A sol-gel-based microfluidics system enhances the efficiency of RNA aptamer selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.     

Methodologies for bulky DNA adduct analysis and biomonitoring of environmental and occupational exposures

It is undisputed that DNA adduct formation is one of the key processes in early carcinogenesis. Therefore, analysis of DNA adduct levels may be one of the best tools available to characterize exposure to complex mixtures of genotoxic chemicals as occurring in different environmental and occupational exposure settings. However, from an analytical point of view the detection and quantification of DNA adducts is a challenging enterprise as extremely high sensitivity and selectivity are required. The entire spectrum of chromatographic techniques, including thin-layer chromatography (TLC), gas and liquid chromatography as well as capillary electrophoresis has been used in combination with different detection systems, all with their own specific characteristics. Among the various combinations of techniques, the TLC-(32)P-postlabeling combination appears to meet best with criteria of sensitivity and requirements of minimal amounts of material. Recent developments in the application of capillary electrophoresis in combination with either immunochemical or mass spectrometric detection techniques may offer new and promising approaches, with higher selectivity as compared to TLC-(32)P postlabeling. The applicability of these new techniques in biomonitoring studies aiming at the exposure and risk assessment of low and chronic exposures remains to be determined. In this paper we compare and discuss the advantages and limitations of different techniques used in DNA adduct analysis, with specific emphasis on those adducts formed by the polycyclic aromatic hydrocarbons and heterocyclic aromatic amines.     

Advances in SELEX and application of aptamers in the central nervous system

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a screening technique that involves the progressive selection of highly specific ligands by repeated rounds of partition and amplification from a large combinatorial nucleic acid library. The products of the selection are called aptamers, which are short single stranded DNA or RNA molecules, binding with high affinity, attributed to their specific three-dimensional shapes, to a large variety of targets, ranging from small molecules to complex mixtures. Various improvement of the original SELEX method described in 1990 have been obtained recently, such as capillary electrophoresis SELEX, Toggle-SELEX, Tailored-SELEX, Photo-SELEX, and others. These new variants greatly shorten time of selection and improve aptamer affinity and specificity. Such aptamers have great potential as detecting and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, and are considered as potent therapeutic lead structures evaluated in preclinical disease models. Recently, one aptamer has been approved by Food and Drug Administration of US for treating age-related macular degeneration. This review presents recent advances in the field of SELEX with special emphasis on applications of aptamers as analytical, diagnostic and therapeutic tools in the central nervous system.     

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands

SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.     

一种结合SELEX与二代测序技术筛选RNA结合蛋白特异识别序列新方法的建立

RNA结合蛋白通过特异识别RNA底物发挥重要的生物学作用。指数富集的配体系统进化(Systematic evolution of ligands by exponential enrichment,SELEX)技术是一种体外筛选核酸底物的基本方法,SELEX技术通过重复多轮筛选从随机核酸序列库中筛选出特异性与靶物质高度亲和的核酸底物,本研究将利用该技术与二代高通量测序(NGS)相结合,体外合成含有20个随机碱基的RNA文库,将所要研究的蛋白构建到带有可被链亲和酶素磁珠捕获的SBP标记的载体上去,显著提高筛选效率,仅需1轮筛选即可获得所需RNA底物motif。通过该方法获得了人的hn RNP A1的UP1结构域特异识别AGG和AG二种RNA序列,并通过EMSA实验证实其可以与获得的RNA motif结合。这一方法的建立对于研究RNA结合蛋白识别底物的序列特异性,并进一步了解其在生物体内的调控机制有重要意义。     

139 Development and application of crop exudate specific aptamers

The fertilizer industry is lucrative, though it faces environmental challenges. The amount of nitrogen applied to crops far exceeds the nitrogen utilized by crops leading to excess nitrogen in the form of nitrates, gaseous ammonia, and nitrogen oxides (DeRossa et al., 2010). This excess nitrogen can spread into groundwater contaminating drinking water and causing excess algal growth. Developments in nanotechnology may alleviate some of these environmental challenges. Although there are examples of nanotechnology being utilized for fertilizer products, none of these methods are able to respond to the specific nutrient needs of the plant. This project aims to produce a nanofertilizer product that can synchronize the release of its nutrients with the uptake of nutrients by the plant (DeRossa et al., 2010). Aptamers are synthetic molecules of DNA or RNA that can form 3-D shapes, which are capable of strongly and selectively binding a target of interest. Aptamers have been found to have binding affinities similar to, if not surpassing, those of monoclonal antibodies (Sultan et al., 2009). The goal of this project is to use polyelectrolyte microcapsules containing aptamers in their walls that are specific for key plant signals. This will allow the delivery of nutrient molecules from inside the microcapsules only when required by the plants. Root exudate specific aptamers are being developed using SELEX (Systematic Evolution of Ligands through Exponential enrichment) from a random DNA pool, as well as from an existing aptamer pool. These aptamers will act as molecular recognition probes in the development of an intelligent fertilizer system. Progress from these selections will be presented.     

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.

RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX. Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome. The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein. These include a tetraloop of primary sequence AUCA and a variable-length stem with a bulged adenosine residue at a specific stem position. The predicted consensus agrees well with the highest-affinity RNA binding site of the R17 coat protein, identified through classical but laborious techniques. These results demonstrate the value of SELEX as a tool for isolating high affinity RNA ligands to a specific target protein, and the further value of those ligands to point the researcher toward natural sequences for that target protein.     

Sequence-constructive SELEX: A new strategy for screening DNA aptamer binding to Globo H

We proposed to use a novel stepwise sequence-constructive SELEX method to develop DNA aptamers that can recognize Globo H which is a tumor-associated carbohydrate antigen. A combinatorial synthetic library that consisted of DNA molecules with randomized regions of 15-bases was used as the starting library for the first SELEX procedure. The input DNA library for the second round of SELEX consisted of the extension of the 5′ and 3′-ends with 7-bases that were randomized from four selected aptamers. The third round of SELEX was performed following the same procedures as described for the second round of SELEX. The experimental results indicate that the binding affinity of DNA aptamers to Globo H was enhanced when using the sequence-constructive SELEX approach. The selectivity of the DNA aptamers for related disaccharides, mannose derivatives, and Globo H analogs demonstrated the ability of the DNA aptamers to discriminate the presence of various glycans with different structures.     

Dose-dependent reduction of 3,2'-dimethyl-4-aminobiphenyl-derived DNA adducts in colon and liver of rats administered celecoxib

Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-week old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated (32)P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N(2)-DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35-40% with 500 ppm and 65-70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer.     

Immunocytochemical analysis of in vivo DNA modification

In the past decades a large number of DNA adducts induced in the intact animal by alkylating agents have been identified. The formation and repair of these adducts are important determinants, not only of mutagenesis, tumor initiation and DNA-mediated toxicity but probably also of tumor progression. Most studies on in vivo DNA modification have been performed on isolated bulk DNA.

More recently, methods have been developed to study the distribution of DNA adducts at the level of either the individual gene or the individual cell. This paper reviews immunocytochemical methods to study the formation and repair of DNA adducts and other DNA modifications at the level of the individual cell. DNA modifications induced by alkylating agents and a variety of other agents including ultraviolet radiation, aromatic amines, polycyclic aromatic hydrocarbons and platinum anti-cancer drugs will be discussed.

Up to now, immunocytochemical analysis of in vivo modified DNA has largely concentrated on experimental animals. These studies have revealed striking heterogeneities with regard to formation and/or repair of DNA adducts in tissues from rat, hamster and mouse. Immunocytochemical adduct analysis can be used to identify in a convenient, fast and detailed way cell types, cell stages and sites in which biological effects of the adducts might be expressed. More recently, immunocytochemical analysis of DNA adducts also proved to be feasible on in situ exposed human samples.

A number of existing and potential applications in the field of chemical carcinogenesis, experimental chemotherapy and molecular epidemiology are discussed.     

Libraries for genomic SELEX.

An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo. A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism. As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids. The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism. We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase. We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX.     

Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.     

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.     

Complex Relationships between Occupation,Environment, DNA Adducts,Genetic Polymorphisms and Bladder Cancer in a Case-Control Study Using a Structural Equation Modeling

DNA adducts are considered an integrate measure of carcinogen exposure and the initial step of carcinogenesis. Their levels in more accessible peripheral blood lymphocytes (PBLs) mirror that in the bladder tissue. In this study we explore whether the formation of PBL DNA adducts may be associated with bladder cancer (BC) risk, and how this relationship is modulated by genetic polymorphisms, environmental and occupational risk factors for BC. These complex interrelationships, including direct and indirect effects of each variable, were appraised using the structural equation modeling (SEM) analysis. Within the framework of a hospital-based case/control study, study population included 199 BC cases and 213 non-cancer controls, all Caucasian males. Data were collected on lifetime smoking, coffee drinking, dietary habits and lifetime occupation, with particular reference to exposure to aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs). No indirect paths were found, disproving hypothesis on association between PBL DNA adducts and BC risk. DNA adducts were instead positively associated with occupational cumulative exposure to AAs (p = 0.028), whereas XRCC1 Arg 399 (p<0.006) was related with a decreased adduct levels, but with no impact on BC risk. Previous findings on increased BC risk by packyears (p<0.001), coffee (p<0.001), cumulative AAs exposure (p = 0.041) and MnSOD (p = 0.009) and a decreased risk by MPO (p<0.008) were also confirmed by SEM analysis. Our results for the first time make evident an association between occupational cumulative exposure to AAs with DNA adducts and BC risk, strengthening the central role of AAs in bladder carcinogenesis. However the lack of an association between PBL DNA adducts and BC risk advises that these snapshot measurements are not representative of relevant exposures. This would envisage new scenarios for biomarker discovery and new challenges such as repeated measurements at different critical life stages.     

In vitro selection of high-affinity nucleic acid ligands to parasite target molecules

The logic of using nucleic acids as pharmaceutical reagents is in part based on their capacity to interact with high affinity and specificity with other biological components. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for 'systematic evolution of ligands by exponential enrichment'. The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognise ligands of different chemical origin. The method is fast, it does not require special equipment and the selected aptamers typically bind their target with high affinity and high specificity. Here we summarise the recent examples of the SELEX technique within the context of identifying high-affinity ligands against parasite target molecules.     

Carcinogen DNA adducts and the risk of colon cancer: case–control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case–control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833–15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

Comparison of DNA adduct levels associated with exogenous and endogenous exposures in human pancreas in relation to metabolic genotype

Recently, we examined normal human pancreas tissue for DNA adducts derived from either exogenous chemical exposure and/or endogenous agents. In an effort to explain the different types and levels of DNA adducts formed in the context of individual susceptibility to cancer, we have focused on gene-environment interactions. Here, we report on the levels of hydrophobic aromatic amines (AAs), specifically those derived from 4-aminobiphenyl (ABP), and DNA adducts associated with oxidative stress in human pancreas. Although these adducts have been reported in several human tissues by different laboratories, a comparison of the levels of these adducts in the same tissue samples has not been performed. Using the same DNA, the genotypes were determined for N-acetyltransferase 1 (NAT1), the glutathione S-transferase (GST) M1, GSTP1, GSTT1, and NAD(P)H quinone reductase-1 (NQO1) as possible modulators of adduct levels because their gene products are involved in the detoxification of AAs, lipid peroxidation products and in redox cycling. These results indicate that ABP-DNA adducts, malondialdehyde-DNA adducts, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) adducts are present at similar levels. Of the metabolic genotypes examined, the presence of ABP-DNA adducts was strongly associated with the putative slow NAT1*4/*4 genotype, suggesting a role for this pathway in ABP detoxification.