Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based ...selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers.
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In order to construct an aptasensor, aptamers that show high affinity for target molecules are required. While the systematic evolution of ligands by exponential enrichment (SELEX) is an efficient ...method for selecting aptamers, it sometimes fails to obtain aptamers with high affinity and so additional improvements are required. We applied a genetic algorithm (GA) to post-SELEX screening as an
in silico maturation of aptamers. First, we pre-selected DNA aptamers against prostate specific antigen (PSA) through three rounds of SELEX. To improve the PSA-binding ability of the aptamers, we carried out post-SELEX screening using GA with the pre-selected oligonucleotide sequences. For screening using GA, we replicated the oligonucleotide sequences obtained through SELEX, crossed over and mutated
in silico resulting in 20 sequences. Those oligonucleotide sequences were synthesized and assayed
in vitro. Then, the oligonucleotides were ranked according to PSA-binding ability and the top sequences were selected for the next cycle of GA operation. After GA operations, we identified the aptamer showing a 48-fold higher PSA-binding ability than candidates obtained by SELEX. The dissociation constant (
K
D) of the obtained aptamer was estimated to be several tens of nM. We demonstrated sensing of PSA using the obtained aptamer and succeeded in sensing PSA concentrations between 40 and 100
nM. This is the first report of a DNA aptamer against PSA and its application to PSA sensing.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Systematic evolution of ligands by exponential enrichment (SELEX) is an efficient method to identify aptamers; however, it sometimes fails to identify aptamers that bind to their target with high ...affinity. Thus, post-SELEX optimization of aptamers is required to improve aptamer binding affinity. We developed in silico maturation based on a genetic algorithm as an efficient mutagenesis method to improve aptamer binding affinity. In silico maturation was performed to improve a VEGF-binding DNA aptamer (VEap121). The VEap121 aptamer is considered to fold into a G-quadruplex structure and this structure may be important for VEGF recognition. Using in silico maturation, VEap121 was mutated with the exception of the guanine tracts that are considered to form the G-quartet. As a result, four aptamers were obtained that showed higher affinity compared with VEap121. The dissociation constant (K d) of the most improved aptamer (3R02) was 300 pM. The affinity of 3R02 was 16-fold higher than that of VEap121. Moreover, a bivalent aptamer was constructed by connecting two identical 3R02s through a 10-mer thymine linker for further improvement of affinity. The bivalent aptamer (3R02 Bivalent) bound to VEGF with a K d value of 30 pM. Finally, by constructing a VEGF-detection system using a VEGF antibody as the capture molecule and monovalent 3R02 as the detection molecule, a more sensitive assay was developed compared with the system using VEap121. These results indicate that in silico maturation could be an efficient method to improve aptamer affinity for construction of sensitive detection systems.
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Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, ...and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.
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Proteinase 3 antineutrophil cytoplasmic antibody (PR3-ANCA) is a serologic marker for granulomatosis with polyangiitis. However, recent studies have also shown their role as diagnostic markers for ...ulcerative colitis (UC). This study was performed to investigate the clinical roles of PR3-ANCAs in the disease severity, disease extension, and clinical course of UC.
Serum PR3-ANCAs were measured in 173 UC patients including 77 patients with new-onset patients UC diagnosed within 1 month, 110 patients with Crohn's disease, 48 patients with other intestinal diseases, and 71 healthy controls. Associations between the PR3-ANCA titer and clinical data, such as disease severity, disease extension, and clinical course, were assessed. The clinical utility of PR3-ANCA measurement was evaluated by receiver operating characteristic (ROC) analysis.
PR3-ANCA ≥3.5 U/mL demonstrated 44.5% sensitivity and 95.6% specificity for the diagnosis of UC in all patients. PR3-ANCA positivity was more prevalent in the 77 new-onset UC patients (58.4%). In this group, the disease severity and extension were more severe in PR3-ANCA positive patients than in PR3-ANCA negative group (p<0.001). After treatment, the partial Mayo scores were significantly decreased with the PR3-ANCA titers. The proportion of patients who required steroids for induction therapy was significantly higher among PR3-ANCA positive than negative group. ROC analysis revealed that PR3-ANCA ≥3.5 U/mL had 75% sensitivity and 69.0% specificity for steroid requirement in new-onset UC patients.
Our results indicate that PR3-ANCA measurement is useful not only for diagnosing UC but also for evaluating disease severity and extension and predicting the clinical course.
Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a ...1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.
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A 60-year-old Japanese woman was diagnosed with celiac disease (CeD) and treated with a gluten-free diet. For five years, she had a good clinical course. However, she complained of inappetence and ...nausea. Colonoscopy revealed ulcerative tumors in the terminal ileum. A histological examination of biopsy specimens from the ulcerative tumor showed diffuse infiltration of large atypical lymphocytes. Immunohistologically, the atypical lymphoid cells were positive for cluster of differentiation (CD) 10 and CD20. Many Epstein-Barr virus-encoded small RNA (EBER)-positive atypical lymphocytes were detected by in situ hybridization. This represents the first reported case of Epstein-Barr virus-positive intestinal diffuse large B-cell lymphoma complicated with CeD.
The prevalence of Crohnʼs disease (CD) has been increasing in Japan. If clinicians suspect a case of CD based on clinical symptoms and inflammatory findings on biochemical examination, the final ...diagnosis is confirmed by various morphological and histopathological findings. Although the process of diagnosis has not changed significantly, it has varied owing to the development of new diagnostic tools such as capsule endoscopy and balloon-assisted small bowel endoscopy. The common treatment options mainly include nutritional therapy and drug therapy. However, in recent years, the number of drug therapy options has increased with the addition of biologics and molecular targeted drugs. Shared decision making with patients is necessary to select effective and satisfactory treatment options. In addition, the current treatment of CD requires tight monitoring or “Treat to Target.”
We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n ...denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-D-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein.
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