Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats.
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ABSTRACT:
SUBMITTER: Dua P
PROVIDER: S-EPMC5125936 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface.
| S-EPMC5282457 | biostudies-literature
Project description:Aflatoxins, especially aflatoxin B1 (AFB1), are the most prevalent mycotoxins in nature. They contaminate various crops and cause global food and feed safety concerns. Therefore, a simple, rapid, sensitive, and specific AFB1 detection tool is urgently needed. Aptamers generated by SELEX technology can specifically bind the desired targets with high affinity. The broad range of targets expands the scope of applications for aptamers. We used an AFB1-immobilized magnetic nanoparticle for SELEX to select AFB1-specific aptamers. One aptamer, fl-2CS1, revealed a dissociation constant (Kd = 2.5 μM) with AFB1 determined by isothermal titration calorimetry. Furthermore, no interaction was shown with other toxins (AFB2, AFG1, AFG2, OTA, and FB1). According to structural prediction and analysis, we identified a short version of the AFB1-specific aptamer, fl-2CS1/core, with a minimum length of 39-mer used in the AFB1-aptasensor system by real-time qPCR. The aptasensor showed a broad range of detection from 50 ppt to 50 ppb with an accuracy of 90% in the spiked peanut extract samples. With the application of the AFB1-aptasensor we have constructed, a wide range detection tool with high accuracy might be developed as a point-of-care testing tool in agriculture.
| S-EPMC9599246 | biostudies-literature
Project description:BackgroundAn aptamer based biosensor (aptasensor) was developed and evaluated for rapid colorimetric detection of Escherichia coli (E. coli) O157:H7.Methodology/principal findingsThe aptasensor was assembled by modifying the truncated lipopolysaccharides (LPS)-binding aptamer on the surface of nanoscale polydiacetylene (PDA) vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM) was used to confirm the specific interactions between the truncated aptamer and E. coli O157:H7. The aptasensor could detect cellular concentrations in a range of 10(4)~ 10(8) colony-forming units (CFU)/ml within 2 hours and its specificity was 100% for detection of E. coli O157:H7. Compared with the standard culture method, the correspondent rate was 98.5% for the detection of E. coli O157:H7 on 203 clinical fecal specimens with our aptasensor.ConclusionsThe new aptasensor represents a significant advancement in detection capabilities based on the combination of nucleic acid aptamer with PDA vesicle, and offers a specific and convenient screening method for the detection of pathogenic bacteria. This technic could also be applied in areas from clinical analysis to biological terrorism defense, especially in low-resource settings.
| S-EPMC3492178 | biostudies-literature
Project description:Aptamers are widely used in numerous biochemical, bioanalytical, and biological studies. Most aptamers are developed through an in vitro selection process called SELEX against either purified targets or living cells expressing targets of interest. We report here an in vivo SELEX in mice using a PEGylated RNA library for the identification of a 2'-F RNA aptamer (RA16) that specifically binds to NCI-H460 non-small-cell lung cancer cells with an affinity (KD) of 9 ± 2 nM. Interestingly, RA16 potently inhibited cancer cell proliferation in a dose-dependent manner with an IC50 of 116.7 nM. When tested in vivo in xenografted mice, RA16 showed gradual migration toward tumor and accumulation at tumor site over time. An in vivo anti-cancer study showed that the average inhibition rate for mouse tumors in the RA16-treated group was 54.26% ± 5.87% on day 16 versus the control group. The aptamer RA16 adducted with epirubicin (RA16-epirubicin) showed significantly higher toxicity against targeted NCI-H460 cells and low toxicity against non-targeted tumor cells. Furthermore, RA16-epirubicin adduct exhibited in vivo anti-cancer efficacy, with an inhibition rate of 64.38% ± 7.92% when administrated in H460 xenograft mouse model. In summary, a specific bi-functional RNA aptamer RA16 was selected targeting and inhibiting toward NCI-H460 in vitro and in vivo.
| S-EPMC5752333 | biostudies-literature
Project description:TIM3 belongs to a family of receptors that are involved in T-cell exhaustion and Treg functions. The development of new therapeutic agents to block this type of receptors is opening a new avenue in cancer immunotherapy. There are currently several clinical trials ongoing to combine different immune-checkpoint blockades to improve the outcome of cancer patients. Among these combinations we should underline PD1:PDL1 axis and TIM3 blockade, which have shown very promising results in preclinical settings. Most of these types of therapeutic agents are protein cell-derived products, which, although broadly used in clinical settings, are still subject to important limitations. In this work we identify by HT-SELEX TIM3 non-antigenic oligonucleotide aptamers (TIM3Apt) that bind with high affinity and specificity to the extracellular motives of TIM3 on the cell surface. The TIM3Apt1 in its monomeric form displays a potent antagonist capacity on TIM3-expressing lymphocytes, determining the increase of IFN-γ secretion. In colon carcinoma tumor-bearing mice, the combinatorial treatment of TIM3Apt1 and PDL1-antibody blockade is synergistic with a remarkable antitumor effect. Immunotherapeutic aptamers could represent an attractive alternative to monoclonal antibodies, as they exhibit important advantages; namely, lower antigenicity, being chemically synthesized agents with a lower price of manufacture, providing higher malleability, and antidote availability.
| S-EPMC4826223 | biostudies-literature
Project description:We report here a new small molecule fluorogen and RNA aptamer pair for RNA labeling. The small-molecule fluorogen is designed on the basis of fluorescently quenched sulforhodamine dye. The SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure and fluorescence screening in E. coli have been applied to discover the aptamer that can specifically activate the fluorogen with micromolar binding affinity. The systematic mutation and truncation study on the aptamer structure determined the minimum binding domain of the aptamer. A series of rationally modified fluorogen analogues have been made to probe the interacting groups of fluorogen with the aptamer. These results led to the design of a much improved fluorogen ASR 7 that displayed a 33-fold increase in the binding affinity for the selected aptamer in comparison to the original ASR 1 and an 88-fold increase in the fluorescence emission after the aptamer binding. This study demonstrates the value of combining in vitro SELEX and E. coli fluorescence screening with rational modifications in discovering and optimizing new fluorogen-RNA aptamer labeling pairs.
| S-EPMC3044212 | biostudies-literature
Project description:Hepatocellular carcinoma (HCC) is one of the most deadly human cancers due to its ability of invasion and metastasis. Thus, the approaches to identify potential compounds that inhibit invasion and metastasis of HCC are critical for treatment of this disease. In the present study, we used HCCLM9 cells with high metastatic potential and MHCC97L with low metastatic potential as a model system to study the molecular mechanisms of HCC metastasis. By applying cell- Systematic Evolution of Ligands by Exponential enrichment (SELEX) against living cells, we used HCCLM9 as target cells and MHCC97L cells as control to screen a group of HCC metastasis- and cell-specific DNA aptamers. One of selected aptamers, LY-1, could specifically bind to metastatic HCC with a dissociation constant (Kd) in nanomolar range. In vitro studies demonstrated that LY-1 can recognize and bind to membrane protein of metastatic HCC cells. Furthermore, QD605 labeled LY-1 aptamer could recognize HCC cells in both local liver cancer tissues and pulmonary metastatic sites in a xenograft model of HCC with pulmonary metastasis. Further biochemical and immunostaining studies showed that LY-1 could selectively bind to a subpopulation of more metastatic cells in HCCLM9 cells, which express more CK19 and vimentin. Finally, treatment of highly metastatic cells with LY-1 led to reduced migration and invasiveness of HCCLM9 cells in vitro and suppression of xenograft growth in vivo. Taken together, the present study demonstrated the tumor targeting and tumor suppressive effects of LY-1, which could be a promising molecular probe for metastatic HCC and a potential candidate of chemotherapy for metastatic HCC.
| S-EPMC4884992 | biostudies-literature
Project description:Aptamers are short single-stranded oligonucleotides capable of binding to various targets with high specificity and affinity. This study aimed to identify an aptamer against mouse interleukin-2 (mIL-2) as one of the most important cytokines in autoimmune diseases for diagnostic and therapeutic purposes. For this purpose, 14 SELEX rounds were performed on recombinant mIL-2 with high stringency. The dot blot and flow cytometry techniques were conducted to determine affinity, dissociation constant (Kd), specificity, and SELEX rounds screening. The stringency of rounds was considered based on aptamer/target incubation time, washing steps, and target proteins. Finally, the aptamer's structure was mapped and predicted by M-fold and QGRS Mapper web-based software. After 14 rounds, the flow cytometry analysis revealed that the 11th round was a proper round. The high-affinity aptamers M20 and M15 were chosen for their ability to bind mIL-2. According to DNA folding software, M20 and M15 aptamers had G-quadruplex and stem-loop structures, respectively. The M20 aptamer affinity was greater than M15, and its predicted Kd was 91 nM. A simple SELEX protocol with round stringency was explained to identify DNA aptamers against protein targets. The reported G-quadruplex aptamer might have potential diagnostic or therapeutic application in IL-2–related disorders. Interleukin-2; Aptamer; SELEX stringency; G-quadruplex; Dot blot assay.
| S-EPMC9218155 | biostudies-literature
Project description:BACKGROUND: The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ? RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ? in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential ? decoys in two large ? RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ? decoy that competitively inhibits P protein binding to the authentic ? signal on pgRNA. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the first successful identification of human HBV ? aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ? decoy RNAs to interfere with viral P-? complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.
| S-EPMC3220704 | biostudies-literature
Project description:The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 μM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.
| S-EPMC7855447 | biostudies-literature