Development of single-stranded DNA aptamers for specific Bisphenol a detection

The development of reagents with high affinity and specificity to small molecules is crucial for the high-throughput detection of chemical compounds, such as toxicants or pollutants. Aptamers are short and single-stranded (ss) oligonucleotides able to recognize target molecules with high affinity. Here, we report the selection of ssDNA aptamers that bind to Bisphenol A (BPA), an environmental hormone. Using SELEX process, we isolated high affinity aptamers to BPA from a 10(15) random library of 60 mer ssDNAs. The selected aptamers bound specifically to BPA, but not to structurally similar molecules, such as Bisphenol B with one methyl group difference, or 4,4'-Bisphenol with 2 methyl groups difference. Using these aptamers, we developed an aptamer-based sol-gel biochip and detected BPA dissolved in water. This novel BPA aptamer-based detection can be further applied to the universal and high-specificity detection of small molecules.     

Development of models for specific crop calendar events

In order to test growth prediction and yield prediction equations on historical weather data for areas where planting dates were not published, or for areas where dates of planting will not be as readily accessible as weather data, there is a need for a model to predict the date of planting. Such a model was developed by regressing reported percentages of the wheat crop planted on various weather variables and their transformations. The model for predicting percentage of wheat acreage planted in North Dakota was tested on data not used in formulating the coefficients of the model. Although the predictions may have errors of several days the model is considered to be an improvement over a system utilizing a constant year-to-year average planting date.The research was supported by funds supplied by the National Aeronautics and Space Administration under contract No. NAS 9-14006.Presented at the Seventh International Biometeorological Congress, 17–23 August 1975, College Park, Maryland, USA.     

RNA aptamers specific for bovine thrombin

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a K(d) of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished.     

Single-stranded DNA aptamers specific for antibiotics tetracyclines

Tetracyclines (TCs) are a group of antibiotics comprising of a common tetracycline (TET) nucleus with variable X(1) and X(2) positions on 5 and 6 carbon atoms, such as oxytetracycline (OTC) and doxycycline (DOX). In this study, the tetracycline group specific (TGS) ssDNA aptamers were identified by modified SELEX method by employing tosylactivated magnetic beads (TMB) coated with OTC, TET, and DOX, respectively, as targets and counter targets. Twenty TGS-aptamers were selected, of which seven aptamers, designated as T7, T15, T19, T20, T22, T23, and T24, showed high affinity to the basic TET backbone (K(d)=63-483 nM). The specificity of these TGS-aptamers to structural analogues followed the order in which the TCs was employed during SELEX process (OTC>TET>DOX) except aptamer T22, which was highly specific to TET than OTC or DOX. Aptamers that were specific to one target molecule but fail to bind the other structurally related TCs were eliminated during counter selection steps. Three aptamers, T7, T19, and T23 contained palindromic consensus sequence motif GGTGTGG. The remaining TGS-aptamers showed many consensus sequences that are truncated forms of this palindrome forming mirror image or inverted sequences. For example, GTGG or its inverted form, GGTG motif was found in all TGS-aptamers. A consensus sequence motif TGTGCT or its truncated terminal T-residue was found in most TGS-aptamers, which is predicted to be essential for high affinity and group specificity. These TGS-aptamers have potential applications such as target drug delivery, and detection of TCs in pharmaceutical preparations and contaminated food products.     

Peptide aptamers: specific inhibitors of protein function

In recent years, peptide aptamers have emerged as novel molecular tools that are useful for both basic and applied aspects of molecular medicine. Due to their ability to specifically bind to and inactivate a given target protein at the intracellular level, they provide a new experimental strategy for functional protein analyses, both in vitro and in vivo. In addition, by using peptide aptamers as "pertubagens", they can be employed for genetic analyses, in order to identify biochemical pathways, and their components, that are associated with the induction of distinct cellular phenotypes. Furthermore, peptide aptamers may be developed into diagnostic tools for the detection of a given target protein or for the generation of high-throughput protein arrays. Finally, the peptide aptamer technology has direct therapeutic implications. Peptide aptamers can be used in order to validate therapeutic targets at the intracellular level. Moreover, the peptide aptamer molecules themselves should possess therapeutic potential, both as lead structures for drug design and as a basis for the development of protein drugs.     

The selection of aptamers specific for membrane molecular targets

A growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.     

Selection of RNA aptamers specific to active prostate-specific antigen

A counter-SELEX procedure with recombinant purified active prostate specific antigen (PSA) was used to identify specific RNA aptamers against the active PSA. We developed two different kinds of counter-SELEX methods; one includes pre-clearance step with inactive proPSA protein, and the other with tagged GST protein. After 9 iterative selection cycles, several identical RNA aptamers can be identified from both counter-SELEX methods. Real-time PCR analysis and gel retardation experiment showed that the aptamers have a specific binding activity against the active PSA, but not for GST or proPSA. These aptamers could be of potential use as specific diagnostic, imaging and/or therapeutic agents against prostate cancer.     

CK-MB特异性单抗的制备方法及其应用

本研究拟建立肌酸激酶同工酶MB(CK-MB)特异性单克隆抗体(m Ab)的研制方法,对抗CK-MB单抗进行评价分类及性质鉴定,并初步建立CK-MB定量检测试剂。以CK-MB抗原免疫BALB/c小鼠,利用常规单抗制备技术,使用间接和捕获ELISA差异筛选法筛选单抗。利用肌酸激酶同工酶(CK-MM/BB/MB)抗原对所制备单抗的抗原识别表位进行鉴定,另通过免疫印迹法及合成CK-MM、CK-BB差异性的线性表位肽鉴定对所制备的单抗进行评价分类。使用双抗体夹心ELISA方法筛选检测CK-MB抗原的配对m Ab,并初步建立CK-MB定量检测试剂。使用74例临床标本初步评价该试剂与罗氏试剂的检测一致性。最终,我们成功筛选到22株稳定分泌抗CK-MB抗体的杂交瘤细胞株,这些单抗可以分为线性、偏构象的CK-MB和CK-MM或者CK-BB交叉的单抗以及与CK-MB特异反应的偏构象型单抗,并使用偏构象型单抗研制出CK-MB定量检测试剂,该试剂与罗氏试剂相关系数r达到0.930 9。综上所述,本研究建立了研制CK-MB偏构象型特异性单抗的筛选方法,通过对所筛选的单抗进行分析鉴定并建立了CK-MB定量检测试剂,与罗氏试剂检测结果符合率高。     

Production and characterization of RNA aptamers specific for amyloid fibril epitopes

One of the most fascinating features of amyloid fibrils is their generic cross-beta architecture that can be formed from many different and completely unrelated proteins. Nonetheless, amyloid fibrils with diverse structural and phenotypic properties can form, both in vivo and in vitro, from the same protein sequence. Here, we have exploited the power of RNA selection techniques to isolate small, structured, single-stranded RNA molecules known as aptamers that were targeted specifically to amyloid-like fibrils formed in vitro from beta(2)-microglobulin (beta(2)m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent K(D) approximately nm) to beta(2)m fibrils with diverse morphologies generated under different conditions in vitro, as well as to amyloid fibrils isolated from tissues of dialysis-related amyloidosis patients, demonstrating that they can detect conserved epitopes between different fibrillar species of beta(2)m. Interestingly, the aptamers also recognize some other, but not all, amyloid fibrils generated in vitro or isolated from ex vivo sources. Based on these observations, we have shown that although amyloid fibrils share many common structural properties, they also have features that are unique to individual fibril types.     

Selection and characterization of DNA aptamers specific for Listeria species

Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37 °C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays.     

Development of specific Rho-kinase inhibitors and their clinical application

Hexahydro-1-(isoquinoline-5-sulfonyl)-1H-1,4-diazepine, HA-1077, is a known selective inhibitor of Rho-kinase. Although its IC(50) value against Rho-kinase is more than 10 times lower than those for kinases such as PKA, PKB, PKC, PKG, MLCK, CaMKII and others, the molecule still retains relative potent inhibition activities against these kinases. In order to produce highly specific Rho-kinase inhibitors, several HA-1077 analogs were synthesized and their kinase inhibition properties evaluated. (S)-Hexahydro-1-(4-ethenylisoquinoline-5-sulfonyl)-2-methyl-1H-1,4-diazepine was found to be a potent Rho-kinase inhibitor. The IC50 value against Rho-kinase was 6 nM, while those against other kinases remained at almost the same level as that of HA-1077. Furthermore, we designed HA-1077 analogs on the basis of the complex structure of PKA and HA-1077. Amongst these, (S)-hexahydro-4-glycyl-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1H-1,4-diazepine and other glycine derivatives were found to be highly specific Rho-kinase inhibitors. These Rho-kinase specific inhibitors were applied to rabbit ocular hypertensive models and were shown to reduce intraocular pressure. These results demonstrate that the new 5-isoquinolinesulfonylamides are not only potent ROCK selective compounds, but are also useful compounds for clinical applications.     

Selection of aptamers against pathogenic bacteria and their diagnostics application

Aptamers are short nucleotide sequences which can specifically bind to a variety of targets with high affinity. They are identified and selected via systematic evolution of ligands by exponential enrichment (SELEX). Compared to antibodies, aptamers offer several advantages including easy labeling, high stability and lower cost. Those advantages make it possible to be a potential for use as a recognition probe to replace antibody in the diagnostic field. This article is intended to provide a comprehensive review, which is focused on systemizing recent advancements concerning SELEX procedures, with special emphasis on the key steps in SELEX procedures. The principles of various aptamer-based detections of pathogenic bacteria and their application are discussed in detail, including colorimetric detection, fluorescence detection, electrochemical detection, lateral flow strip test, mass sensitive detection and PCR-based aptasensor. By discussing recent research and future trends based on many excellent publications and reviews, we attempt to give the readers a comprehensive view in the field of aptamer selection against pathogenic bacteria and their diagnostics application. Authors hope that this review will promote lively and valuable discussions in order to generate new ideas and approaches towards the development of aptamer-based methods for application in pathogenic bacteria diagnosis.     

非洲黑麦染色体特异性标记的建立与应用

以非洲黑麦、小麦-非洲黑麦双二倍体、安岳排灯麦等为材料筛选100条ISSR引物。其中, 引物UBC815可在非洲黑麦中扩增出1条长561 bp的特异性片段(命名为pSaUBC815₅₆₁), 而小麦对照均未扩出该片段。引物UBC815同样能在黑麦属的瓦维洛夫黑麦(Secale vavilovii Grossh.)、森林黑麦(Secale sylvestre Host.)等5个种扩增出pSaUBC815₅₆₁。根据pSaUBC815₅₆₁设计特异PCR引物U815-F、U815-R, 对小麦族多物种进行扩增, 表明pSaUBC815₅₆₁为黑麦属特有。进而利用一套中国春-Imperial黑麦二体附加系及小麦-黑麦异源材料进行扩增, 结果显示, pSaUBC815₅₆₁分布在黑麦整套染色体上, 并且所有后代材料都能扩增出pSaUBC815₅₆₁, 表明pSaUBC815₅₆₁可作为特异性标记用来检测小麦背景中的黑麦染色质。     

Prospects for application of aptamers in diagnostics of bacterial infections

Nucleic acid-based aptamers are widely accepted as promising tools for development of a plethora of diagnostic and therapeutic preparations, as well as means ofenvironmental monitoring. Aptamers can be regarded as fully synthetic analogs of antibodies. At the same time, certain properties ofaptamers render them superior to antibodies in terms of development of new diagnostic and monitoring systems that combine high sensitivity and specificity with high reproducibility and inexpensive manufacturing. In particular, the aptamers tailored to bind biomolecules and live cells can be employed in solving the problem of combining short analysis time with high sensitivity and specificity in detection of pathogenic bacteria. The present review summarizes the current state of the techniques developed for aptamer-based detection of bacteria and their components and discusses the potential of their practical application.     

Expanding the application potential of DNA aptamers by their functionalization

Side-by-side development of two competing technologies for obtaining affinity antibody-based and aptamer-based molecules opens new horizons for the creation of diagnostic and therapeutic agents of extremely high efficiency. Benefits of aptamers, such as relatively small size and selection simplicity, have been jeopardized for a long time by their intrinsic downsides, i.e., obscure process of obtaining aptamers against certain targets because of a low diversity of functional groups (purine and pyrimidine bases) in DNA and RNA aptamers. Another side effect of the aptamer technique inherent to the traditional SELEX method is unspecific enrichment with aptamers with high affinity to off-target reaction components. Today, due to current progress in the development of new technology methods and chemical coupling reactions, the modern aptamer technology helps to avoid its disadvantages and become capable of being the source of new diagnostic and therapeutic tools, which are properly unique in their efficiency. The review focuses on modern methods of increasing efficiency of the aptamer selection and on synthetic nucleotide modifications, which make it possible to prepare high-affinity aptamers against traditionally ‘hard’ targets.     

Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells

Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues.