Advancing Aptamers as Molecular Probes for Cancer Theranostic Applications—The Role of Molecular Dynamics Simulation

Theranostics cover emerging technologies for cell biomarking for disease diagnosis and targeted introduction of drug ingredients to specific malignant sites. Theranostics development has become a significant biomedical research endeavor for effective diagnosis and treatment of diseases, especially cancer. An efficient biomarking and targeted delivery strategy for theranostic applications requires effective molecular coupling of binding ligands with high affinities to specific receptors on the cancer cell surface. Bioaffinity offers a unique mechanism to bind specific target and receptor molecules from a range of non‐targets. The binding efficacy depends on the specificity of the affinity ligand toward the target molecule even at low concentrations. Aptamers are fragments of genetic materials, peptides, or oligonucleotides which possess enhanced specificity in targeting desired cell surface receptor molecules. Aptamer–target binding results from several inter‐molecular interactions including hydrogen bond formation, aromatic stacking of flat moieties, hydrophobic interaction, electrostatic, and van der Waals interactions. Advancements in Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has created the opportunity to artificially generate aptamers that specifically bind to desired cancer and tumor surface receptors with high affinities. This article discusses the potential application of molecular dynamics (MD) simulation to advance aptamer‐mediated receptor targeting in targeted cancer therapy. MD simulation offers real‐time analysis of the molecular drivers of the aptamer‐receptor binding and generate optimal receptor binding conditions for theranostic applications. The article also provides an overview of different cancer types with focus on receptor biomarking and targeted treatment approaches, conventional molecular probes, and aptamers that have been explored for cancer cells targeting.     

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.     

A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone.     

DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes

The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K_d values between 20 and 240 nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.     

Combinatorial Chemistry of Nucleic Acids: SELEX

A method of irrational oligonucleotide design, SELEX, is considered. Individual SELEX products, aptamers, are small molecules (40–100 nt) that have a unique three-dimensional structure, which provides for their specific and high-affinity binding to targets varying from low-molecular-weight ligands to proteins. Thus, the sophisticated biosynthesis of recognizing protein elements, antibodies, can be emulated in vitro via selection and synthesis of principally new recognizing elements based on nucleic acids.     

DNA aptamers developed against a soman derivative cross‐react with the methylphosphonic acid core but not with flanking hydrophobic groups

Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para‐aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase‐based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP‐binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867–876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre‐incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three‐dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p‐aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd.     

In vitro selection of high-affinity DNA aptamers for streptavidin

In this study, we developed a systematic evolution of ligands by exponential enrichment （SELEX） method using a combination of magnetic beads immobilization and flow cytometric measurement. As an example, the selection of streptavidin-specific aptamers was performed. In this protocol, the conventional SELEX procedure was optimized, fiirst using magnetic beads for target immobilization to facilitate highly efficient separation of the binding single-stranded DNA （ssDNA） aptamers from the unbound ssDNAs, and second using flow cytometry and fluorescein labeling to monitor the enrichment. The sensitivity of flow cytometry was adequate for ssDNA quantification during the SELEX procedures. The streptavidin-specific aptamers obtained in this work can be used as tools for characterization of the occupancy of streptavidin-modified surfaces with biotinylated target molecules. The method described in the study is also generally applicable to target molecules other than streptavidin.     

Aptamers: Features,Synthesis and Applications

Aptamers have become a topic of interest among the researchers and scientists since they not only possess all of the benefits of antibodies but also possess special qualities including heat stability, low cost, and limitless uses⋅ Here we give a review about the features, applications, and challenges of aptamers and also how they are beneficial over the antibodies for biomedical applications. Their unique features make aptamers a prominent tool in therapeutics, diagnostics, biosensors and targeted drug delivery. In conclusion, aptamers represent exciting materials for a variety of applications and can be modified to improve their properties and to extend their applications in biomedical field.     

早期胃腺癌原代细胞适配子的筛选与鉴定

胃腺癌是消化道最常见的恶性肿瘤之一,由于没有针对早期胃腺癌有效的诊断方法,目前胃腺癌手术治疗还主要针对中晚期患者,预后差. 本文应用cell-SELEX技术,筛选早期胃腺癌原代细胞的适配子,为早期胃腺癌的诊断提供新的思路. 从早期胃腺癌组织中分离得到早期胃腺癌原代细胞,应用体外合成全长88 bp中间含52 bp随机序列的单链DNA文库,通过对PCR扩增条件的优化,借助生物素-链霉亲和素磁珠系统,经cell-SELEX反复筛选,可获得针对早期胃腺癌原代细胞的特异性适配子.经12轮cell-SELEX筛选,ssDNA文库与早期胃腺癌原代细胞的亲和力由1 560上升到4 336,表明亲和力较高的适配子得到逐步富集. 经克隆和测序,应用软件分析可知,30个克隆子中编号为C17和C27的2个序列完全一致,具同源性,二级结构预测可知单链DNA形成不同的茎环结构可能是适配子与早期胃腺癌原代细胞作用的结构基础. 特异性分析显示,胃腺癌原代细胞组与正常胃粘膜上皮细胞、空白对照组之间荧光强度值差异非常显著（P＜001）;正常胃粘膜上皮细胞组与空白对照组之间差异不显著（P＞005）. 经亲和力测定,各适配子与早期胃腺癌原代细胞的解离系数达到nmol/L,具有很高的亲和力.利用cell-SELEX技术成功筛选到早期胃腺癌原代细胞的适配子,为胃腺癌的早期诊断与治疗药物靶点方面的研究奠定了实验基础.     

RNA-aptamers that modulate the RhoGEF activity of Tiam1

Rho GTPases regulate the actin cytoskeleton and thereby control cell migration, cell morphology, cell motility, and other cellular functions. The gene product of the oncogene Tiam1 acts as a guanine nucleotide exchange factor (GEF) for the Rho GTPase Rac. Like other RhoGEFs, Tiam1 is involved in cancer progression, but it also counteracts invasion in different cancer cell types. Hence, further investigations are required to unravel the functions of Tiam1 in the context of cancer initiation and progression, which appear to be cell specific. Although RhoGEFs in general seem to be attractive therapeutic targets, not many inhibitors have been described, yet. Here we report the identification and characterization of inhibitory RNA aptamers that specifically target Tiam1. After 16 selection rounds three aptamers sharing a 15 nucleotides consensus motif were identified. The clones K91 and K11 inhibited the Tiam1-mediated activation of the GTPase Rac2 in vitro. The tightest binder K91 neither bound the Rho GEF Vav1 nor the Arf GEF Cytohesin-2. In the presence of Rac1, the binding of K91 to Tiam1 was impaired indicating that the binding motif on Tiam1 overlaps with the GTPase binding site. K91 and K11 are the first reported inhibitory molecules targeting the GEF function of Tiam1. Due to their specificity over related GEF proteins they may represent promising tools for further elucidation of the biological functions of Tiam1. We anticipated that these aptamers will prove useful in validating the ambiguous roles of Tiam1 in cancer biology.