DNA aptamers as potential anti-HIV agents

Guanine (G)-rich DNA sequences can adopt stable G-quadruplex structures by G-tetrad hydrogen-bonding and hydrophobic stacking. Recently, it has been shown that a DNA sequence forms an aptamer (termed 93del) and adopts a novel dimeric quadruplex folding topology in K+ solution. This aptamer exhibits anti-HIV1 integrase activity in the nanomolar range in vitro. A docking-based model of the 93del-integrase complex positions the DNA aptamer within a channel of the tetrameric integrase. This mutual fitting blocks several catalytic amino acid residues that are essential for integrase function, and accounts for the anti-HIV1 activity of the 93del aptamer.     

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.     

Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers

High sensitivity and specificity of two modified ssDNA aptamers capable of photocross-linking recombinant human basic fibroblast growth factor (bFGF((155))) were demonstrated. The aptamers were identified through a novel, covalent, in vitro selection methodology called photochemical systematic evolution of ligands by exponential enrichment (PhotoSELEX). The aptamers exhibited high sensitivity for bFGF((155)) comparable with commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF((155)) under prevailing test conditions. The aptamers exquisitely distinguished bFGF((155)) from consanguine proteins, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). A commercially viable diagnostic system incorporating PhotoSELEX-evolved aptamers capable of simultaneous quantification of a large number of analyte molecules is also described. Such a system benefits from covalent bonding of aptamer to target protein allowing vigorous washing with denaturants to improve signal to noise.     

Selection of DNA aptamers against polychlorinated biphenyls as potential biorecognition elements for environmental analysis

Polychlorinated biphenyls (PCBs) have been of major concerns for decades due to their potential toxicity to human health. To trace the PCBs efficiently and sensitively, many detection methods have been developed. Aptamers, a new class of diagnostic tools, are considered to be such additional candidates for detection of pollutants. In the current study, we report the DNA aptamers, isolated by FluMag-SELEX (a modified SELEX [systematic evolution of ligands by exponential enrichment] technology), that recognize PCBs with the dissociation constants (Kd values) down to the micromolar range. Using the selected aptamers, a highly sensitive aptamer-based fluorescent assay for detection of PCBs was established using gold nanoparticles, with a widely linear range from 0.1 to 100 ng/ml. Moreover, our aptamer-based gold nanoprobe displays specificity toward 3,3',4,4'-tetrachlorobiphenyl (PCB77) compared with a few common PCB77 structural analogs. These results open the possibility of using aptamers as biorecognition elements for easy and fast environmental monitoring.     

SELEX法筛选宫颈癌前病变核酸适配子

构建随机ssDNA文库,通过SELEX技术,以正常、炎性宫颈脱落细胞为反筛细胞,以上皮内低级别病变(CIN1)、上皮内高级别病变(CIN2、CIN3)和鳞状细胞癌脱落细胞为正筛细胞,经过12轮筛选特异性适配子高度富集得到宫颈癌前病变适配子库,经特异性、亲和力分析和细胞免疫荧光确立高特异性适配子CIN-Ap4可作为诊断宫颈癌前病变生物标志物,为宫颈癌前病变分子诊断奠定理论基础,提供新思路。利用Prime Premier 5.0设计构建了随机ssDNA文库并根据文库两端固定序列设计引物,对对称PCR和间接不对称PCR中的退火温度、循环数以及上、下游引物浓度比等条件进行优化,分析确定50μL反应体系中对称PCR的最佳反应条件为:最佳退火温度为49.5℃,最佳循环数为15个循环;间接不对称PCR的最佳反应条件为:50μL反应体系中上、下游引物浓度的最佳比例为80∶1,最佳循环数为35个循环。实验结果表明成功构建了寡核苷酸文库,在最适PCR条件下可获得理想的dsDNA和ssDNA,并具有良好的重复性,为顺利筛选适配子提供保证。     

Characterization and application of aptamers for Taq DNA polymerase selected using an evolution-mimicking algorithm

Using an evolution-mimicking algorithm (EMA), we have recently identified DNA aptamers that inhibit Taq DNA polymerase. In the present study, we have attempted to improve further the inhibitory activities of aptamers, as well as to characterize those aptamers with the most potent inhibitory activities. To characterize the most potent aptamer and demonstrate its applicability, the abilities to inhibit Tth DNA polymerase and to modulate specific amplification in PCR were investigated. This aptamer inhibited both Tth DNA polymerase and Taq DNA polymerase and improved the specificity of detection of a low-copy-number target gene in PCR using these DNA polymerases.     

Selection of DNA aptamers for breast cancer

A method of selection of DNA aptamers to breast tumor tissue based on the use of postoperative material has been developed. Breast cancer tissues were used as the positive target; the negative targets included benign tumor tissue, adjacent healthy tissues, breast tissues from mastopathy patients, and also tissues of other types of malignant tumors. During selection a pool of DNA aptamers demonstrating selective binding to breast cancer cells and tissues and insignificant binding to breast benign tissues has been obtained. These DNA aptamers can be used for identification of protein markers, breast cancer diagnostics, and targeted delivery of anticancer drugs.     

Selection of thrombin-binding aptamers by using computational approach for aptasensor application

The possibility of introducing a computationally assisted method to study aptamer-protein interaction was evaluated with the aim of streamlining the screening and selection of new aptamers. Starting from information on the 15-mer (5'-GGTTGGTGTGGTTGG-3') thrombin binding aptamer (TBA), a library of mutated DNA sequences (994 elements) was generated and screened using shapegauss a shape-based scoring function from openeye software to generate computationally derived binding scores. The TBA and three other mutated oligonucleotides, selected on the basis of their binding score (best, medium, worst), were incorporated into surface plasmon resonance (SPR) biosensors. By reducing the ionic strength (binding buffer, 50 mM TrisHCl pH 7.4, 140 mM NaCl, 1mM MgCl?, diluted 1:50) in order to match the simulated condition, the analytical performances of the four oligonucleotide sequences were compared using signal amplitude, sensitivity (slope), linearity (R2) and reproducibility (CVav %). The experimental results were in agreement with the simulation findings.     

DNA aptamers against the Lup an 1 food allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs.     

Dihydrochelerythrine and its derivatives: Synthesis and their application as potential G-quadruplex DNA stabilizing agents

A convenient route was envisaged toward the synthesis of dihydrochelerythrine (DHCHL), 4 by intramolecular Suzuki coupling of 2-bromo-N-(2-bromobenzyl)-naphthalen-1-amine derivative 5 via in situ generated arylborane. This compound was converted to (±)-6-acetonyldihydrochelerythrine (ADC), 3 which was then resolved by chiral prep-HPLC. Efficiency of DHCHL for the stabilization of promoter quadruplex DNA structures and a comparison study with the parent natural alkaloid chelerythrine (CHL), 1 was performed. A thorough investigation was carried out to assess the quadruplex binding affinity by using various biophysical and biochemical studies and the binding mode was explained by using molecular modeling and dynamics studies. Results clearly indicate that DHCHL is a strong G-quadruplex stabilizer with affinity similar to that of the parent alkaloid CHL. Compounds ADC and DHCHL were also screened against different human cancer cell lines. Among the cancer cells, (±)-ADC and its enantiomers showed varied (15–48%) inhibition against human colorectal cell line HCT116 and breast cancer cell line MDA-MB-231 albeit low enantio-specificity in the inhibitory effect; whereas DHCHL showed 30% inhibition against A431 cell line only, suggesting the compounds are indeed cancer tissue specific.     

Selective inhibitory DNA aptamers of the human RNase H1

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA–DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K_d values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5′ and 3′ tails that form a stem of six base pairs. Base pairing between the 5′ and 3′ tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC₅₀ ranging from 50 to 100 nM.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

Kinetic characterization of inhibition of human thrombin with DNA aptamers by turbidimetric assay

A sensitive turbidimetric method for detecting fibrin association was used to study the kinetics of fibrinogen hydrolysis with thrombin. The data were complemented by high-performance liquid chromatography (HPLC) measurements of the peptide products, fibrinopeptides released during hydrolysis. Atomic force microscopy (AFM) data showed that the fibril diameter is the main geometric parameter influencing the turbidity. The turbidimetric assay was validated using thrombin with the standard activity. To study thrombin inhibitors, a kinetic model that allows estimating the inhibition constants and the type of inhibition was proposed. The kinetic model was used to study the inhibitory activity of the two DNA aptamers 15-TBA (thrombin-binding aptamer) and 31-TBA, which bind to thrombin exosites. For the first time, 31-TBA was shown to possess the competitive inhibition type, whereas the shortened aptamer 15-TBA has the noncompetitive inhibition type.     

Deconvolution of a complex target using DNA aptamers

In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets. We propose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to the analysis of other complex biological targets.     

In vitro selection of DNA aptamers binding ethanolamine

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.     

In vitro selection of hematoporphyrin binding DNA aptamers

DNA aptamers that bind to hematoporphyrin IX (HPIX) were isolated using an in vitro selection technique. Most aptamers obtained after the 7th and 10th rounds contained guanine-rich sequences. Binding assay using fluorescence polarization technique and structural analysis by CD spectra revealed that the parallel guanine-quartet structure of the aptamer participates in the recognition of HPIX.     

Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination

Human Rad51 (HsRad51), a key element of the homologous recombination repair pathway, is related to the resistance of cancer cells to chemo- and radio-therapies. This protein is thus a good target for the development of anti-cancer treatments. We have searched for new inhibitors directed against HsRad51 using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach. We have selected three aptamers displaying strong effects on strand exchange activity. Analysis by circular dichroism shows that they are highly structured DNA molecules. Our results also show that they affect the first step of the strand exchange reaction by promoting the dissociation of DNA from the ATP/HsRad51/DNA complex. Moreover, these inhibitors bind only weakly to RecA, a prokaryotic ortholog of HsRad51. Both the specificity and the efficiency of their inhibition of recombinase activity offer an analytical tool based on molecular recognition and the prospect of developing new therapeutic agents.