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Abstract:Molecular medicine focus on understanding the diseases based on molecular level, and developing personalized medicine strategies for diagnostics and therapeutics. However, powerful molecular recognition tool is still limited for cancer diagnosis and therapy, which impeding cancer research. Aptamers are generated from systematic evolution of ligands by exponential enrichment (SELEX) also known as in vitro selection, ranging from synthetic single-stranded DNA, RNA or XNA (enhanced modified nucleotides), HNA (nucleotides of specific structures such as G quadruplex). The main advantages of aptamers including high specificity, high affinity, simple and rapid synthesis, easy chemical modification, wide target range, good tissue penetration and low immunogenicity. As a molecular recognition tool in molecular medicine, aptamer shows wide applications in developing personalized prediction, diagnosis and therapeutics for its high specificity and high affinity against target. This review discusses the applications of aptamers in disease diagnosis, including aptamer-based tumor marker discovery, liquid biopsy, and molecular imaging, Moreover, the applications of aptamer-based cancer therapy are reviewed, including aptamer-based inhibitors, aptameric drug conjugates, nanomedicines, and aptamer-mediated immunotherapy. Finally, it is promising aptamer will be extensively employed in the future including fundamental research, diagnosis and therapeutics. However, following issues are still need to be addressed. First, the application scenarios-dependent SELEX procedures lack studying which limits the clinical applications of aptamers. Second, the structure of aptamer-target complex has not been fully elucidated, which restricts the precise regulation of aptamers. Third, aptamer is easily degraded by enzymes in vivo and has a short half-life period, which hinders the applications of aptamer-drug conjugates in the development of targeted drugs. With the advancement of screening technology and the further enhancement of aptamer performance, it is expected that aptamers will find more extensive utilization in the field of molecular medicine in the future.

Abstract:Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules with specific molecular recognition capability, obtained by a process called systematic evolution of ligands by exponential enrichment (SELEX). They have the advantages of high thermal stability, ease of chemical synthesis and modification, and low immunogenicity compared to antibodies, and have attracted widespread interest in many fields such as bioanalysis, biomedicine, and biotechnology. High-quality aptamers are the basis of applications, however, the number of them that meet requirements of practical applications is very limited. How to obtain aptamers with high affinity, high specificity, and high in vivo stability is the technical bottleneck in the field of aptamers. Firstly, this review briefly introduces the basic theory of SELEX and its critical experimental steps including design of nucleic acid library, monitoring selection process, preparation of secondary library, sequencing and screening of candidate aptamers. The six main research directions of SELEX during the past thirty years are then concluded. They are respectively (1) how to improve the specificity of aptamers, (2) how to improve the stability of aptamers against nuclease degradation, (3) rapid SELEX, (4) how to isolate aptamers for complex targets, (5) how to isolate small molecule-binding aptamers, and (6) how to isolate high affinity aptamers. The development of rapid SELEX technologies has attracted tremendous attention and almost all physical separation methods have been applied to improve the SELEX efficiency. Very recently, several methods involving the highly efficient chemical reactions have been reported, providing novel strategies for the rapid isolation of aptamers. The key research progresses of SELEX technologies suitable for the isolation of small molecule-binding aptamers are subsequently reviewed and the challenges of each method are critically commented. There are three types of SELEX methods including the target-immobilized SELEX, library-immobilized SELEX (Capture-SELEX), and homogeneous SELEX (GO-SELEX). Even though the target-immobilized SELEX suffers from many issues such as steric hindrance, it is still a popularly used method due to its simplicity. In recent years, Capture-SELEX has been widely applied. The experimental conditions of Capture-SELEX (concentration of positive-SELEX target, choice of negative-SELEX targets and their concentrations) and the affinity (K_D, dissociation constant) and the specificity of the isolated aptamers for the 36 targets are listed in a table. Based on the information from the table, the effect of the experimental conditions on the affinity and the specificity is discussed. The statistical data indicates that the lower concentration of the positive-SELEX targets favors the isolation of the higher affinity aptamers, while it is not a necessary condition. Negative-SELEX is currently the dominant strategy to improve the specificity of aptamers. However, the specificity of many aptamers cannot meet the requirement for practical applications. The choice of negative-SELEX targets and their concentrations in each case are quite different. In 20 out of the 36 targets, no negative-SELEX was performed for the aptamer isolation. How to obtain the aptamers with high specificity is the most difficult challenge for small molecule targets. It is in urgent need to establish novel strategies beyond negative-SELEX to improve the specificity of aptamers. The experimental conditions of GO-SELEX and the K_D and the specificity of the isolated aptamers for the 13 small molecule targets are also list for comparison. The comparison data shows the less numbers of the enrichment cycles required for GO-SELEX than Capture-SELEX, while the obtained aptamers all commonly have K_D in the nanomolar range. The lower enrichment efficiency of Capture-SELEX should be due to the self-dissociation of the immobilized library. The affinity evaluation is the important part of the characterization of aptamer structure and performance. More than ten affinity assays are frequently used for aptamer characterization, which are roughly divided into three categories: separation-based, immobilization-based, and homogeneous methods. All techniques could generate false-positive and false-negative results. Taking gold nanoparticle-based colorimetric assay and isothermal thermal titration as examples, we review the technical progresses and comment on the fundamental reasons resulting in the inconsistent results when the different affinity assays are conducted. The final part of this review provides an outlook on the future trends of aptamer isolation technologies, affinity characterization techniques, and the technical standardization.

Abstract:Renal carcinoma is one of the most common tumors in the urinary system, and its incidence is on the rise. Renal cell carcinoma, as the main type of renal tumor, has a high frequency of local invasion and distant metastasis, and about 33% to 50% of patients with renal cell carcinoma have metastases at the time of discovery. Since there are no specific signs and symptoms in the early stage of renal cell carcinoma, the main treatment is surgical resection, insensitive to radiotherapy and chemotherapy, and the therapeutic means are limited, the early diagnosis of renal cell carcinoma can greatly improve the chances of effective treatment of renal cell carcinoma, which is of great significance for the effective treatment of renal cell carcinoma. Aptamers are oligonucleotide fragments obtained from nucleic acid molecular libraries by systematic evolution of ligands by exponential enrichment (SELEX). It can selectively bind to small molecular ligands or high affinity protein targets, and has high affinity and specificity to target molecules or cells, and has been widely used in tumor imaging diagnosis and targeted therapy. This article mainly reviews the aptamers related to renal cancer, and summarizes and discusses the application of aptamers in the diagnosis and treatment of renal carcinoma.

Abstract:Traditional tumor treatment methods lack sufficient targeting and can cause serious side effects. Exosomes are natural nanovesicles that carries a variety of biomolecules such as nucleic acids, proteins, metabolites, etc. They participate in intercellular communication and serve as drug delivery carriers with exceptional performance advantages, including low immunogenicity, low toxicity, and the ability to traverse natural barriers. However, targeting ability of exosome-based drug delivery systems is still insufficient. Aptamer is a class of chemically synthesized single-stranded nucleic acid molecules, which have the characteristics of small molecular mass, easy modification and low immunogenicity, and can be used as affinity ligands to specifically bind to targeted molecules. Aptamer-modified exosomes can precisely deliver drugs to the site where tumor cells occur, thereby achieving targeted treatment of tumors, enhancing the effectiveness of tumor therapy, and reducing adverse effects. Aptamers can modify exosomes in a variety of ways, mainly including receptor-ligand binding, hydrophobic interactions, DSPE-PEG2000-Mal conjugation, amide bonding, click chemistry, which lays a solid foundation for the development and application of aptamer functionalized exosome drug delivery systems. At present, the aptamer functionalized exosome drug delivery system still faces the following problems, such as the difficulty of aptamer screening, the low efficiency of exosome isolation and purification, and the lack of safety research on the use of aptamer-functionalized exosomes as drug-targeting delivery systems. As long as the above problems are solved, the role of aptamer functionalized exosomes in the treatment of tumors can be maximized. This review will focus on the application of aptamer functionalized exosome drug targeting delivery systems in various tumor treatments, and elaborate on their future challenges and opportunities.

Abstract:Extracellular vesicles (EVs) play an important role in many physiological and pathological processes by participating in intercellular communication. Therefore, the isolation and analysis of EVs have a great value for understanding their biological functions and developing EV-based diagnosis and treatment methods for diseases. The efficient isolation and highly sensitive and reliable detection of EVs largely depend on the recognition ligands. Aptamers are a type of single stranded oligonucleotides that can efficiently and specifically bind to their targets. Their merits of easy modification and programmability make them ideal recognition ligands for EV isolation and analysis. To improve the isolation efficiency of EVs, various strategies have been proposed to enhance the affinity of aptamers and the contact probability between interfaces and EVs. In addition, the isolation of EV subtypes helps to understand the biological significance of EVs. In terms of EV analysis, methods such as electrochemistry, visualization, surface enhanced Raman spectroscopy (SERS) and fluorescence were developed, according to the transduction modes of the recognition signals of aptamers and EVs. This review summarizes recent progress, challenges, and future directions in the selection of aptamers and their applications in EV isolation and analysis.

Abstract:Point-of-care testing (POCT) is an innovative diagnostic technology that provides cost-effective and rapid analysis, as well as accurate diagnostics. It enables patients to obtain clinically relevant results through self-testing. This technology has played a vital role in clinical diagnosis, disease monitoring, and early detection of infectious diseases. Nucleic acid aptamers, which are molecular probes capable of specifically recognizing multiple targets, have emerged as valuable components in biomedical sensors for molecular recognition. They offer advantages such as easy synthesis, good stability, and signal amplification. In recent years, research on aptamer-based POCT technology has garnered widespread attention in the world. The key issues in current research include obtaining more high-affinity aptamers to meet the detection needs of various targets, improving detection sensitivity through signal amplification, and integrating with different sensors to meet the requirements of POCT products. In this review, we first briefly introduce the selection process and the targets used for systematic evolution of ligands by exponential enrichment (SELEX). We discuss new SELEX strategies that have been developed to improve the selection efficiency and enhance the affinity of aptamers. Furthermore, we analyze 4 commonly used signal amplification strategies in aptamer-based POCT sensors. Among these methods, nucleic acid signal amplification and self-assembly signal amplification techniques are commonly used due to their low cost and wide applicability. The combination of these two techniques has also been developed to improve detection sensitivity and shorten reaction time. Coupling aptamers with enzyme-based reactions is the simplest method to improve signal amplification in POCT sensors. Various nanomaterials, such as metal nanoparticles, graphene, carbon nanotubes, and metal-organic frameworks, are widely used to improve the detection sensitivity. The combination of multi-functional nanomaterials for signal amplification has also been introduced in this part. Additionally, we introduce a strategy that involves the use of aptamers to initiate the activation of CRISPR-associated proteins, resulting in the cleavage of DNA or RNA molecular beacons and leading to signal amplification. Furthermore, we also introduce the most recent advances in the development of various aptamer-based electrochemical sensors and optical sensors in the field of POCT. Aptamer-based electrochemical sensors offer advantages such as fast response, easy operation, and portability. In this part, we highlight a series of blood glucose meter based aptasensors used to quantify a variety of biomarkers. For the importance of research on continuous detection device, we review recent progress in the development of aptamer-based continues electrochemical testing devices. In aptamer-based optical POCT techniques, the recent development of colorimetry, lateral flow assay (LFA), fluorescence, surface-enhanced Raman scattering (SERS), surface plasmon resonance (SPR), and evanescent wave fiber optic sensors are introduced, with a focus on comparing the performance characteristics of each sensor. Finally, this review presents a summary and future challenges in the research and commercialization of aptamer-based POCT sensors. To simplify the aptamers selection process, it is crucial to invest in studying the molecular recognition mechanisms of aptamers and developing artificial intelligence-based methods for aptamer selection. Additionally, integrating aptamers with advanced sensor technologies like microfluidic chips and wearable devices can greatly enhance detection sensitivity and stability. From a commercial perspective, current aptamer-based POCT products mostly comprise fluorescent or colorimetric assay kits and lateral flow test strips. However, to garner more attention in the competitive POCT market, aptamer-based POCT sensors have an edge in small molecules detection and multi-channel detection.

Abstract:Abnormal glycosylation of tumor cells is a sign of cancer, and it plays a vital role in malignant transformation and cancer progression. Tumor-associated carbohydrate antigens (TACAs) caused by different mechanisms have been suggested as biomarkers for clinical oncology diagnosis, as well as specific targets for therapeutic interventions. For both aspects, the development of TACA-specific binders with high affinity and specificity is of essential significance. Lectins and antibodies are the major biological tools for the recognition of specific glycans. However, due to the complex structural homology and low immunogenicity of glycans, the recognition capability of lectins and preparation of sugar-specific antibodies are facing distinct challenges. Aptamers, which are short single-stranded DNA/RNA oligonucleotides capable of recognizing a range of chemical and biological species, seem to be a potential solution. They exhibit several significant advantages, such as smaller size, better stability, easier synthesis, facile modification, lower toxicity, and immunogenicity, for in vivo utilization. In recent years, aptamers have attracted increasing attention in the recognition of carbohydrates, but review literatures on aptamers targeting glycans are lag behind. This review focuses on the current development of TACA-binding aptamers. Firstly, we present a brief overview of the role of glycosylation changes in cancer growth, and cite some frequent TACAs as recognized hallmark traits. Secondly, we discuss the major challenges that hinder the exploration of glycan recognition receptors, and compare the strengths and weaknesses of lectins and antibodies. Thirdly, we underline the unique advantages of aptamers, and summarize the available or improved TACA-binding aptamers. According to the target sources in the aptamer screening procedure, 3 kinds of targets including purified carbohydrate molecules, glycan epitope of proteins, and serum carbohydrate antigens are described. We highlight specific examples to emphasize the progress in terms of screening methods, aptamer performance and applicabilities. Finally, we conclude the main contents, and provide the suggestions and directions for developing more valuable, effective, and high-performance TACA-targeted aptamers in the future. In contrast with lectins and antibodies, aptamers are still a newly emerging force, and the aptamer-based scientific research and translational applications have experienced rapid expansion recently. It is worth noting that only a few aptamers with sufficient affinity, specificity, and stability could be used for practical applications, and the number of aptamers available for glycan recognition is even more limited. Until now, most aptamers against glycoprotein biomarkers have been obtained without directing the selection towards any specific region of the target. Consequently, in most cases it is not known whether glycan, peptide or both are involved in the binding. It has still remained a great challenge to screen glycan-specific aptamers, and more efforts are needed to elevate the application of aptamers in cancer diagnosis and treatment to a new level. With the continuous advancement of aptamer screening technology, we believe that more new types of TACA aptamers will be generated, and their specificities will be further improved. Therefore, nucleic acid aptamers hold great prospect and strong market in the future clinical practice, and the detection of glycoforms of current biomarkers is a promising approach to improve sensitivity and specificity in early cancer diagnosis.

Abstract:Aptamers, as novel recognition molecules, have great potential in the field of analysis and detection. Steroid hormones are a kind of hormones with cyclopentane polyhydrophene as the mother nucleus, including sex hormones and corticosteroids, which have extremely important medical value in maintaining life and regulating physiological state. It is of great significance to monitor the content of steroids in vivo and in vitro for disease monitoring and environmental protection. The aptamer isolation of steroid hormones is the prerequisite for the application of aptamers to the analysis and detection of steroidal hormones. So far researchers have isolated aptamers for estrogens such as estradiol (E2) and estrone (E1), androgens such as testosterone (TES) and nortestosterone, and progesterone (P4) and corticosteroids such as cortisol (COR). This paper summarizes the isolation principles and strategies of the reported steroid hormone aptamers. The aptamer sequence, equilibrium dissociation constants (K_D) and determination methods are summarized briefly. The new ideas of computer simulation technology in aptamer isolation and optimization are introduced. The various steroid hormone aptamer sensors developed at present are briefly introduced in order to provide reference for the follow-up research.

Abstract:The puffer fish has won the hearts of diners because of its rich nutrition and delicious meat. However, tetrodoxin (TTX) is extremely toxic and has stable physical and chemical properties. It is difficult to inactivate it by conventional cooking methods. If it is inadvertently eaten or improperly handled, it can easily lead to poisoning accidents. TTX poses a great threat to ecological security, food safety and public safety. Therefore, rapid, efficient, sensitive and specific detection technology is an important measure to effectively prevent and respond to TTX poisoning incidents. After years of exploration and research, researchers have gradually formed traditional and classic bioassay methods, immunoassay methods and chemical assay methods. These three types of detection methods have their own advantages and disadvantages. According to the actual application scenarios and combined with their detection characteristics, the three types of methods have been used independently or in combination to varying degrees. The difficulty of TTX detection lies in the small content and many interference factors in the sample. The traditional detection methods still have the disadvantages of weak anti-interference ability and insufficient sensitivity. The emergence of aptamers, a new molecular recognition element, has opened up a new path for TTX detection. In particular, the combination of aptamers with biosensor platforms such as fluorescence technology and SERS technology makes it possible to achieve rapid, ultra-sensitive and real-time detection of TTX.

Abstract:Objective To investigate the target RNA cleavage activity of V4-2-Dz a conjugate formed by coupling the aptamer V4-2 of the hepatocellular carcinoma cell-specific membrane protein VASN (vasorin) with the DNAzyme (Dz) targeting the telomerase reverse transcriptase (hTERT), in vitro and in hepatocellular carcinoma cells, and to establish a new strategy for DNAzyme delivery.Methods The secondary structure of V4-2-Dz was predicted by using the Mfold website and the cleavage activity of V4-2-Dz was examined by in vitro cleavage assay. And then, the ability of V4-2-Dz binding to hepatocellular carcinoma HepG2 cells was examined by flow cytometry and laser confocal assay. The effects of V4-2-Dz on the hTERT mRNA expression and cell proliferation of HepG2 cells were analyzed by using RT-qPCR and MTT assays.Results In vitro cleavage assays showed that V4-2-Dz has hTERT RNA cleavage activity. Flow cytometry and laser confocal results showed that V4-2-Dz specifically binds to HepG2 cells and could reduce the hTERT mRNA levels and inhibit cell proliferation significantly.Conclusion The conjugate of aptamer V4-2 with DNAzyme of telomerase reverse transcriptase, V4-2-Dz, has targeted cellular delivery and cleavage activity. The conjugate based on the aptamer and DNazyme provides a new idea for antitumor drug research.

Abstract:Objective To construct a novel electrochemical aptasensor for L-lactate detection.Methods Based on gold-palladium-doped nitrogen-rich multi-walled carbon nanotube nanocomposite (Au/Pd-N-MWCNTs) modified glassy carbon electrodes, a triple-helix molecular switch (THMS) triggers the cyclic shearing effect of Pb²⁺-assisted deoxyribozyme (DNAzyme) with RNA-cleaving activity on the signal probes that are loaded on the electrode surface, used for ultrasensitive electrochemical detection of L-lactate. Differential pulse voltammetry (DPV) records the current signal changes.Results The signal probes concentration of 4 μmol/L, the Pb²⁺ concentration of 4 μmol/L, and the DNAzyme shear incubation time of 60 min are the optimal test conditions for the sensor. Under optimal experimental conditions, the L-lactate sensor’s linear range is 1-20 mmol/L, and the detection limit is 0.51 mmol/L. In addition, the aptasensor has excellent stability (RSD=1.04%), reproducibility (RSD=2.80%) and selectivity. When detecting L-lactate in human serum samples, the recoveries were 105.60%- 110.80%, and the RSDs were 2.35%-4.56%, which has high consistency with the traditional method.Conclusion The aptasensor can realize ultrasensitive L-lactate detection and has broad application prospects in biomedical diagnosis, the food industry, and environmental monitoring.

Abstract:Objective Vibrio anguillarum is an important pathogen in aquaculture, which can infect salmon, eel, perch and flounder. Rapid detection is necessary to ensure aquaculture and food safety.Methods Based on the strong affinity between V. anguillarum and its aptamers, the bacterium can capture its aptamer from the surface of colloidal gold particles and change the absorbance of the colloidal gold solution. Thus, a colorimetric method for the quantitative detection of V. anguillarum was established.Results The optical density for V. anguillarum by the present method was much higher than those of non-target bacteria such as Vibrio alginolyticus, Pseudomonas aeruginosa, Pseudomonas plecoglossicida, Aeromonas hydrophila and Edwardsiella tarda (P<0.01). The detection method had good linear relationship in the range of 1-10⁵ CFU/ml. The seawater samples with different salinities and fish body tissue samples were detected for the application, and the results showed that the recoveries and relative standard deviations of these samples were in line with the corresponding detection standards.Conclusion The present detection method has good specificity for V. anguillarum and can be used for the quantitative detection of the bacterium in aquatic products or food.

Abstract:Objective To study the effectiveness of DNA-RNA molecules linked with aptamers as hybrid vectors to target tumor cells and introduce functional RNA molecules into cells, as well as their effects on tumor cells.Methods Design and synthesize short complementary DNA and RNA molecules, and assemble them into DNA-RNA hybrid chains; connect AS1411 aptamer as targeting molecule, and then connect p21 saRNA and TIGIT siRNA as drug molecules, denoted as P21 saRNA and TIGIT siRNA , constitute a hybrid vector, the general structural formula is AS1411-DNA/RNA-sxRNA; detect whether AS1411-DNA/RNA-sxRNA can target and enter tumor cells and its effect on the survival, migration, invasion and apoptosis of tumor cells.Results Equimolar parts of the designed hybrid vector were added to the hybridization buffer system and incubated at a specific temperature. TBM polyacrylamide gel electrophoresis detected the successful assembly of AS1411-DNA/RNA-sxRNA; AS1411-DNA/RNA-sxRNA hybridized carriers are also showed good anti-degradation stability under the condition of 10% serum; observed under fluorescence microscope and laser confocal microscope, there were a large number of green fluorescent signals on the surface and in the cell of SKOV3 cells, and the hybrid carrier successfully entered the tumor cells. After the hybrid carrier incubated, at the mRNA level, the expression of p21 gene (2.14±0.25) was nearly 2 times that of the control group (1.02±0.10), P<0.05, the expression of TIGIT gene (0.63±0.09) was lower than that of the control group (1.09±0.15), P<0.05, at the protein level, the expression of p21 gene (1.57±0.16) was more than 1.5 times that of the control group (1.10±0.09), P<0.05, the expression of TIGIT gene (0.61±0.12) was lower than that of the control group (1.01±0.07) , P<0.05. The CCK-8 experiment showed that the proliferation ability of ovarian cancer cells decreased significantly (P<0.05) in the P21 saRNA (3.10±0.13) and TIGIT siRNA (2.91±0.13) groups, compared with the blank control group (3.67±0.15); the experimental results showed that the healing rate of P21 saRNA group ((42.53±2.90)%) and TIGIT siRNA group ((36.23±3.43)%) were significantly lower than those of the blank control group ((76.47±3.64)%), P<0.05; Transwell assay showed that: P21 saRNA group (128.25±5.36), TIGIT siRNA group (119.50±8.79) were lower than the control group (186.5±8.56), P<0.05. And the invasion ability: P21 saRNA group (145.5±9.45), TIGIT siRNA group (112.25±5.63) were also significantly lower than the control group (202.50±10.12), P<0.05; apoptosis rate: P21 saRNA group ((11.74±2.47)%) and TIGIT siRNA group ((17.12±2.04)%) were significantly higher than the control group ((5.66±1.44)%), P<0.05.Conclusion The prepared AS1411-DNA/RNA-sxRNA hybrid vector can effectively target tumor cells, carry functional small RNAs into tumor cells, regulate the expression of target genes, and inhibit the proliferation, invasion and migration of tumor cells. The results provide an experimental basis for using DNA-RNA conjugated AS1411 aptamer as a hybrid carrier as a targeting tool to target and kill tumor cells expressing NCL protein on the surface.

Abstract:Objective Colon cancer is a common malignant tumor of the gastrointestinal tract and the second leading cause of cancer-related death. It is a type of cancer which is difficult to detect in early stage and easy to metastasize in late stage operation. The aim of this work is to solve the difficult problem of early diagnosis of cancer and realize the early detection of colon cancer and provide a new idea for early diagnosis of colon cancer.Methods Serum-SELEX technique was used to screen the specific aptamers of colon cancer serum. The aptamer sequence with higher affinity and more stable properties can be obtained through the cyclic process of prophase positive screening for colon cancer and prophase positive screening for colon cancer and prophase positive screening for other cancers, and gradually increasing the screening pressure, such as reducing the incubation time of positive screening targets, increasing the incubation time of reverse screening targets and elution steps. qPCR was used to quantitatively monitor the positive screen template obtained to ensure that the aptamer pool was enriched. In this process, the aptamers of common protein binding of negative and positive targets were continuously reduced, so that the sequences in the pool had high specificity. After 16 rounds of screening, a total of four candidate aptamers were selected.Results The results showed that APT-1, APT-2 and APT-5 had high family homology, and the four candidate aptamers mainly formed stem ring and pseudojunction. Affinity was tested by qPCR. The K_d values of the four candidate aptamers binding to the serum of colon cancer patients were (4.466±0.384), (6.122±0.615), (5.610±0.202) and (6.964±0.181) nmol/L, respectively, which were all smaller than 10 nmol/L. The specificity of the serum of about 60 patients was analyzed. It was found that candidate APT-2 had good specificity, and the detection rate of this method was about 82.5%.Conclusion An excellent functional APT-2 was selected, and the APT-2 could be specifically bound to the serum of colon cancer patients with good affinity, so it has a good development prospect for the early diagnosis of colon cancer.

Abstract:Objective The purpose of this study is to improve the affinity and selectivity of aptamers of progesterone (P4).Methods The computer-aided optimization strategy (in silico maturation, ISM) based on genetic algorithm (GA) was used. The 4 rounds of GA (including crossover mutation, single point mutation and double point mutation operations) were performed to construct the initial library and G1, G2 and G3 generation ssDNA oligonucleotides as new candidate aptamer libraries. The candidate aptamers were isolated and analyzed by molecular docking, and continuously optimized using an iterative strategy. In addition, a new strategy is proposed to predict the tertiary structure of ssDNA more accurately. Their secondary and tertiary structures were modeled by Mfold and RNA composer respectively. Output PDB files were modified from RNA to DNA in the Discovery Studio software. Finally, the structure is energy minimized by Molecular Operating Environment.Results In the second generation, P4S-0 sequence was optimized in the local search space, and P4G1-14, P4G2-20, P4G1-6, P4G1-7 and P4G2-14 were selected as the best aptamers for P4. AuNPs colorimetric method was applied to verify the affinity of the optimized aptamer. And then a fluorescence method based on the structure switch of the aptamer was constructed to determine the dissociation constant (K_D) of the aptamer, and the selectivity of the aptamer (for bisphenol A, estradiol, testosterone and cortisol) was evaluated.Conclusion The aptamer optimized by ISM has a greater affinity for P4 than the original aptamer, and still retains the selectivity for recognizing molecules with similar structure.

Abstract:Objective To obtain the finest aptamers with stronger affinity and shortest sequences from the combination of molecular docking simulation and surface plasmon resonance (SPR) evaluation experimentation.Methods Towards three previously screened single-stranded DNA aptamers against ricin with 80 nt, L14, P3, L7, on the confirmation basis of binding ability between their 2D stem-loop sequences of random region with target proteins, the H-DOCK molecular docking was performed to guide the determination of their minimum aptamer binding motifs (MABM), and an elongated stepping sequence cluster was sequentially constructed. The affinity and kinetic parameters of the designed clusters were measured by SPR, in which the binding key structures of the selected aptamers were depicted, and finally the finest aptamers could be selected.Results All three random region aptamers, L14r, P3r and L7r, can form some certain hairpin structures, and the affinity of L14r is increased by nine times than L14, L7r increased by 2 times, and P3r kept the same. The results of molecular docking between the random region aptamers and ricin showed that, the docking scores of L14r, P3r and L7r were all lower than negative sequence of 40T, the number of key binding amino acids were 11, 8 and 9, and the predicted binding sites with distances less than 5 ? were 20, 12 and 15, respectively, indicating good binding ability with ricin. Further, the sequence composition of MABM, L14rm, P3rm and L7rm, were deduced from the binding structures confined in the ricin active pockets, and the elongated stepping sequence cluster was built. On the parameters including the number of key binding amino acids, binding sites, the docking scores, as well as the results of SPR evaluation, the finest aptamers were evolved, in which the affinity of L14rm and L7rm-2 continued to be increased by 1-2 times.Conclusion The random region aptamer can effectively bind ricin with stronger affinity than the full-length aptamer. Molecular docking and stepping sequence clusters design can aid the fast evolution of three finest aptamers from only 17 sequences as well as the investigation of binding interaction. The K_D values of the three finest aptamers against ricin, L14rm, P3r and L7rm-2, were (64±30), (167±19) and (120±1) nmol/L, respectively, and the affinity was increased to 14, 1 and 4 times of the full-length aptamers.