等温滴定量热法研究核糖开关aac与氨基糖苷类抗生素的相互作用

核糖开关(riboswitch)是位于m RNA非编码区域的RNA元件,可与小分子配体结合导致结构转变,进而调控下游基因的表达。核糖开关aac能够特异性结合氨基糖苷类抗生素,从而调控氨基糖苷类抗生素抗性基因的表达。目前,核糖开关aac与氨基糖苷类抗生素相互作用的位点和机制尚不清楚。作者利用等温滴定量热法对核糖开关aac与氨基糖苷类抗生素的结合亲和力及结合热力学性质进行了研究,并初步探讨了点突变对相互作用的影响。结果发现,西索米星、庆大霉素、G418、奈替霉素和巴龙霉素能够特异性结合核糖开关aac,而阿米卡星、卡那霉素A、链霉素、链丝菌素、新霉胺、新霉素及核糖霉素与核糖开关aac无特异性相互作用;核糖开关aac与氨基糖苷类抗生素的结合热力学性质表明二者通过氢键和范德华力发生相互作用。西索米星滴定核糖开关aac突变体的结果表明,U68和A18位点很可能与氨基糖苷类抗生素形成氢键,A13和A44位点对于相互作用也有影响。这些结果对进一步研究核糖开关aac与氨基糖苷类抗生素相互作用的位点和机制具有重要的参考价值。     

氨基糖苷类抗生素的抗HIV潜力

以新霉素为代表的氨基糖苷类抗生素通过与HIV RNA的RRE结构域结合,对HIV的RRE—Rev相互作用产生抑制作用．从而具有阻遏HIV复制的活性。人们通过对各种氨基糖苷类抗生素单体及其多种衍生物与RRE结合的实验研究,对它们之间的相互作用有了越来越多的了解。     

DNA功能化纳米金结合基因芯片可视化检测microRNAs

开发了一种纳米金探针结合基因芯片用于micro RNAs(mi RNAs)检测的高灵敏度便携式生物传感器.靶标mi RNAs特异性捕获探针固定在芯片上,通过与纳米金探针杂交进行检测,最后利用过氧化氢(H2O2)还原四氯金酸(HAu Cl4)来放大检测信号.利用单个纳米金探针结合基因芯片可以检测到10 pmol/L的靶标mi RNAs,测得mi R-126在胎牛血清中的回收率为81.5%~109.1%.用这种生物传感器检测肺癌组织样本总RNA中的mi R-126,结果与定量PCR结果具有一致性.利用双纳米金探针进一步提高了检测灵敏度,可以检测到1 fmol/L的mi R-125a-5p.整个分析时间不超过1 h,并且实验结果可以用肉眼观察.这个平台可以同时检测肺癌相关mi R-126和mi R-125a-5p,并且具有费用低、快速和便捷的优势,有望用于mi RNAs的超灵敏可视化检测.     

HPV病毒检测分型芯片的研制和优化

研制和优化寡核苷酸芯片以初步实现对多种常见HPV(Human papillomavirus)病毒的分型检测.应用生物学软件对四型常见HPV病毒(6、11、16、18型)的全基因组序列进行分析,设计具有型特异性、熔解温度(Tm)相近的～60 mer寡核苷酸探针,对玻片片基进行优化处理后,点样制备成寡核苷酸基因芯片.将含HPV全长基因序列的质粒作为阳性标准品,利用梯度限制性荧光标记技术对其进行荧光标记,标记好的样品与芯片杂交.结果显示HPV样品与相应的型特异性探针杂交有明显的荧光信号,而与阴性对照探针和空白对照探针没有杂交信号.通过对芯片片基处理和样品荧光标记方法的优化,可以提高芯片检测的杂交特异性和荧光信号强度.     

cDNA芯片阳性对照的制备及在芯片敏感性分析中的应用

cDNA芯片是一种高通量基因表达谱分析技术,在生理病理条件下细胞基因表达谱分析,新基因发现和功能研究等方面具有广阔应用前景。CDNA芯片阳性对照的选取以及CDNA芯片检测敏感性是芯片成功应用的关键问题之一。以在系统发育上与人类基因同源性小的荧火虫荧光素酶基因材料,制备了用于人类和其他动物基因表达谱CDNA芯片的通用型阳性对照探针和相应的mRNA参照物,经反转录对mRNA参照物进行Cy3荧光标记并与DNA芯片杂交后发现,mRNA参照物能特异性地与荧光酶基因cDNA片断杂交,而与人β-肌动蛋白基因,人G3PDH基因以及λDNA/HINDⅢ无杂交反应。把mRNA参照物以不同比例加入HepG2总RNA中,以反转录荧光标记后与CDNA芯片杂交,结果发现当总RNA中的MRNA含量为1/10^4稀释（即mRNA分子个数约为10^8个）时,CDNA芯片基本检测不出mRNA标记产物的杂交信号。而且,cDNA芯片检测的信号强度与芯片上固定的探针浓度密切相关,当探针浓度为2g/L时,杂交信号最强,随着探针浓度下降芯片的杂交信号趋于减弱。CDNA芯片通用型阳性参照物的制备以及应用于CDNA芯片检测敏感性研究为CDNA芯片应用于人和其他动物基因表达谱高通量分析和新基因功能研究提供了技术基础和理论依据。     

DNA芯片制作原理及其杂交信号检测方法

文章讨论了ＤＮＡ芯片的制作原理和杂交信号的检测方法。依其结构,ＤＮＡ芯片可分为两种形式,ＤＮＡ阵列和寡核苷酸微芯片。ＤＮＡ芯片的制作方法主要有光导原位合成法和自动化点样法。ＤＮＡ芯片与标记的探针或ＤＮＡ样品杂交,并通过探测杂交信号谱型业实现ＤＮＡ序列或基因表达的分析。适应于ＤＮＡ芯片的发展,同时出现了许多新型的杂交信号检测方法。主要有激光荧光扫描显微镜、激光扫描共焦显微镜、结合作用ＣＣＤ相机的荧光     

双色荧光杂交芯片在检测 P 1A 1M P I 基C 因多态性中的应用

目的：应用一种新的高通量SNP检测方法-双色荧光杂交芯片技术检测CYPIA1 MspI基因多态性。方法：收集江苏汉族人群原发性肺癌患者75例和相应对照77例,应用双色荧光杂交芯片技术检测了152例样本的CYPIAI基因MspI基因多态性,并应用PCR-RFLP技术验证双色荧光杂交芯片的特异性。结果：152例样本的CYPIAI基因双色荧光杂交芯片技术分型结果与PCR-RFLP结果完全相符,两种方法的基因型分型结果具有很好的一致性。结论：双色荧光杂交芯片技术是一个高通量SNP检测的良好工具,特异性高,在大规模人群SNP筛检中具有良好的发展前案。     

DNA芯片制作原理及其杂交信号检测方法

文章讨论了DNA芯片的制作原理和杂交信号的检测方法。依其结构,DNA芯片可分为两种形式,DNA阵列和寡核苷酸微芯片。DNA芯片的制作方法主要有光导原位合成法和自动化点样法。DNA芯片与标记的探针或DNA样品杂交,并通过探测杂交信号谱型来实现DNA序列或基因表达的分析。适应于DNA芯片的发展,同时出现了许多新型的杂交信号检测方法。主要有激光荧光扫描显微镜、激光扫描共焦显微镜、结合使用CCD相机的荧光显微镜、光纤生物传感器、化学发生法、光激发磷光物质存储屏法、光散射法等。     

口蹄疫等5种动物病毒基因芯片检测技术的研究

用分子克隆方法获得口蹄疫病毒、水泡性口炎病毒、蓝舌病病毒、鹿流行性出血热病毒和赤羽病病毒各一段高度保守的基因片段,用芯片点样仪点样到包被过的玻璃片上,制备成检测芯片。提取样品中的RNA,进行反转录和荧光标记后滴加到芯片上进行特异性杂交,对杂交结果进行扫描检测,可同时诊断上述5种动物传染病,此方法不但快速、准确、敏感,而且可同时进行多种病毒的检测,达到大批动物高通量检疫的目的。     

糖芯片的制备和检测应用研究进展

近数十年来,糖芯片逐渐成为分析糖介导的识别和结合作用的强有力工具,具有样品检测用量少、特异性强和高通量等优点,可以大大提高糖生物学研究的效率。主要介绍了通过糖库的构建、共价结合和非共价吸附法等方法制备糖芯片的过程,糖芯片的检测方法及其在生物学研究和生物医学领域的应用,以期为糖芯片相关研究提供参考。     

Defining RNA motif–aminoglycoside interactions via two-dimensional combinatorial screening and structure–activity relationships through sequencing

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3 × 3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure–activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif–aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site.     

Aminoglycoside binding to human and bacterial A-Site rRNA decoding region constructs

The 16S bacterial ribosomal A-site decoding rRNA region is thought to be the pharmacological target for the aminoglycoside antibiotics. The clinical utility of aminoglycosides could possibly depend on the preferential binding of these drugs to the prokaryotic A-site versus the corresponding A-site from eukaryotes. However, quantitative aminoglycoside binding experiments reported here on prokaryotic and eukaryotic A-site RNA constructs show that there is little in the way of differential binding affinities of aminoglycosides for the two targets. The largest difference in affinity is 4-fold in the case of neomycin, with the prokaryotic A-site construct exhibiting the higher binding affinity. Mutational studies revealed that decoding region constructs retaining elements of non-Watson-Crick (WC) base pairing, specifically bound aminoglycosides with affinities in the muM range. These studies are consistent with the idea that aminoglycoside antibiotics can specifically bind to RNA molecules as long as the latter have non-A form structural elements allowing access of aminoglycosides to the narrow major groove.     

Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray

Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2′) from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2′). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2′) is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.     

Which aminoglycoside ring is most important for binding? A hydropathic analysis of gentamicin, paromomycin, and analogues

The NMR structures of gentamicin and paromomycin in complex with the A-site of Escherichia coli 16S ribosomal RNA were modified with molecular modeling to 12 analogues. The intermolecular interactions between these molecules and RNA were examined using the HINT (Hydropathic INTeractions) computational model to obtain interaction scores that have been shown previously to be related to free energy. The calculations correlated well with experimental binding data, and the interaction scores were used to analyze the specific structural features of each aminoglycoside that contribute to the overall binding with the 16S rRNA. Our calculations indicate that, while ring I binds to the main binding pocket of the rRNA A-site, ring IV of paromomycin-based aminoglycosides contributes significantly to the overall binding.     

Genetic reconstruction of protozoan rRNA decoding sites provides a rationale for paromomycin activity against Leishmania and Trypanosoma

Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics.     

An aminoglycoside microarray platform for directly monitoring and studying antibiotic resistance

Antibiotic resistance is a major threat to human health. Since resistance to the aminoglycoside class of antibiotics is most commonly caused by enzymatic modification, we developed a high-throughput microarray platform for directly assaying resistance enzyme activity on aminoglycosides. After modification, the array can be hybridized with the therapeutic target, a bacterial rRNA A-site mimic, to study the effect that modification has on binding. Such studies will help identify important factors that contribute to high-affinity recognition of therapeutic targets and low-affinity recognition of and modification by resistance enzymes. This platform may also be useful for screening chemical libraries to discover new antibiotics that evade resistance.     

Enhanced binding of aminoglycoside dimers to a "dimerized" A-site 16S rRNA construct

In this work, we investigated the binding of a series of dimeric aminoglycoside molecules to (i) a 27 nt A-site 16S rRNA construct, and (ii) an artificially grafted 46 nt 'dimerized' A-site 16S rRNA construct. It was observed that the dissociation constants of dimeric aminoglycosides to the dimerized A-site 16S rRNA construct can achieve up to approximately 19-fold enhancement compared to the monomeric aminoglycoside molecules.     

Electrostatic Interactions in Aminoglycoside-RNA Complexes

Electrostatic interactions often play key roles in the recognition of small molecules by nucleic acids. An example is aminoglycoside antibiotics, which by binding to ribosomal RNA (rRNA) affect bacterial protein synthesis. These antibiotics remain one of the few valid treatments against hospital-acquired infections by Gram-negative bacteria. It is necessary to understand the amplitude of electrostatic interactions between aminoglycosides and their rRNA targets to introduce aminoglycoside modifications that would enhance their binding or to design new scaffolds. Here, we calculated the electrostatic energy of interactions and its per-ring contributions between aminoglycosides and their primary rRNA binding site. We applied either the methodology based on the exact potential multipole moment (EPMM) or classical molecular mechanics force field single-point partial charges with Coulomb formula. For EPMM, we first reconstructed the aspherical electron density of 12 aminoglycoside-RNA complexes from the atomic parameters deposited in the University at Buffalo Databank. The University at Buffalo Databank concept assumes transferability of electron density between atoms in chemically equivalent vicinities and allows reconstruction of the electron densities from experimental structural data. From the electron density, we then calculated the electrostatic energy of interaction using EPMM. Finally, we compared the two approaches. The calculated electrostatic interaction energies between various aminoglycosides and their binding sites correlate with experimentally obtained binding free energies. Based on the calculated energetic contributions of water molecules mediating the interactions between the antibiotic and rRNA, we suggest possible modifications that could enhance aminoglycoside binding affinity.     

Enhanced RNA binding of dimerized aminoglycosides.

Aminoglycoside antibiotics have recently emerged as an intriguing family of RNA binding molecules and they became leading structures for the design of novel RNA ligands. The demystification of the aminoglycoside-RNA recognition phenomenon is required for the development of superior binders. To explore the existence of multiple binding sites in a large RNA molecule, we have synthesized covalently linked symmetrical and nonsymmetrical dimeric aminoglycosides. These unnatural derivatives were compared to their natural "monomeric" counterparts in their ability to inhibit the Tetrahymena ribozyme. The dimeric aminoglycosides inhibit ribozyme function 20 to 1.2 x 10(3) fold more effectively than their natural parent compounds. The inhibition curves of dimeric aminoglycosides have characteristic shapes suggesting the presence of at least two high affinity-binding sites within the ribozyme's three-dimensional fold. The interaction of a dimeric aminoglycoside with two complementary sites of the RNA molecule is proposed. This binding motif may have implications on the development of new drugs targeting pivotal RNA molecules of bacterial and viral pathogens.     

Thermodynamics of aminoglycoside-rRNA recognition: the binding of neomycin-class aminoglycosides to the A site of 16S rRNA

We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA. Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration. (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups. (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH. Specifically, increasing the pH results in a corresponding increase in the number of linked protons. (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH. (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration. In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH. Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases. (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycin > paromomycin > ribostamycin. The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin. (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA. In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide. Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.