利用糖芯片技术检测氨基糖苷类抗生素与RNAs和蛋白质之间的相互作用

王雪玉1 王晓丽1 张蓓蕾1 胡静2 尹健1

(1.江南大学生物工程学院糖化学与生物技术教育部重点实验室, 江苏无锡 214122)
(2.江南大学无锡医学院, 江苏无锡 214122)

【摘要】氨基糖苷类抗生素是一类广谱型抗细菌感染药物,其不断增加的细菌耐药性很大程度上限制了它的临床应用,研究和开发新型氨基糖苷类抗生素具有重要意义。将氨基糖苷类抗生素固定到玻璃片基上,制成糖芯片,再分别与荧光标记的RNAs和蛋白质杂交,通过分析杂交后的荧光信号强度检测它们之间的相互作用。结果显示,氨基糖苷类抗生素芯片可以特异性地与r RNA的A位点模拟物、I型核酶和蛋白酶结合。因此糖芯片技术不仅可以检测氨基糖苷类抗生素与r RNAs的特异性结合,而且可以应用于寻找新型RNA结合配体的研究,为快速鉴定和筛选可紧密结合RNA靶标且毒性较低的新型氨基糖苷类抗生素奠定了一定的基础。

【关键词】 氨基糖苷类抗生素芯片; rRNA的A位点模拟物; I型核酶; Klenow DNA聚合酶; 磷脂酶C;

【DOI】

【基金资助】 国家自然科学基金(No.21502071) National Natural Science Foundation of China(No.21502071) 江苏省自然科学基金(Nos.BK20140154,BK20150140) Natural Science Foundation of Jiangsu Province,China(Nos.BK20140154,BK20150140) 江南大学公共卫生研究中心项目(No.JUPH201502)资助 the Public Health Research Center at Jiangnan University(No.JUPH201502)

Download this article

    References

    [1]Kondo S,Hotta K.Semisynthetic aminoglycoside antibiotics:development and enzymatic modifications.J Infect Chemother,1999,5(1):1-9.

    [2]Magnet S,Blanchard JS.Molecular insights into aminoglycoside action and resistance.Chem Rev,2005,105(2):477-498.

    [3]Ding YL,Hofstadler SA,Swayze EE,et al.Design and synthesis of paromomycin-related heterocyclesubstituted aminoglycoside mimetics based on a mass spectrometry RNA-binding assay.Angew Chem Int Ed,2003,42(29):3409-3412.

    [4]Shandrick S,Zhao Q,Han Q,et al.Monitoring molecular recognition of the ribosomal decoding site.Angew Chem Int Ed,2004,116(24):3239-3244.

    [5]Verhelst SHL,Michiels PJA,van der Marel GA,et al.Surface plasmon resonance evaluation of various aminoglycoside–RNA hairpin interactions reveals low degree of selectivity.Chem Bio Chem,2004,5(7):937-942.

    [6]Wang DN.Carbohydrate microarrays.Proteomics,2003,3(11):2167-2175.

    [7]Horlacher T,Seeberger PH.Carbohydrate arrays as tools for research and diagnostics.Chem Soc Rev,2008,37(7):1414-1422.

    [8]Park S,Gildersleeve JC,Blixt O,et al.Carbohydrate microarrays.Chem Soc Rev,2013,42(10):4310-4326.

    [9]Yang J,Moraillon A,Siriwardena A,et al.Carbohydrate microarray for the detection of glycan-protein interactions using metal-enhanced fluorescence.Anal Chem,2015,87(7):3721-3728.

    [10]Childs RA,Palma AS,Wharton S,et al.Receptor-binding specificity of pandemic influenza A(H1N1)2009 virus determined by carbohydrate microarray.Nat Biotechnol,2009,27(9):797-799.

    [11]Rogers CJ,Clark PM,Tully SE,et al.Elucidating glycosaminoglycan-protein-protein interactions using carbohydrate microarray and computational approaches.Proc Natl Acad Sci USA,2011,108(24):9747-9752.

    [12]Mendell JT,Olson EN.Micro RNAs in stress signaling and human disease.Cell,2012,148(6):1172-1187.

    [13]Wong SC,Klein JJ,Hamilton HL,et al.Co-injection of a targeted,reversibly masked endosomolytic polymer dramatically improves the efficacy of cholesterol-conjugated small interfering RNAs in vivo.Nucleic Acid Ther,2012,22(6):380-390.

    [14]Piñeiro D,Martinez-Salas E.RNA structural elements of hepatitis C virus controlling viral RNA translation and the implications for viral pathogenesis.Viruses,2012,4(10):2233-2250.

    [15]Disney MD,Magnet S,Blanchard JS,et al.Aminoglycoside microarrays to study antibiotic resistance.Angew Chem Int Ed,2004,43(12):1591-1594.

    [16]Mingeot-Leclercq MP,Brasseur R,Schanck A.Molecular parameters involved in aminoglycoside nephrotoxicity.J Toxicol Environ Health,1995,44(3):263-300.

    [17]Ren YG,Martínez J,Kirsebom LA,et al.Inhibition of klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides.RNA,2002,8(11):1393-1400.

    [18]Morris JC,Lei PS,Zhai HX,et al.Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418.J Biol Chem,1996,271(26):15468-15477.

    [19]Dal Pozzo A,Vanini L,Fagnoni M,et al.Preparation and characterization of poly(ethylene glycol)-crosslinked reacetylated chitosans.Carbohydr Polym,2000,42(2):201-206.

    [20]Muthusamy S,Gnanaprakasam B,Suresh E.Desymmetrization of cyclic anhydrides using dihydroxy compounds:selective synthesis of macrocyclic tetralactones.Org Lett,2006,8(9):1913-1916.

    [21]Disney MD,Haidaris CG,Turner DH.Recognition elements for 5′exon substrate binding to the Candida albicans group I intron.Biochemistry,2001,40(21):6507-6519.

    [22]Wong CH,Hendrix M,Priestley ES,et al.Specificity of aminoglycoside antibiotics for the A-site of the decoding region of ribosomal RNA.Chem Biol,1998,5(7):397-406.

    [23]Wong CH,Hendrix M,Manning DD,et al.A library approach to the discovery of small molecules that recognize RNA:use of a 1,3-hydroxyamine motif as core.J Am Chem Soc,1998,120(33):8319-8327.

    [24]Battaglia C,Salani G,Consolandi C,et al.Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II.Biotechniques,2000,29(1):78-81.

    [25]Disney MD,Seeberger PH.Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins.Chem A Eur J,2004,10(13):3308-3314.

    [26]Griffey RH,Hofstadler SA,Sannes-Lowery KA,et al.Determinants of aminoglycoside-binding specificity for r RNA by using mass spectrometry.Proc Natl Acad Sci USA,1999,96(18):10129-10133.

    [27]Ryu,DH,Rando RR.Aminoglycoside binding to human and bacterial A-site r RNA decoding region constructs.Bioorg Med Chem,2001,9(10):2601-2608.

    [28]Llano-Sotelo B,Azucena EF,Kotra LP,et al.Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-t RNA site.Chem Biol,2002,9(4):455-463.

    [29]Mei HY,Mack DP,Galan AA,et al.Discovery of selective,small-molecule inhibitors of RNA complexes-1.The tat protein/TAR RNA complexes required for HIV-1 transcription.Bioorg Med Chem,1997,5(6):1173-1184.

    [30]Otto GA,Puglisi JD.The pathway of HCV IRES-mediated translation initiation.Cell,2004,119(3):369-380.

    [31]Michael K,Wang H,Tor Y.Enhanced RNA binding of dimerized aminoglycosides.Bioorg Med Chem,1999,7(7):1361-1371.

    [32]Velagapudi SP,Seedhouse SJ,Disney MD.Structure-activity relationships through sequencing(St ARTS)defines optimal and suboptimal RNA motif targets for small molecules.Angew Chem Int Ed,2010,49(22):3816-3818.

    [33]Mikkelsen NE,Johansson K,Virtanen A,et al.Aminoglycoside binding displaces a divalent metal ion in a t RNA-neomycin B complex.Nat Struct Biol,2001,8(6):510-514.

    [34]Mingeot-Leclercq M-P,Tulkens PM.Aminoglycosides:nephrotoxicity.Antimicrob Agents Chemother,1999,43(5):1003-1012.

This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 10, Pages 1362-1371

October 2016

Downloads:0

Share
Article Outline

知识点

摘要

  • 1材料与方法
  • 2结果与分析
  • 3结论
  • 参考文献