Patent application WO2006078640 reports the introduction of substances into cells causing gene alteration, however, carbon nanotubes are not used.
  • Rust control is accomplished through the use of disease resistant cultivars, crop practices and / or the application of pesticides.
  • the existence of a large variability of circulating U. appendi- culatus breeds makes rust control difficult.
  • This fact, associated with the use of pesticides that lead to the selection of phytopathogenic microorganisms resistant to available compounds shows the need to look for more effective and less polluting alternatives for the control of rust in plants.
  • the internalization of antisense oligonucleotides, using the nanotube-oligonucleotide conjugate, represents an efficient alternative for functional analysis of genes in filamentous fungi, especially those with limitations with conventional techniques, as well as a tool for the development of new disease control strategies in the field.
  • Figure 1 Atomic Force Microscopy Ultrasound of (a) INF24 antisense adsorbed to carbon nanotubes and (b) INF24 sense oligonucleotides adsorbed to carbon nanotubes.
  • Figure 2 Bean leaves inoculated with the fungus Uromyces appendiculatus after treatment with (a) carbon nanotube, (b) water, (c) INF24 antisense oligonucleotides adsorbed on carbon nanotubes and (d) INF24 sensory oligonucleotides adsorbed on carbon nanotubes.
  • the present invention relates a non-covalent immobilization process of a specific gene sequence to carbon nanotubes (NTC) thus producing a carbon nanotube-oligonucleotide hybrid system for use as a gene internalizing agent in the fight against plant pathogens.
  • NTC carbon nanotubes
  • the purpose of this internalization is to allow the oligonucleotide to pass into the pathogen's cytoplasm and interfere with the mechanisms of protein synthesis regulated by the pathogen's messenger RNA, which as a result leads to inhibition of infection structures, causing pathogen death or diminished effects. harm from the aggressor to the host.
  • This conjugated hybrid system has high bioactivity, high selectivity and specificity as it is constructed by immobilizing a specific oligonucleotide to the carbon nanotube.
  • Oligonucleotide and NTC conjugates were obtained from a functionalized INF24 and NTC oligonucleotide solution (SWNTf or MWNTf) which was sonicated in bath ultrasound for 30 minutes at room temperature and stored at -20 ° C until use in the experiments. .
  • Example 2 Atomic Force Microscopy Analysis of Conjugates
  • SWNTf or MWNTf was poured onto urediniospores deposited on plastic membranes containing inductive topography or bean leaf discs. Inoculated substrates were incubated for 4 h at 18 ° C, fixed and the number of appressoria formed in 100 germ tubes. In the case of bean leaf discs the number of appressoria was determined in randomly selected germ tubes and from these, the germ tubes that grew on stomata.
  • UWN appendiculatus urediniospores treated with INF24 sense oligonucleotide conjugated mean appressorium formation values of 29.17% ⁇ 8.06%; 32.33% ⁇ 17.98%; 26.33% ⁇ 9.62% and 26.50% ⁇ 8.04%, in the proportions of 1: 0.5; 1: 1; 1: 2 and 1: 3 respectively. However, these values did not differ statistically from each other, from control and from MWNT.
  • MWNTf-treated urediniospores conjugated with antisense INF24 oligonucleotide had inhibited appressor formation.
  • 1: 0.5; 1: 1; 1: 2 and 1: 3 MWNTf: I F24
  • the 1: 1 treatment showed a significant difference (P ⁇ 0.05) when compared to the control treatments, MWNTf and 1: 0.5.
  • MWNTf In the 1: 2 treatment, a statistically significant difference was observed when compared to the control and 1: 0.5 (P ⁇ 0.01) and MWNTf (P ⁇ 0.05).
  • the 1: 3 treatment presented the lowest percentage of appressory formation and differed from the control, MWNTf and 1: 0.5 (P ⁇ 0.001).
  • the 1: 0.5 ratio did not differ significantly from control and MWNTf treatments.
  • the data show that the ratio between NTC and oligonucleotides influences the efficiency of inhibiting the formation of infection structures. No sign of cell morbidity was observed in any of the treatments (cytoplasmic granulation, vacuolization, etc.).
  • U24 appendiculatus urediniospores treated with INF24 sense oligonucleotide conjugate showed mean appressorium formation values of 51.44% ⁇ 9.00%; 56.00% ⁇ 5.78%; 51, 78% ⁇ 4.55% and 49.11% ⁇ 4.00%, at doses of 50; 25; 12.5 and 6.25% (v / v) respectively. However, these values did not differ statistically from each other, from control and from SWNTf.
  • U. appendiculatus urediniospores infect bean plants and develop leaf surface lesions 7 days after inoculation. Yellow spots, characteristic of the early stage of disease development are observed during this period. These points evolve to coalescent, brown-colored, powdery lesions with release of U. appendiculatus urediniospores in susceptible plants without control treatment.
  • Inhibition of rust caused by U. appendiculatus was performed by pouring suspension of the oligonucleotide conjugate INF24 and NTC onto the adaxial face of primary bean plant leaves 24 h before urediniospore inoculation. Inoculated plants were incubated under leaf wetness for 18 h at room temperature. After the onset of symptoms characteristic of the disease, the number of lesions was determined. These points evolve to coalescent, brown-colored powdery lesions with release of U. appendiculatus urediniospores in susceptible plants without control treatment. These symptoms are also observed in plants treated with sense-conjugated MWNTf and INFWN-conjugated MWNTf and inoculated with U. appendiculatus urediniospores, showing that these treatments do not interfere with the developmental process. plant infection.

Abstract

The present invention describes the construction and use of a hybrid system involving the conjugation of carbon nanotubes and oligonucleotides. The more general aspect of said invention relates to the process and methodology for inhibiting or controlling pests and pathogenic infections in plants, especially in commercially important crops, such as beans, soya, coffee and eucalyptus. The oligonucleotide-carbon nanotube conjugate is used as a cell internalization agent carrying a specific nucleic acid sequence, also termed oligonucleotide, from outside into the pathogen cell cytoplasm.

Description

"CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS".  "CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES".
CAMPO DA INVENÇÃO  FIELD OF INVENTION
A presente invenção descreve a construção e uso de um siste- ma híbrido envolvendo a conjugação de nanotubos de carbono e oligonucle- otídeos. Em seu aspecto mais geral relata processo e metodologia para a inibição ou controle de pragas e infecções de patógenos em vegetais, em especial, em culturas de importante interesse comercial tais como: feijão, soja, café e eucalipto. O conjugado oligonucleotídeo-nanotubos de carbono é utilizado como agente de internalização celular carreando uma seqiiência específica de ácido nucléico, também denominado de oligonucleotídeo, de fora para dentro do citoplasma da célula do microrganismo. O objetivo desta internalização é permitir que o oligonucleotídeo passe para o citoplasma do microrganismo e interfira nos mecanismos de síntese protéica regulada pelo RNA mensageiro do microrganismo que como resultado leva a inibição de estruturas de infecção, causando a morte do microrganismo ou diminuição dos efeitos nocivos do agressor ao hospedeiro.  The present invention describes the construction and use of a hybrid system involving the conjugation of carbon nanotubes and oligonucleotides. In its most general aspect reports process and methodology for the inhibition or control of pests and pathogen infections in vegetables, especially in crops of important commercial interest such as: beans, soy, coffee and eucalyptus. The oligonucleotide-carbon nanotube conjugate is used as a cell internalizing agent carrying a specific nucleic acid sequence, also called an oligonucleotide, from outside into the microorganism cell cytoplasm. The purpose of this internalization is to allow the oligonucleotide to pass into the microorganism's cytoplasm and interfere with the protein synthesis mechanisms regulated by the microorganism's messenger RNA, which as a result leads to inhibition of infection structures, causing the microorganism to die or to reduce the harmful effects of the microorganism. aggressor to the host.
ESTADO DA TÉCNICA TECHNICAL STATE
Nanotubos de Carbono são estruturas quase unidimensionais formadas por ligações carbono-carbono em hibridização sp2 na forma de tubos cujo diâmetro pode variar de 1 nm (10"9 m) a 100 nm e comprimento típico da ordem de 104 vezes seu diâmetro (HERBST, M.H.; MACEDO, M.I.F. & ROCCO, A.M. Tecnologia dos Nanotubos de carbono: tendências e perspectivas de uma área multidisciplinar. Quím.Nova. 27 (6): 986-992. 2004; IIJIMA, S & ICHIHASHI, T. Single-shell carbon nanotubes of 1-nm di- ameter. Nature. 363: 603-605. 1993; IIJIMA, S. Helical microtubules of gra- phitic carbon. Nature. 354: 56-58. 1991). Os nanotubos de carbono podem ser produzidos com uma única parede de carbono sendo denominados de nanotubos de carbono de parede única (SWNT) ou com múltiplas paredes de carbono concêntricas denominados de nanotubos de carbono de múltiplas paredes (MWNT) (SINHA, N. & YEOW, J.T.W. Carbon Nanotubes for biomedical applications. IEEE Transactions on Nanobioscience. 4(2): 180- 195. 2005). Os nanotubos de carbono possuem alta rigidez estrutural, alta biocompatibilidade, baixa citotoxicidade e com seu diâmetro na faixa de alguns nanômetros são pequenos o suficiente para atravessar passivamente a parede celular e membrana citoplasmática de células, carreando assim bio- moléculas ligadas a sua superfície do meio extracelular para o intracelular (KOSTARELOS, K.; LACERDA, L; PASTORIN, G.; WU, W.; WIECKOWSKI, S.; LUANGSIVILAY, J.; GODEFROY, S.; PANTAROTTO, D.; BRIAND, J.; MULLER.S.; PRATO, M. & BIANCO, A. Cellular uptake of functionalized car- bon nanotubes is independent of functional group and cell type. Nature. 2: 108-113. 2007; ZHANG, Z.; YANG, X.; ZHANG, Y.; ZENG, B.; WANG, S.; ZHU, T.; RODEN, R.B.S.; CHEN, Y. & YANG, R. Delivery of telomerase re- verse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clin. Câncer Res. 12 (16): 4933-4939. 2006; KAM, N.W.S. & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, N.W.S.; JESSOP, T.C.; WENDER, P.A. & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004). A ligação ou imobilização de biomoléculas a parede externa de nanotubos de carbono pode ser feita através de dois processos: Carbon nanotubes are quasi one-dimensional structures formed by carbon-carbon bonds in sp 2 hybridization in the form of tubes whose diameter can range from 1 nm (10 "9 M) at 100 nm and typical length of approximately 10 4 times its diameter (HERBST , MH; MACEDO, MIF & ROCCO, AM Carbon Nanotube Technology: Trends and Perspectives in a Multidisciplinary Area Chem.Nova. 27 (6): 986-992. 2004; IIJIMA, S & ICHIHASHI, T. Single-shell carbon nanotubes of 1-nm dimeter Nature 363: 603-605 1993; IIJIMA, S. Helical microtubules of graphical carbon Nature 354: 56-58 1991) Carbon nanotubes can be produced. single-walled carbon nanotubes (SWNT) or concentric multiple-walled carbon nanotubes (MWNT) (SINHA, N. & YEOW, JTW Carbon Nanotubes for biomedical applications IEEE Transactions on Nanobioscience 4 (2): 180- 195. 2005). Carbon nanotubes have high structural rigidity, high biocompatibility, low cytotoxicity, and their diameter in the range of a few nanometers are small enough to passively pass through the cell wall and cytoplasmic membrane of cells, thus carrying bio-molecules attached to their middle surface. extracellular to intracellular (KOSTARELOS, K .; LACERDA, L; PASTORIN, G .; WU, W .; WIECKOWSKI, S.; LUANGSIVILAY, J .; GODEFROY, S .; PANTAROTTO, D .; BRIAND, J .; MULLER PRATO, M. & BIANCO, A. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type Nature 2: 108-113. 2007; ZHANG, Z .; YANG, X .; ZHANG, Y .; ZENG, B.; WANG, S .; ZHU, T.; RODEN, RBS; CHEN, Y. & YANG, R. Delivery of telomerase transcriptase small interfering RNA in complex with positively charged single- walled carbon nanotubes suppresses tumor growth Cancer Clin Res 12 (16): 4933-4939 2006; KAM, NWS & DAI, H. Carbon nanotubes as intrace llular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, NWS; JESSOP, TC; WENDER, PA & DAI, H. Nanotube molecular transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004). The attachment or immobilization of biomolecules to the outer wall of carbon nanotubes can be accomplished by two processes:
- Imobilização covalente - neste caso uma molécula ponte tem uma de suas extremidades ligada covalentemente à parede do nanotubo de carbono e a biomolécula ligada covalentemente à outra extremidade da molécula ponte (CHEN, S.; SHEN, W.; WU, G.; CHEN, D. & JIANG, M. A new approach to the functionalization of single-walled carbon nanotubes with both alkyl and carboxyl groups. Chem.Phys.Letters. 402: 312-317. 2005; HE, P. & URBAN, M.W. Controlled phospholipids functionalization of single-walled carbon nanotubes. Biomacromolecules. 6: 2455-2457. 2005; WANG, Y.; IQBAL, Z. & MALHOTRA, S.V. Functionalization of carbon nanotubes with amines and enzymes. Chem.Phys.Letters. 402: 96-101. 2005; PANTAROTTO, D.; PARTIDOS, C.D.; GRAFF, R.;HOEBEKE, J.; BRIAND, J.; PRATO, M. & BIANCO, A. Synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides. J.Am. Chem. Soe. 125 (20): 6160-6164. 2003; POMPEO, F. & RESASCO, D.E. Water solubilization of single-walled carbon nanotubes by functionalization with glucosamine. Nano- letters. 2 (4): 369-373. 2002). Covalent immobilization - in this case a bridge molecule has one end covalently attached to the carbon nanotube wall and the biomolecule covalently linked to the other end of the bridge molecule (CHEN, S .; SHEN, W .; WU, G .; CHEN , D. & JIANG, M. A new approach to the functionalization of single-walled carbon nanotubes with both alkyl and carboxyl groups Chem.Phys.Letters 402: 312-317 2005; HE, P. & URBAN, MW Controlled phospholipids functionalization of single-walled carbon nanotubes Biomacromolecules 6: 2455-2457 2005; WANG, Y; IQBAL, Z. & MALHOTRA, SV Functionalization of carbon nanotubes with amines and enzymes Chem.Phys.Letters 402: 96 -101. 2005; PANTAROTTO, D .; PARTIES, CD; GRAFF, R.; HOEBEKE, J .; BRIAND, J .; PRATO, M. & BIANCO, A. Synthesis, structural characterization, and immunological properties of carbon nanotubes functionalized with peptides. J.Am. Chem. Sound. 125 (20): 6160-6164. 2003; POMPEO, F. & RESASCO, DE Water solubilization of single-walled carbon nanotubes by functionalization with glucosamine. Nano letters. 2 (4): 369-373. 2002).
- Imobilização não-covalente - neste caso a biomolécula adsor- ve à parede do nanotubo de carbono de modo não-covalente ficando fracamente aderida a sua parede por forças de Van der Walls (BECKER, M.L.; FAGAN, J.A.; GALLANT, N.D.; BAUER, B.J.; BAJPAI, V.; HOBBIE, E.K.; LACERDA, S.H.; MIGLER, K.B. & JAKUPCIAK, J.P. Length-dependent upta- ke of DNA-wrapped single-walled carbon nanotubes. Adv.Mater. 19: 939- 945. 2007; GIGLIOTTI, B.; SAKIZZIE, B.; BETHUNE, D.S.; SHELBY, R.M. & CHA, J.N. Sequence-independent helical wrapping of single-walled carbon nanotubes by long genomic DNA. Nanoletters. 6 (2): 159-164. 2006; ZHANG, Z.; YANG, X.; ZHANG, Y.; ZENG, B.; WANG, S.; ZHU, T.; RODEN, R.B.S.; CHEN, Y. & YANG, R. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clin. Câncer Res. 12 (16): 4933-4939. 2006; KAM, N.W.S. & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am. Chem. Soe. 127: 6021-6026. 2005; SIRDESHMUKH, R.; TEKER, K. & PANCHAPAKESAN, B. Biological functionalization of carbon nanotubes. Mat. Res. Soe. Symp. Proc. 823: W4.1-.1-W4.1.6. 2004; CHEN, R.J.; BANGSARUNTIP, S.; DROUVALA- KIS, K.A.; KAM, N.W.S.; SHIM, M.; LI, Y.; KIM.W.; UTZ, P.J. & DAI, H. Non- covalent functionalization of carbon nanotubes for highiy specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).  - Non-covalent immobilization - in this case the biomolecule non-covalently adsorbs to the carbon nanotube wall and is weakly adhered to its wall by Van der Walls forces (BECKER, ML; FAGAN, JA; GALLANT, ND; BAUER , BJ; BAJPAI, V.; HOBBIE, EK; LACERDA, SH; MIGLER, KB & JAKUPCIAK, JP Length-dependent uptake of DNA-wrapped single-walled carbon nanotubes Adv.Mater. 19: 939-945. 2007 ; GIGLIOTTI, B.; SAKIZZIE, B .; BETHUNE, DS; SHELBY, RM & CHA, JN Sequence-independent helical wrapping of single-walled carbon nanotubes by long genomic DNA. 6 (2): 159-164. 2006 ; ZHANG, Z .; YANG, X .; ZHANG, Y .; ZENG, B.; WANG, S .; ZHU, T .; RODEN, RBS; CHEN, Y. & YANG, R. Delivery of reverse transcriptase telomerase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth.Clin Cancer Res. 12 (16): 4933-4939. 2006; KAM, NWS & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological functionality. J.Am. Chem. Sound. 127: 6021-6026. 2005; SIRDESHMUKH, R .; TEKER, K. & PANCHAPAKESAN, B. Biological functionalization of carbon nanotubes. Mat. Res. Soc. Symp. Proc. 823: W4.1-1.1-W4.1.6. 2004; CHEN, R.J .; BANGSARUNTIP, S .; DROUVALA-KIS, K.A .; KAM, N.W.S .; SHIM, M .; LI, Y .; KIM.W .; UTZ, P.J. & DAI, H. Non-covalent functionalization of carbon nanotubes for high specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).
O feijoeiro é cultivado em todas as unidades da federação, sendo o quarto produto agrícola em área plantada e o sexto em valor da produção de grãos do país (IBGE - Fundação Instituto Brasileiro de Geografia e Estatística. Levantamento sistemático da produção agrícola: pesquisa men- sal de previsão e acompanhamento das safras agrícolas no ano civil. Rio de Janeiro, v.21 , n.1 , 79p, 2009; ZIMMERMANN, MJ. DE; ROCHA, M.; YAMA- DA, T. Cultura do feijoeiro. Piracicaba: Associação Brasileira para a Pesqui- sa da Potassa e do Fosfato (POTAFOS), 689p. 1988). Entretanto, os Estados do Paraná, Minas Gerais, Bahia e São Paulo são, em ordem decrescente, os principais produtores do país (IBGE - Fundação Instituto Brasileiro de Geografia e Estatística. Levantamento sistemático da produção agrícola: pesquisa mensal de previsão e acompanhamento das safras agrícolas no ano civil. Rio de Janeiro, v.21 , n.1, 79p, 2009). The common bean is grown in all units of the federation, being the fourth agricultural product in planted area and the sixth in value of the country's grain production (IBGE - Brazilian Institute of Geography and Statistics Foundation. Systematic survey of agricultural production: men- salt forecasting and monitoring of the crop in the calendar year Rio de Janeiro, v.21, n.1, 79p, 2009; ZIMMERMANN, MJ DE; ROCHA, M .; YAMADA, T. Bean culture. Piracicaba : Brazilian Association for Research Potash and Phosphate (POTAFOS), 689p. 1988). However, the states of Paraná, Minas Gerais, Bahia and São Paulo are, in decreasing order, the main producers of the country (IBGE - Brazilian Institute of Geography and Statistics Foundation. Systematic survey of agricultural production: monthly forecast and follow-up survey of crops in the calendar year (Rio de Janeiro, v.21, n.1, 79p, 2009).
Dentre os fatores responsáveis por baixar a produtividade do feijoeiro estão as doenças, das quais a ferrugem [Uromyces appendiculatus (Pers.) Unger] é considerada como de grande importância, causando danos da ordem de 45% podendo chegar a 100%, o que vai estar diretamente relacionado à severidade precoce da infecção (SANNAZZARO, A.M.; OLIVEIRA, S.H.F.; WUTKE, E.B.; CASTRO, J.L; GALLO, P.B.; MARTINS, A.L.M.; BORTOLETO, N.; SABINO, J.C.; SILVEIRA, L.C.P.; SAKAI.M.; SAES, L.A.; PAULO, E.M.; KASAI, F.S.; DORNELLES, C.R.F. & BACCHI, G.S. Severi- dade de ferrugem em cultivares de feijoeiro no Estado de São Paulo. Arq.Inst.Biol. 70 (3): 323-329. 2003; COELHO, R.R.; VALE F.X.R.; JESUS JÚNIOR, W.C.; PAUL, P.A.; ZAMBOLIM, L. & BARRETO, R.W. Determinação das condições climáticas que favorecem o desenvolvimento da ferrugem e da mancha angular do feijoeiro. Fitopatol.Bras. 28 (5): 508-514. 2003; JE- SUS JÚNIOR, W.C.; VALE, F.X.R.; COELHO, R.R.; HAU, B.; ZOMBOLIM, L; COSTA, L.C. & BERGAMIN FILHO, A. Effects of angular leaf spot and rust on yield loss of Phaseolus vulgaris. Phytopathology. 91 (11): 1045-1053. 2001 ; FALEIRO, F.G.; RAGAGNIN, V.A.; VINHADELLI, W.S.; MOREIRA, M.A.; STAVELY, J.R. & BARROS, E.G. Resistência de linhagens de feijoeiro a quatro raças de Uromyces appendiculatus isoladas em Minas Gerais, Brasil. Fitopatol.Bras. 26 (1): 77-80. 2001 ; RODRIGUES, F.A.; FERNANDES, J.J. & MARTINS, M. Influência de semeaduras sucessivas de feijoeiro na severidade da mancha-angular e ferrugem e perdas na produção. Pesq. Agropec. Bras. 34 (8): 1373-1378. 1999; HALL, R. Compendium of bean dis- ease. St. Paul: The American Phytopathological Society, 73p. 1991).  Among the factors responsible for lowering bean yield are diseases, of which rust [Uromyces appendiculatus (Pers.) Unger] is considered to be of great importance, causing damage of 45% and may reach 100%, which will be directly related to the early severity of the infection (SANNAZZARO, AM; OLIVEIRA, SHF; WUTKE, EB; CASTRO, JL; GALLO, PB; MARTINS, ALM; BORTOLETO, N .; SABINO, JC; SILVEIRA, LCP; SAKAI.M. ; SAES, LA; PAULO, EM; KASAI, FS; DORNELLES, CRF & BACCHI, GS Severity of rust in common bean cultivars in the State of São Paulo Arq.Inst.Biol. 70 (3): 323-329. 2003; COELHO, RR; VALE FXR; JESUS JÚNIOR, WC; PAUL, PA; ZAMBOLIM, L. & BARRETO, RW Determination of climatic conditions that favor the development of bean rust and angular leaf spot Fitopatol.Bras. 28 (5 ): 508-514. 2003; JESUS JUNIOR, WC; VALLEY, FXR; RABBIT, RR; HAU, B.; ZOMBOLIM, L; COSTA, LC & B ERGAMIN FILHO, A. Effects of angular leaf spot and rust on yield loss of Phaseolus vulgaris. Phytopathology. 91 (11): 1045-1053. 2001; FALEIRO, F.G .; RAGAGNIN, V.A .; VINHADELLI, W.S .; MOREIRA, M.A .; STAVELY, J.R. & BARROS, E.G. Resistance of common bean strains to four Uromyces appendiculatus breeds isolated in Minas Gerais, Brazil. Phytopathol.Bras. 26 (1): 77-80. 2001; RODRIGUES, F.A .; FERNANDES, J.J. & MARTINS, M. Influence of successive bean plantings on angular leaf spot and rust severity and yield losses. Search Agriculture Bras. 34 (8): 1373-1378. 1999; HALL, R. Compendium of bean dis- ease. St. Paul: The American Phytopathological Society, 73p. 1991).
U. appendiculatus é um fungo parasita obrigatório que se caracteriza por apresentar alta variabilidade patogênica. No mundo inteiro, mais de 250 raças já foram identificadas (HALEY, S.D.; MIKLAS, P.N.; AFANA- DOR, L; KELLY, J.D. Random amplified polymorphic DNA (RAPD) marker variability between and within gene pools of common bean. Journal of the American Society for Horticultural Science, 119: 122-125, 1994). No Brasil várias raças fisiológicas diferentes já foram encontradas, confirmando sua variabilidade nas populações deste fungo presentes em nosso país (SAN- NAZZARO, A.M.; OLIVEIRA, S.H.F.; WUTKE, E.B.; CASTRO, J.L; GALLO, P.B.; MARTINS, A.LM.; BORTOLETO, N.; SABINO, J.C.; SILVEIRA, L.C.P.; SAKAI.M.; SAES, LA; PAULO, E.M.; KASAI, F.S.; DORNELLES, C.R.F. & BACCHI, G.S. Severidade de ferrugem em cultivares de feijoeiro no Estado de São Paulo. Arq.lnst.Biol. 70 (3): 323-329. 2003; FALEI RO, F.G.; RA- GAGNIN, V.A.; VINHADELLI, W.S.; MOREIRA, M.A.; STAVELY, J.R. & BARROS, E.G. Resistência de linhagens de feijoeiro a quatro raças de U- romyces appendiculatus isoladas em Minas Gerais, Brasil. Fitopatol.Bras. 26 (1): 77-80. 2001 ; RIOS, G.P.; ANDRADE, E.M. & COSTA, J.LS. Avaliação da resistência de cultivares e linhagens do feijoeiro comum a diferentes populações de Uromyces appendiculatus. Fitopatol.Bras. 26 (2): 128-133. 2001 ; RODRIGUES, F.A.; FERNANDES, J.J. & MARTINS, M. Influência de semeaduras sucessivas de feijoeiro na severidade da mancha-angular e fer- rugem e perdas na produção. Pesq. Agropec. Bras. 34 (8): 1373-1378. 1999). U. appendiculatus is a obligate parasitic fungus characterized by high pathogenic variability. Worldwide, more 250 races have been identified (HALEY, SD; MIKLAS, PN; AFANADOR, L; KELLY, JD) Random amplified polymorphic DNA (RAPD) marker variability between and within common bean pools. Journal of the American Society for Horticultural Science , 119: 122-125, 1994). In Brazil several different physiological races have been found, confirming their variability in the populations of this fungus present in our country (SANNAZZARO, AM; OLIVEIRA, SHF; WUTKE, EB; CASTRO, JL; GALLO, PB; MARTINS, A.LM. ; BORTOLETO, N .; SABINO, JC; SILVEIRA, LCP; SAKAI.M .; SAES, LA; PAULO, EM; KASAI, FS; DORNELLES, CRF & BACCHI, GS Severity of rust in common bean cultivars in São Paulo State 70. (3): 323-329. 2003; FALI RO, FG; RAGAGNIN, VA; VINHADELLI, WS; MOREIRA, MA; STAVELY, JR & BARROS, EG Resistance of bean strains to four breeds of U-romyces appendiculatus isolated in Minas Gerais, Brazil Fitopatol.Bras. 26 (1): 77-80. 2001; RIOS, GP; ANDRADE, EM & COSTA, J.LS. Resistance evaluation of cultivars and strains of common bean to different populations of Uromyces appendiculatus Fitopatol.Bras 26 (2): 128-133 2001; RODRIGUES, FA; FERNANDES, JJ & MARTINS, M. Influence of successive bean sowing the severity of angular leaf spot and rust and production losses. Search Agropec. Bras. 34 (8): 1373-1378. 1999).
O processo de infecção da ferrugem-do-feijão é complexo e depende do reconhecimento por parte do patógeno de estruturas particulares da superfície do hospedeiro. Um apressório deve ser produzido após o con- tato da ponta do tubo germinativo com o lábio do estômato que é a estrutura apropriada à penetração (CORRÊA JR., A. & HOCH, H.C. Identification of thigmoresponsive loci for cell differentiation in Uromyces germlings. Protop- lasma. 186: 34-40. 1995; TERHUNE, B.T.; BOJKO, RJ. & HOCH, H.C. De- formation of stomatal guard cell lips and microfabricated artificial topogra- phies during appressorium formation by Uromyces. Experimental Mycology. 17: 70-78. 1993; WYNN, E.K. Appressorium formation over stomates by the bean rust fungus: Response to a surface contact stimulus. Phytopathol. 66:136-146. 1976). O apressório é então o produto do reconhecimento do sítio de infecção e o seu correto posicionamento no hospedeiro é um estágio crucial para o sucesso da infecção e consequentemente o estabelecimento da doença (ALLEN, E. A., HAZEN, B. E., HOCH, H. C, KWON, Y., LEINHOS, G. M. E., STAPLES, R. C, STUMPF, M. A. & TERHUNE, B. T. Apressorium for- matium in response to topographical signals by 27 rust species. Phytopathol. 81 :323-331. 1991 ; HOCH, H. C. & STAPLES, R. C. Structural and chemical changes among the rust fungi during appressorium formation. Annual Review of Phytopathology. 25:231-247. 1987; STAPLES, R. C, MACKO, V., WYNN, W. K. & HOCH, H. C. Terminology to describe the differentiation response by germlings of fungai spores. Phytopathol. 73: 380. 1983). Desta forma, qualquer interrupção no processo de reconhecimento do sítio de infecção ou desenvolvimento do apressório afeta o estabelecimento da doença. The process of bean rust infection is complex and depends on the pathogen's recognition of particular host surface structures. An appressorium must be produced after contacting the tip of the germ tube with the stomatal lip which is the appropriate structure for penetration (CORRÊA JR., A. & HOCH, HC Identification of thigmoresponsive loci for cell differentiation in Uromyces germlings. - lasma 186: 34-40 1995; TERHUNE, BT; BOJKO, RJ. & HOCH, HC Deformation of stomatal guard cell lips and artificial microfabricated topographies during appressorium formation by Uromyces Experimental Mycology 17: 70- 78. 1993; WYNN, EK Appressorium formation over stomates by the bean rust fungus: Response to a surface contact stimulus Phytopathol. 66: 136-146. 1976). The appressory is therefore the product of recognition of the site of infection and its correct positioning in the host is a crucial stage for the success of the infection and consequently the establishment of the disease (ALLEN, EA, HAZEN, BE, HOCH, H. C, KWON , Y., LEINOS, GME, STAPLES, R. C, STUMPF, MA & TERHUNE, BT Apressorium formalin in response to topographical signals by 27 rust species Phytopathol 81: 323-331. 1991; HOCH, HC & STAPLES , RC Structural and chemical changes among the rust fungi during appressorium formation.Analysis of Phytopathology.25: 231-247. 1987; STAPLES, R. C, MACKO, V., WYNN, WK & HOCH, HC Terminology to describe the differentiation response by germlings of fungal spores (Phytopathol 73: 380, 1983). Thus, any interruption in the process of recognition of the site of infection or development of the appressorium affects the establishment of the disease.
Durante o desenvolvimento de um apressório em ferrugens, vários genes são transcritos (KULKARNI, R.D. & DEAN, R.A. Identification of proteins that interact with two regulators of appressorium development, ade- nylate cyclase and cAMP-dependent protein kinase A, in the rice blast fungus Magnaporthe grisea. Mol. Gen. Genomics. 270: 497-508. 2004; KWON, Y.H.; HOCH, H.C. & AIST, J.R. Initiation of appressorium formation in Uro- myces appendiculatus: organization of the apex, and the responses involving microtubules and apical vesicles. Can. J. Bot. 69: 2560-2573. 1991). Em U. appendiculatus (Pers.:Pers.) Ungler diversos estudos foram realizados, tentando investigar moléculas envolvidas no processo de diferenciação do tubo germinativo em apressório (YANIV, Z. & STAPLES, R. C. The purification and properties of the aminoacyltRNA from bean rust urediniospores. Biochem. Biophys. Acta. 232:717-725. 1971 ; RAMAKRISHNAN, L. & STAPLES, R. C. Evidence for a template RNA in resting urediniospores of the bean rust fun- gus. Contrib. B. Thompson Instit. 24:1197-1202. 1970). Uma família de genes expressos entre o período coincidente com a formação do apressório foi caracterizada (XUEI, X.; BHAIRI, S.; STAPLES, R.C. & YODER, O.C. Cha- racterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus. Gene. 110: 49-55. 1992; BHAIRI, S.M.; STAPLES, R.C.; FREVE, P. & YODER, O.C. Characterization of an infection structure-specific gene from the rust fungus, Uromyces appendiculatus. Gene. 81 : 237-243. 1989). Entretanto, estudos de função e identificação desses genes são de difícil execução, uma vez que o isolamento tradicional de transform antes para características de infecção gera obrigatoriamente células incapazes de infectar, o que no caso de parasitas obrigatórios resulta em letalidade, pois estes fungos se multiplicam apenas nos tecidos vivos do hospedeiro. During the development of a rust appressorium, several genes are transcribed (KULKARNI, RD & DEAN, RA Identification of proteins that interact with two regulators of appressorium development, adenylate cyclase and cAMP-dependent protein kinase A, in the rice blast fungus). Magnaporthe grisea Mol. Gen. Genomics 270: 497-508 2004; KWON, YH; HOCH, HC & AIST, JR Initiation of appressorium formation in Uroomyces appendiculatus: organization of the apex, and the responses involving microtubules and apical Canes J. Bot. 69: 2560-2573. 1991). In U. appendiculatus (Pers.:Pers.) Ungler several studies have been performed, trying to investigate molecules involved in the process of differentiation of the germinal tube in appressorium (YANIV, Z. & STAPLES, RC. The purification and properties of the aminoacyltRNA from bean rust urediniospores. Biochem Biophys Acta 232: 717-725 1971 RAMAKRISHNAN, L. & STAPLES, RC Evidence for an RNA template in resting urediniospores of the bean rust fungi Contrib. 1202. 1970). A family of genes expressed between the period coincident with the formation of the appressorium was characterized (XUEI, X .; BHAIRI, S .; STAPLES, RC & YODER, OC. Characterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus. Gene 110: 49-55 1992; BHAIRI, SM; STAPLES, RC; FREVE, P. & YODER, OC Characterization of a structure-specific gene infection from the rust fungus, Uromyces appendiculatus. Gene. 81: 237-243. 1989). However, studies of function and identification of these genes are difficult to perform, since the traditional isolation of transform prior to infection characteristics necessarily generates cells unable to infect, which in the case of obligate parasites results in lethality, as these fungi multiply only in the living tissues of the host.
A técnica de oligonucleotídeo anti-senso é usada extensivamen- te como ferramenta para inibir a expressão de mRNA alvos e permitir a elucidação da função de genes, ambos in vitro e in vivo (OEKELEN, D.V.; LUY- TEN, W.H.M.L. AND LEYSEN, J.E. Ten years of antisense inhibition of brain G-protein-coupled receptor function. Brain Res. Rev. 42: 123-142. 2003). Foi demonstrado previamente, por um membro da equipe que apresenta este pedido de patente, que a microinjeção de sequências em anti-senso do gene INF24 resulta na inibição da formação de apressórios de U. appendiculatus in vitro (BARJA, F.; CORRÊA JR., A.; STAPLES, R.C. AND HOCH, H.C. Mi- croinjected antisense Inf 24 oligonucleotides inhibit appressorium develop- ment in Uromyces. Mycol.Res. 102 (12): 1513-1518. 1998). No entanto, a técnica de microinjeção é de difícil execução e com baixa rentabilidade, o que inviabiliza a sua utilização em protocolos de controle de doença.  The antisense oligonucleotide technique is extensively used as a tool to inhibit expression of target mRNAs and to enable elucidation of gene function both in vitro and in vivo (OEKELEN, DV; LUYTEN, WHML AND LEYSEN, JE). Ten years of antisense inhibition of brain G-protein-coupled receptor function (Brain Res. Rev. 42: 123-142. 2003). It has been previously demonstrated by a member of the team filing this patent that microinjection of antisense sequences of the INF24 gene results in inhibition of U. appendiculatus appressorium formation in vitro (BARJA, F .; CORRÊA JR. STAPLES, RC AND HOCH, HC Microinjected antisense Inf 24 oligonucleotides inhibit appressorium development in Uromyces. Mycol Res. 102 (12): 1513-1518 (1998). However, the microinjection technique is difficult to perform and with low profitability, which makes its use in disease control protocols impossible.
É conhecida a propriedade de nanotubos de carbono de parede única (SWNT) ou de múltiplas paredes (MWNT) em penetrar no interior de células de mamíferos, plantas, bactérias e fungos (LIU, Q.; CHEN, B.; WANG, Q.; SHI, X.; XIAO, Z.; LIN, J. & FANG, X. Carbon Nanotubes as Molecular Transporters for Walled Plant Celis. Nano Lett. 9 (3): 1007-1010. 2009; KOSTARELOS, K.; LACERDA, L; PASTORIN, G.; WU, W.; WIECK- OWSKI, S.; LUANGSIVILAY, J.; GODEFROY, S.; PANTAROTTO, D.; BRIAND, J.; MULLER, S.; PRATO, M. & BIANCO, A. Cellular uptake of func- tionalized carbon nanotubes is independent of functional group and cell type. Nature. 2: 108-113. 2007; KAM, N.W.S.; LIU, Z. & DAÍ, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J.Am.Chem.Soc. 127: 12492- 12493. 2005; KAM, N.W.S.; JESSOP, T.C.; WENDER, RA. & DAI, H. Nano- tube molecular transporters: Internalization of carbon nanotube-protein con- jugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004). Essa propriedade permite a utilização dessa ferramenta como promissores carre- adores de biomoléculas para o interior de células vivas e o estudo de sua função. The property of single wall (SWNT) or multiple wall (MWNT) carbon nanotubes is known to penetrate into mammalian, plant, bacterial and fungal cells (LIU, Q .; CHEN, B .; WANG, Q. ; SHI, X .; XIAO, Z .; LIN, J. & FANG, X. Carbon Nanotubes as Molecular Transporters for Walled Plant Celis. Nano Lett. 9 (3): 1007-1010. 2009; KOSTARELOS, K .; LACERDA; L; PASTORIN, G .; WU, W .; WIECK-OWSKI, S.; LUANGSIVILAY, J .; GODEFROY, S .; PANTAROTTO, D .; BRIAND, J .; MULLER, S .; PRATO, M. &. BIANCO, A. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type Nature 2: 108-113. 2007; KAM, NWS; LIU, Z. & DAI, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J.Am.Chem.Soc. 127: 12492-124949. 2005; KAM, NWS; JESSOP, TC; WENDER, RA. & DAI, H. Nano-molecular tube transporters: Internalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004). This property allows the use of this tool as promising biomolecule loaders into living cells and the study of their function.
Moléculas biológicas, tais como proteína e ácidos nucléicos, interagem com facilidade à superfície de NTC e têm sido usadas como surfac- tantes dessas nanoestruturas com sucesso (ZHAO, X. & JOHNSON, J.K. Simulation of adsorption of DNA on carbon nanotubes. J.Am.Chem.Soc. 129: 10438-10445. 2007; MALIK, S.; VOGEL, S; RÕSNER, H.; ARNOLD, K.; HENNRICH, R; KOHLER, A.; RICHERT, C. & KAPPES, M.M. Physical Chemical characterization of DNA-SWNT suspensions and associated composites. Composites Science and Technology. 67: 916-921. 2007; GIGLIOTTI, B.; SAKIZZIE, B.; BETHUNE, D.S.; SHELBY, R.M. & CHA, J.N. Sequence- independent helical wrapping of single-walled carbon nanotubes by long ge- nomic DNA. Nanoletters. 6 (2): 159-164. 2006; KAM, N.W.S.; LIU, Z. & DAÍ, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J.Am.Chem.Soc. 127: 12492-12493. 2005; KAM, N.W.S. & DAI, H. Carbon nanotubes as intracellular protein transporters: generality and biological func- tionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, N.W.S.; JESSOP, T.C.; WENDER, RA. & DAI, H. Nanotube molecular transporters: Internaliza- tion of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004; CHEN, R.J.; BANGSARUNTIP, S.; DROUVALAKIS, KA; KAM, N.W.S.; SHIM, M.; LI, Y; KIM,W.; UTZ, PJ. & DAI, H. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).  Biological molecules, such as protein and nucleic acids, interact easily with the surface of NTC and have been successfully used as surfactants of these nanostructures (ZHAO, X. & JOHNSON, JK. Simulation of DNA adsorption on carbon nanotubes. J.Am 129: 10438-10445 2007; MALIK, S.; VOGEL, S.; ROSNER, H.; ARNOLD, K.; HENNRICH, R.; KOHLER, A.; RICHERT, C. & KAPPES, MM Physical Chemical characterization of DNA-SWNT suspensions and associated composites Composites Science and Technology 67: 916-921 2007. GIGLIOTTI, B; SAKIZZIE, B; BETHUNE, DS; SHELBY, RM & CHA, JN Sequence-independent helical wrapping of single-walled carbon nanotubes by long genomic DNA Nanoletters 6 (2): 159-164. 2006; KAM, NWS; LIU, Z. & DAI, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing J.Am.Chem.Soc 127: 12492-12493 2005; KAM, NWS & DAI, H. Carbon nanotubes as intr acellular protein transporters: generality and biological functionality. J.Am.Chem.Soc. 127: 6021-6026. 2005; KAM, N.W.S .; JESSOP, T.C .; WENDER, RA. & DAI, H. Nanotube molecular transporters: Internationalization of carbon nanotube-protein conjugates into mammalian cells. J.Am.Chem.Soc. 126: 6850-6851. 2004; CHEN, R.J .; BANGSARUNTIP, S .; DROUVALAKIS, KA; KAM, N.W.S .; SHIM, M .; LI, Y; KIM, W .; UTZ, PJ. & DAI, H. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. PNAS. 100 (9): 4984-4989. 2003).
Foram encontradas algumas patentes relacionadas à invenção: Some patents related to the invention were found:
O pedido de patente WO2005104179 descreve o uso de nano- tubos de carbono para análises de amostras químicas e biológicas, porém não relata o uso de nanotubos para introduzir substâncias no interior do citoplasma de células. Patent application WO2005104179 describes the use of carbon nanotubes for analysis of chemical and biological samples, but does not report the use of nanotubes to introduce substances into the cytoplasm of cells.
O pedido de patente WO2006078640 relata a introdução de substâncias em células causando alteração gênica, entretanto, não são utili- zados nanotubos de carbono.  Patent application WO2006078640 reports the introduction of substances into cells causing gene alteration, however, carbon nanotubes are not used.
Todavia, não foi descrito no estado da técnica a inibição de fito- patógenos utilizando oligonucleotídeos conjugados com nanotubos de carbono.  However, inhibition of phytopathogens using carbon nanotube conjugated oligonucleotides has not been described in the art.
PROBLEMAS DO ESTADO DA TÉCNICA  TECHNICAL STATE PROBLEMS
O controle da ferrugem é realizado através da utilização de cultivares resistentes à doença, práticas culturais e/ou a aplicação de defensivos agrícolas. A existência de uma grande variabilidade de raças de U. appendi- culatus circulante dificulta o controle da ferrugem. Esse fato, associado à utilização de defensivos agrícolas que levam a seleção de microrganismos fitopatogênicos resistentes aos compostos disponíveis mostra a necessidade de se buscar alternativas mais eficazes e menos poluentes para o controle da ferrugem em vegetais.  Rust control is accomplished through the use of disease resistant cultivars, crop practices and / or the application of pesticides. The existence of a large variability of circulating U. appendi- culatus breeds makes rust control difficult. This fact, associated with the use of pesticides that lead to the selection of phytopathogenic microorganisms resistant to available compounds shows the need to look for more effective and less polluting alternatives for the control of rust in plants.
VANTAGENS DA TECNOLOGIA TECHNOLOGY ADVANTAGES
A compreensão dos mecanismos de patogênese e sua manipu- lação abrem caminho para a obtenção de estratégias que permitem uma solução mais específica e menos impactante para o meio ambiente.  Understanding the pathogenesis mechanisms and their manipulation opens the way to obtain strategies that allow a more specific and less impacting solution for the environment.
A internalização de oligonucleotídeos anti-senso, através da utilização do conjugado nanotubo-oligonucleotídeo, representa uma alternativa eficiente para análise funcional de genes em fungos filamentosos, especial- mente daqueles que apresentam limitações com as técnicas convencionais, bem como uma ferramenta para o desenvolvimento de novas estratégias de controle da doença no campo.  The internalization of antisense oligonucleotides, using the nanotube-oligonucleotide conjugate, represents an efficient alternative for functional analysis of genes in filamentous fungi, especially those with limitations with conventional techniques, as well as a tool for the development of new disease control strategies in the field.
BREVE DECRIÇÃO DAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1 - Ultramicrografias de Microscopia de Força Atómica de (a) INF24 anti-senso adsorvidos em nanotubos de carbono e (b) oligonucleotídeos INF24 senso adsorvidos a nanotubos de carbono.  Figure 1 - Atomic Force Microscopy Ultrasound of (a) INF24 antisense adsorbed to carbon nanotubes and (b) INF24 sense oligonucleotides adsorbed to carbon nanotubes.
Figura 2 - Folhas de feijoeiro inoculadas com o fungo Uromyces appendiculatus após tratamento com (a) nanotubo de carbono, (b) água, (c) oligonucieotídeos INF24 anti-senso adsorvidos em nanotubos de carbono e (d) oligonucieotídeos INF24 senso adsorvidos a nanotubos de carbono. Figure 2 - Bean leaves inoculated with the fungus Uromyces appendiculatus after treatment with (a) carbon nanotube, (b) water, (c) INF24 antisense oligonucleotides adsorbed on carbon nanotubes and (d) INF24 sensory oligonucleotides adsorbed on carbon nanotubes.
DESCRIÇÃO DETALHADA DA TECNOLOGIA DETAILED DESCRIPTION OF TECHNOLOGY
A presente invenção relata um processo de imobilização não covalente de uma sequência especifica de genes a nanotubos de carbono (NTC) produzindo assim um sistema híbrido oligonucleotídeos-nanotubo de carbono para ser utilizado como agente de internalização gênica no combate de fitopatógenos. O objetivo desta internalização é permitir que o oligonu- cleotídeo passe para o citoplasma do patôgeno e interfira nos mecanismos de síntese protéica regulada pelo RNA mensageiro do patógeno que como resultado leva a inibição de estruturas de infecção, causando a morte do patôgeno ou diminuição dos efeitos nocivos do agressor ao hospedeiro.  The present invention relates a non-covalent immobilization process of a specific gene sequence to carbon nanotubes (NTC) thus producing a carbon nanotube-oligonucleotide hybrid system for use as a gene internalizing agent in the fight against plant pathogens. The purpose of this internalization is to allow the oligonucleotide to pass into the pathogen's cytoplasm and interfere with the mechanisms of protein synthesis regulated by the pathogen's messenger RNA, which as a result leads to inhibition of infection structures, causing pathogen death or diminished effects. harm from the aggressor to the host.
Este sistema híbrido conjugado possui alta bioatividade, alta bio- seletividade e especificidade uma vez que é construído pela imobilização de um oligonucleotídeo específico ao nanotubo de carbono.  This conjugated hybrid system has high bioactivity, high selectivity and specificity as it is constructed by immobilizing a specific oligonucleotide to the carbon nanotube.
Como modelo biológico para comprovar e aferir a eficácia da invenção utilizou-se o fungo fitopatogênico Uromyces appendiculatus (Pers.: Pers.) Ungler que é um fungo parasita obrigatório, causador da ferrugem-do- feijoeiro (Phaseolus vulgaris L.).  As a biological model to prove and measure the effectiveness of the invention we used the phytopathogenic fungus Uromyces appendiculatus (Pers .: Pers.) Ungler which is a obligate parasitic fungus that causes bean rust (Phaseolus vulgaris L.).
A presente invenção pode ser mais bem entendida através dos seguintes exemplos, não limitantes de tecnologia:  The present invention may be better understood by the following non-limiting examples of technology:
Exemplo 1 : Preparação dos conjugados oligonucleotídeo-NTC Example 1: Preparation of oligonucleotide-NTC Conjugates
Os conjugados oligonucleotídeo e NTC foram obtidos a partir de uma solução de oligonucleotídeo INF24 e NTC funcionalizados (SWNTf ou MWNTf) que foi sonicada em ultra-som de banho por trinta minutos à temperatura ambiente e armazenada à -20 °C até a utilização nos experimentos. Exemplo 2: Análise de Microscopia de Força Atómica dos conjugados  Oligonucleotide and NTC conjugates were obtained from a functionalized INF24 and NTC oligonucleotide solution (SWNTf or MWNTf) which was sonicated in bath ultrasound for 30 minutes at room temperature and stored at -20 ° C until use in the experiments. . Example 2: Atomic Force Microscopy Analysis of Conjugates
As análises das ultramicrografias de Microscopia de Força Atô- mica (Figura 1) demonstraram que SWNTf conjugados com oligonucleotídeo INF24 apresentaram em sua superfície estruturas helicoidais dispostas ordenadamente ao logo de sua superfície longitudinal compatível com a ad- sorção dos oligonucleotídeos sobre a estrutura de carbono. Estas estruturas não são observadas em nanotubos não conjugados com os oligonucleotídeos. Atomic Force Microscopy ultramicrograph analyzes (Figure 1) demonstrated that SWNTf conjugated to INF24 oligonucleotide showed helical structures arranged neatly along their longitudinal surface compatible with oligonucleotide sorption on the carbon structure. These structures are not observed in nanotubes not conjugated with oligonucleotides.
Exemplo 3: Teste in vitro de inibição de apressórios  Example 3: In vitro Appearance Inhibition Test
A suspensão do conjugado oligonucleotídeo INF24 e NTC Suspension of oligonucleotide conjugate INF24 and NTC
(SWNTf ou MWNTf) foi vertida sobre urediniosporos depositados em membranas plásticas contendo topografia indutiva ou discos de folhas de feijoeiro. Os substratos inoculados foram incubados por 4 h a 18 °C, fixados e o número de apressórios formados foi determinado em 100 tubos germinati- vos. No caso dos discos de folhas de feijoeiro o número de apressórios foi determinado em tubos germinativos escolhidos aleatoriamente e destes, os tubos germinativos que cresceram sobre estômatos. (SWNTf or MWNTf) was poured onto urediniospores deposited on plastic membranes containing inductive topography or bean leaf discs. Inoculated substrates were incubated for 4 h at 18 ° C, fixed and the number of appressoria formed in 100 germ tubes. In the case of bean leaf discs the number of appressoria was determined in randomly selected germ tubes and from these, the germ tubes that grew on stomata.
Exemplo 4 - Nanotubo puro (SWNTf) Example 4 - Pure Nanotube (SWNTf)
Urediniosporos de U. appendiculatus tratados com dispersões de SWNT com modificações químicas (SWNTf) germinaram e desenvolve- ram-se adequadamente sobre substrato plástico, mostrando eficiente formação de apressórios de 53,00 % ± 6,56 % e 46,67 % ± 3,05 %, não apresentando diferença estatística significante entre si e o controle (48,67 % ± 3,79 %). Além disso, não foi observado efeito deletério sobre a germinação e vi- abilidade dos tubos germinativos tratados com os diferentes SWNTf. A taxa de germinação, comprimento do tubo germinativo e sua morfologia não diferiram do controle.  U. appendiculatus urediniospores treated with chemically modified SWNT dispersions (SWNTf) germinated and developed properly on plastic substrate, showing efficient appressorium formation of 53.00% ± 6.56% and 46.67% ± 3 , 05%, with no statistically significant difference between themselves and the control (48.67% ± 3.79%). In addition, no deleterious effect on germination and viability of germ tubes treated with the different SWNTf was observed. Germination rate, germ tube length and morphology did not differ from control.
Exemplo 5 - Nanotubo puro (MWNTf)  Example 5 - Pure Nanotube (MWNTf)
Urediniosporos de U. appendiculatus tratados com MWNTf germinaram e cresceram sobre substrato plástico contendo ranhuras e iniciaram a formação de apressórios após contato com a topografia indutiva como relatado previamente e de maneira similar ao observado para tratamentos com SWNTf. O percentual médio de formação de apressórios em U. appendiculatus incubados no controle (água destilada) e no tratamento de MWNTf foi de 27,33 % ± 9,35 % e 23,33 % ± 7,71 % respectivamente. Nenhuma interferência foi observada nos eventos que se processam durante a formação de apressórios após tratamento com MWNTf. Exemplo 6 - Oligonucleotídeos puros MWNTf-treated U. appendiculatus urediniospores germinated and grown on groove-containing plastic substrate and began to form appressoria after contact with inductive topography as previously reported and similar to that observed for SWNTf treatments. The average percentage of appressiculus formation in U. appendiculatus incubated in control (distilled water) and MWNTf treatment was 27.33% ± 9.35% and 23.33% ± 7.71% respectively. No interference was observed in the events that occur during the formation of appressoria after treatment with MWNTf. Example 6 - Pure Oligonucleotides
Urediniosporos de U. appendiculatus tratados somente com oligonucleotídeos INF24 senso e anti-senso emitiram tubo germinativo, cresceram sobre substrato plástico e desenvolveram apressórios, 42,67 % ± 7,02 %, e 49,67 % ± 3,79 % respectivamente. Ambos não diferiram estatisticamente do controle e do tratado com SWNTf. Esses resultados estão em a- cordo com relatos na literatura, uma vez que, oligonucleotídeo anti-senso adicionado diretamente no meio de cultura não apresenta atividade biológica (BENNET & COWSERT, 1999).  U. appendiculatus urediniospores treated only with INF24 sense and antisense oligonucleotides emitted germ tube, grew on plastic substrate and developed appressoria, 42.67% ± 7.02%, and 49.67% ± 3.79% respectively. Both did not differ statistically from control and SWNTf treated. These results are in accordance with reports in the literature, since antisense oligonucleotide added directly to the culture medium has no biological activity (BENNET & COWSERT, 1999).
Exemplo 7 - MWNTf conjugados com oligonucleotídeo INF24 anti-senso Example 7 - MWNTf Conjugated with Antisense INF24 Oligonucleotide
Urediniosporos de U. appendiculatus tratados com MWNTf conjugados com oligonucleotídeo INF24 senso apresentaram valores médios de formação de apressórios de 29,17 % ± 8,06 %; 32,33 % ± 17,98 %; 26,33 % ± 9,62 % e 26,50 % ± 8,04 %, nas proporções de 1 :0,5; 1 :1 ; 1 :2 e 1 :3 res- pectivamente. Contudo, estes valores não diferiram estatisticamente entre si, do controle e do MWNT.  UWN appendiculatus urediniospores treated with INF24 sense oligonucleotide conjugated mean appressorium formation values of 29.17% ± 8.06%; 32.33% ± 17.98%; 26.33% ± 9.62% and 26.50% ± 8.04%, in the proportions of 1: 0.5; 1: 1; 1: 2 and 1: 3 respectively. However, these values did not differ statistically from each other, from control and from MWNT.
Os urediniosporos tratados com MWNTf conjugados com oligonucleotídeo INF24 anti-senso tiveram a formação de apressório inibida. Nas proporções utilizadas, de 1 :0,5; 1 :1 ; 1 :2 e 1 :3 (MWNTf: I F24), os valores médios de formação de apressórios observados foram de 28,33 % ± 11 ,27 %; 13,50 % ± 7,20 %; 10,83 % ± 4,71 % e 2,17 % ± 2,04 %, respectivamente. O tratamento de 1 :1 apresentou diferença significativa (P<0,05) quando comparado com os tratamentos controle, MWNTf e 1 :0,5. No tratamento de 1 :2, observou-se diferença estatística significante quando comparado com o controle e 1 :0,5 (P<0,01) e MWNTf (P<0, 05).  MWNTf-treated urediniospores conjugated with antisense INF24 oligonucleotide had inhibited appressor formation. In the ratios used, 1: 0.5; 1: 1; 1: 2 and 1: 3 (MWNTf: I F24), the mean values of appressorium formation observed were 28.33% ± 11.27%; 13.50% ± 7.20%; 10.83% ± 4.71% and 2.17% ± 2.04%, respectively. The 1: 1 treatment showed a significant difference (P <0.05) when compared to the control treatments, MWNTf and 1: 0.5. In the 1: 2 treatment, a statistically significant difference was observed when compared to the control and 1: 0.5 (P <0.01) and MWNTf (P <0.05).
O tratamento na proporção de 1 :3 apresentou o menor percentual de formação de apressórios e diferiu do controle, MWNTf e 1 :0,5 (P<0,001). A proporção 1 :0,5 não diferiu significativamente dos tratamentos controle e MWNTf. Os dados mostram que a proporção entre NTC e oligo- nucleotídeos influencia a eficiência da inibição da formação de estruturas de infecção. Não se observou em nenhum dos tratamentos sinal de morbidade celular (Granulação citoplasmática, vacuolização etc). Exemplo 8 - MWNTf conjugados com oligonucleotídeo INF24 anti-senso sobre discos de folha The 1: 3 treatment presented the lowest percentage of appressory formation and differed from the control, MWNTf and 1: 0.5 (P <0.001). The 1: 0.5 ratio did not differ significantly from control and MWNTf treatments. The data show that the ratio between NTC and oligonucleotides influences the efficiency of inhibiting the formation of infection structures. No sign of cell morbidity was observed in any of the treatments (cytoplasmic granulation, vacuolization, etc.). Example 8 - MWNTf Conjugated with Antisense Ol24 Nucleotide on Leaf Discs
Urediniosporos de U. appendiculatus tratados com MWNTf conjugado com INF24 senso apresentaram valores médios de formação de a- pressórios de 21 ,3 % ± 3,05 %. Já para aqueles que apresentaram contato com os estômatos os valores percentuais médios de formação de apressó- rios foi de 69,7 % ± 2,76 %. Estes valores não diferiram estatisticamente do controle (água destilada), INF24S, INF24RC e do MWNTf.  U. appendiculatus urediniospores treated with INF24 sense-conjugated MWNTf showed mean values of 21.3% ± 3.05%. For those who had contact with the stomata the average percentage values of appressoric formation was 69.7% ± 2.76%. These values did not differ statistically from control (distilled water), INF24S, INF24RC and MWNTf.
No tratamento com MWNTf conjugado com INF24 anti-senso foi observado o menor percentual de formação de apressórios, com valores de 4,0 % ± 4,58 % e de 14,6 % ± 16,28 %, para o número de apressórios formados para o total de tubos germinativos e para os apressórios formados após o contato do tubo germinativo com o lábio do estômato respectivamente. Nas duas situações foi observado um efeito inibitório na formação de apressório de maneira similar ao descrito previamente (BARJA et al., 1998) e os valores de formação de apressórios diferiu dos observados nos tratamentos controle, INF24RC e MWNTf conjugado com INF24 senso (P<0,05) e do MWNTf (P<0,01) e dos tratamentos controle, INF24RC, MWNTf e MWNTf conjuagados com INF24 senso (P<0,001), respectivamente para as conta- gens aleatórias e aquelas que apresentaram contato com estômatos.  In the treatment with anti-sense INF24 conjugated MWNTf, the lowest percentage of appressorium formation was observed, with values of 4.0% ± 4.58% and 14.6% ± 16.28%, for the number of appressories formed. for total germ tubes and appressories formed after germ tube contact with stomatal lip respectively. In both situations, an inhibitory effect on appressorium formation was observed in a similar manner to that previously described (BARJA et al., 1998) and the values of appressorium formation differed from those observed in the control, INF24RC and MWNTf treatments combined with INF24 sense (P < 0.05) and MWNTf (P <0.01) and control treatments, INF24RC, MWNTf and MWNTf combined with INF24 sense (P <0.001), respectively for random counts and those with stomata contact.
Exemplo 9 - SWNTf conjugados com oligonucleotídeo INF24 senso Example 9 - SWNTf Conjugated with INF24 Sense Oligonucleotide
Urediniosporos de U. appendiculatus tratados com SWNTf conjugados com oligonucleotídeo INF24 senso apresentaram valores médios de formação de apressórios de 51 ,44 % ± 9,00 %; 56,00 % ± 5,78 %; 51 ,78 % ± 4,55 % e 49,11 % ± 4,00 %, nas doses de 50; 25; 12,5 e 6,25 % (v/v) respectivamente. Contudo, estes valores não diferiram estatisticamente entre si, do controle e do SWNTf.  U24 appendiculatus urediniospores treated with INF24 sense oligonucleotide conjugate showed mean appressorium formation values of 51.44% ± 9.00%; 56.00% ± 5.78%; 51, 78% ± 4.55% and 49.11% ± 4.00%, at doses of 50; 25; 12.5 and 6.25% (v / v) respectively. However, these values did not differ statistically from each other, from control and from SWNTf.
Exemplo 10 - SWNTf conjugados com oligonucleotídeo INF24 anti-senso  Example 10 - SWNTf conjugated with antisense INF24 oligonucleotide
Entretanto quando tratamos urediniosporos com SWNTf conju- gados com oligonucleotídeo INF24 anti-senso em diferentes concentrações tiveram a formação de apressório inibida. Também se observou um efeito dose dependente nas concentrações usadas. Os tratamentos de 50 e 12,5 % (v/v) apresentaram valores médios de formação de apressórios de 36,00 % ± 4,98 % e 37,89 % ± 8,38 %, respectivamente. Estes tratamentos apresentaram diferença significativa (P<0,05) quando comparados com os tratamentos controle, SWNTf e 6,25 % (v/v). However, when treating urediniospores with SWNTf conjugated with antisense INF24 oligonucleotide at different concentrations, suppressive formation was inhibited. A dose dependent effect was also observed on the concentrations used. The 50 and 12.5 treatments % (v / v) presented average values of appressory formation of 36.00% ± 4.98% and 37.89% ± 8.38%, respectively. These treatments presented significant difference (P <0.05) when compared with the control treatments, SWNTf and 6.25% (v / v).
No tratamento de 25 % (v/v), observou-se o menor valor médio de formação de apressório, 31 ,44 % ± 1 ,83 %, que apresentou diferença estatisticamente significativa quando comparado com o controle, SWNTf e 6,25 % (P<0,01). A dose de 6,25 % (v/v) apresentou valor médio de apressórios formados de 52,67 % ± 3,46 % e não diferiu significativamente do controle e SWNTf. Os dados mostraram que conjugados SWNTÍ-INF24RC foram eficazes na inibição da formação de apressórios em U. appendiculatus.  In the 25% (v / v) treatment, the lowest mean value of appressorium formation was observed, 31, 44% ± 1, 83%, which showed a statistically significant difference when compared to the control, SWNTf and 6.25%. (P <0.01). The 6.25% (v / v) dose presented mean value of formed appressoria of 52.67% ± 3.46% and did not differ significantly from control and SWNTf. The data showed that SWNTI-INF24RC conjugates were effective in inhibiting the formation of appressories in U. appendiculatus.
Exemplo 11 : Teste de inibição de U. appendiculatus  Example 11: U. appendiculatus inhibition test
In vivo, urediniosporos de U. appendiculatus infectam as plantas de feijão e desenvolvem lesões na superfície das folhas 7 dias após a inoculação. Pontos amarelados, característicos da fase inicial do desenvolvimento da doença são observados nesse período. Esses pontos evoluem para lesões pulverulentas, de coloração marrom, coalescentes e com liberação dos urediniosporos de U. appendiculatus em plantas suceptíveis e sem tra- tamento de controle.  In vivo, U. appendiculatus urediniospores infect bean plants and develop leaf surface lesions 7 days after inoculation. Yellow spots, characteristic of the early stage of disease development are observed during this period. These points evolve to coalescent, brown-colored, powdery lesions with release of U. appendiculatus urediniospores in susceptible plants without control treatment.
A inibição da ferrugem causada por U. appendiculatus foi realizada vertendo suspensão do conjugado oligonucleotídeo INF24 e NTC sobre a face adaxial de folhas primárias de plantas de feijão 24 h antes da inoculação de urediniosporos. As plantas inoculadas foram incubadas sob molha- mento foliar por 18 h a temperatura ambiente, após o aparecimento dos sintomas características da doença o número de lesões foi determinado. Esses pontos evoluem para lesões pulverulentas, de coloração marrom, coalescentes e com liberação dos urediniosporos de U. appendiculatus em plantas suceptíveis e sem tratamento de controle. Esses sintomas também são ob- servados em plantas tratadas com MWNTf e MWNTf conjugado com INF24 senso e inoculadas com urediniosporos de U. appendiculatus, mostrando que esses tratamentos não interferem no processo de desenvolvimento da infecção das plantas. Inhibition of rust caused by U. appendiculatus was performed by pouring suspension of the oligonucleotide conjugate INF24 and NTC onto the adaxial face of primary bean plant leaves 24 h before urediniospore inoculation. Inoculated plants were incubated under leaf wetness for 18 h at room temperature. After the onset of symptoms characteristic of the disease, the number of lesions was determined. These points evolve to coalescent, brown-colored powdery lesions with release of U. appendiculatus urediniospores in susceptible plants without control treatment. These symptoms are also observed in plants treated with sense-conjugated MWNTf and INFWN-conjugated MWNTf and inoculated with U. appendiculatus urediniospores, showing that these treatments do not interfere with the developmental process. plant infection.
Na avaliação visual (Figura 2) das folhas de feijoeiro, as plantas tratadas com MWNTf (a), controle (b) e MWNTf conjugadas com INF24 senso (d) apresentaram quantidade de pústulas/folha de 356,7 ± 367,54; 261 ,0 ± 212,87 e 131 ,8 ± 61 ,97, respectivamente. No entanto, observamos uma diminuição significativa da quantidade da doença nas plantas tratadas com MWNTf conjugada com INF24 anti-senso (Figura 2c), com valor médio de 31 ,5 ± 36,47, que apesar de menor, quando comparados com as folhas tratadas com MWNTf e MWNTf conjugada com INF24 senso ou Controle, não diferiu estatisticamente desses tratamentos. Adicionalmente nenhum efeito tóxico foi observado nas plantas tratadas com MWNTf e os seus conjugados com INF24. As plantas não apresentaram manchas cloróticas, lesões na lâmina foliar ou senescência das folhas tratadas até 8 dias após o tratamento.  In the visual evaluation (Figure 2) of bean leaves, plants treated with MWNTf (a), control (b) and MWNTf conjugated with INF24 sense (d) presented a quantity of pustules / leaf of 356.7 ± 367.54; 261.0 ± 212.87 and 131.8 ± 61.97, respectively. However, we observed a significant decrease in the amount of disease in plants treated with anti-sense INF24-conjugated MWNTf (Figure 2c), with a mean value of 31.5 ± 36.47, which, although smaller, when compared to the treated leaves. with MWNTf and MWNTf conjugated with sense or Control INF24 did not differ statistically from these treatments. Additionally no toxic effects were observed in MWNTf-treated plants and their conjugates with INF24. The plants showed no chlorotic spots, leaf blade lesions or senescence of the leaves treated until 8 days after treatment.

Claims

REIVINDICAÇÕES
1- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, caracterizada por compreender a conjugação de oligonucleotídeos com nano- tubos de carbono.  1- CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES, characterized by the conjugation of oligonucleotides with carbon nanotubes.
2- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com a reivindicação 1 , caracterizado pelos nanotubos serem selecio- nados do grupo compreendendo SWNTf ou MWNTf.  CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES OF VEGETABLES according to claim 1, characterized in that the nanotubes are selected from the group comprising SWNTf or MWNTf.
3- CONJUGADO DE NANOTUBOS DE CARBONO PARA INI¬ 3- CARBON NANOTUBE CONJUGATE FOR INI¬
BIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com as reivindicações 1 e 2, caracterizada pelos nanotubos apresentarem em sua superfície estruturas helicoidais dispostas ordenadamente ao logo de sua superfície longitudinal compatível com a adsorção dos oligonu- cleotídeos sobre a estrutura de carbono. BIR PATHOGEN INFECTION STRUCTURES according to claims 1 and 2, characterized in that the nanotubes have on their surface helical structures arranged neatly along their longitudinal surface compatible with the adsorption of oligonucleotides onto the carbon structure.
4- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com a reivindicação 1 , caracterizado pelos oligonucleotídeos serem selecionados do grupo compreendendo sequências senso e anti-senso do gene INF24.  CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES according to claim 1, characterized in that the oligonucleotides are selected from the group comprising sense and antisense sequences of the INF24 gene.
5- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com as reivindicações 1 a 4, caracterizado por ser capaz de penetrar no citoplasma da células de patógenos.  CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES according to claims 1 to 4, characterized in that it is capable of penetrating the cytoplasm of pathogen cells.
6- CONJUGADO DE NANOTUBOS DE CARBONO PARA INI¬ 6- CARBON NANOTUBE CONJUGATE FOR INI¬
BIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, de acordo com as reivindicações 1 a 5, caracterizada por interferir nos mecanismos de síntese protéica regulada pelo RNA mensageiro do patógeno. BIR PATHOGEN INFECTION STRUCTURES IN VEGETABLES according to claims 1 to 5, characterized in that it interferes with the mechanisms of protein synthesis regulated by the messenger RNA of the pathogen.
7- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS caracterizado pelo seu uso no controle de doenças no campo.  7. CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES characterized by its use in field disease control.
8- CONJUGADO DE NANOTUBOS DE CARBONO PARA INIBIR ESTRUTURAS DE INFECÇÃO DE PATÓGENOS EM VEGETAIS, caracterizado pela inibição do desenvolvimento da doença causada pelo fungo U. appendiculatus inoculado em espécies vegetais susceptíveis. 8- CARBON NANOTUBE CONJUGATE TO INHIBIT PATHOGEN INFECTION STRUCTURES IN VEGETABLES, characterized by the inhibition of disease development caused by the fungus U. appendiculatus inoculated in susceptible plant species.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023168122A1 (en) * 2022-03-04 2023-09-07 The Regents Of The University Of California Compositions and methods for targeted delivery of chemicals and biomolecules to plants and fungi

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2724935A1 (en) * 1994-09-27 1996-03-29 Centre Nat Rech Scient Compsns. contg. ras oncogene anti-sense oligo:nucleotide(s)
WO1998004740A1 (en) * 1996-07-29 1998-02-05 Nanosphere Llc Nanoparticles having oligonucleotides attached thereto and uses therefor
US20060240554A1 (en) * 2005-02-14 2006-10-26 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules
US20080194031A1 (en) * 2005-01-19 2008-08-14 International Technology Center Alterations Utilizing Nanoparticles

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2724935A1 (en) * 1994-09-27 1996-03-29 Centre Nat Rech Scient Compsns. contg. ras oncogene anti-sense oligo:nucleotide(s)
WO1998004740A1 (en) * 1996-07-29 1998-02-05 Nanosphere Llc Nanoparticles having oligonucleotides attached thereto and uses therefor
US20080194031A1 (en) * 2005-01-19 2008-08-14 International Technology Center Alterations Utilizing Nanoparticles
US20060240554A1 (en) * 2005-02-14 2006-10-26 Sirna Therapeutics, Inc. Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HILLAIREAU, H., ET AL.: "Encapsulation of mono- and oligo-nucleotides into aqueous-core nanocapsules in presence of various water-soluble polymers", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 331, no. 2, 26 October 2007 (2007-10-26), pages 148 - 152, XP005745262 *
IKEDA A; ET AL: "Water-Solubilization of Nucleotides-Coated Single-Walled Carbon Nanotubes Using a High-Speed Vibration Milling Technique", ORGANIC LETTERS, vol. 8, no. 6, 18 February 2006 (2006-02-18), pages 1153 - 1156, XP003007326 *
LIU Q; ET AL: "Carbon nanotubes as molecular transporters for walled plant cells", NANO LETTERS, vol. 9, no. 3, 3 February 2009 (2009-02-03), pages 1007 - 1010, XP002601946 *
QIN W J; YUNG L Y L: "Nanoparticle carrying a single probe for target DNA detection and single nucleotide discrimination", BIOSENSORS AND BIOELECTRONICS, vol. 25, no. 2, 17 July 2009 (2009-07-17), pages 313 - 319, XP026600426 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023168122A1 (en) * 2022-03-04 2023-09-07 The Regents Of The University Of California Compositions and methods for targeted delivery of chemicals and biomolecules to plants and fungi
WO2023168125A1 (en) * 2022-03-04 2023-09-07 The Regents Of The University Of California Compositions and methods for targeted delivery of chemicals and biomolecules to plants and fungi

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