Journal of Biological Engineering
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2020
·Vol. 14
·期 1
·1-13
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IF 3.429
Liu Qingxiu,Zhang Wei,Chen Siying,Zhuang Zhenjing,Zhang Yi,Jiang Lingli,LIN Jun Sheng
Background Aptamers, single-stranded DNAs or RNAs, can be selected from a library containing random sequences using a method called Systematic Evolution of Ligands by EXponential Enrichment (SELEX). In SELEX, monitoring the enriching statuses of aptamer candidates during the process is a key step until today. Conformational change of an aptamer caused by target-binding in gel can be used to indicate its statuses of binding. Results In this study, an easy-to-implement gel-based diffusion method (GBDM) was developed to monitor the interaction between enriched aptamer candidates and their targets. In order to prove the concept, characterization of aptamers targeting their targets including protein (thrombin) and non-protein molecules (acetamiprid, ATP, atrazine, profenofos and roxithromycin), respectively, were performed using mini gels. Our method has advantages over the common methods including easy performed with labor- and time- saving in experimental operation. The concept has been proven by monitoring enrichment of dynamic aptamer candidate libraries targeting a small molecule 2,2-bis(4-chlorophenyl) acetic acid (DDA) during SELEX process. A mini gel cassette was designed and fabricated by our laboratory to make mini agarose gels for diffusion with different directions. Conclusions These results indicate that GBDM, in particular, chasing diffusion is suitable for monitoring the interaction between enriched aptamer candidates and their targets. These pioneering efforts are helpful for novel aptamer selection by breaking through the technical bottleneck of aptamer development and helpful for development of novel aptasensors.
研究领域:
分子生物学
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生物化学
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分析化学
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生物技术
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PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2021
·
IF 7.46
Nuo Duan,Changxin Li,Xiaoyin Zhu,Shuo Qi,Zhouping Wang,Shijia Wu
Histamine (HIS) and tryptamine (TRY) are heterocyclic biogenic amines (BAs) often found in food products. The consumption of products containing high levels of BAs results in a wide range of life-threatening physiological reactions. The work described a Simultaneous coupled with Separate selection strategy for the screening of different aptamers against histamine and tryptamine. Aptamer HIS-3 and TRY-2 exhibited a good binding affinity ; specificity and pH stability to histamine and tryptamine were obtained with a dissociation constant of 11.62 ± 3.241 nM and 6.303 ± 1.758 nM, respectively. The preliminary binding mechanism was analyzed using a circular dichromatic (CD) and molecular docking. Finally, the identified aptamers were used for the development of a colorimetric method for the detection of histamine and tryptamine, achieving limits of detection of 1 ng/mL and 10 ng/mL, respectively. This aptamer-based method was further successfully employed to detect the BAs in fish and pork, providing a potential application for sensitive and efficient detecting multiple food risk factors.
研究领域:
生物技术
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食品科学
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分析化学
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Analytical Methods
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2016
·Vol. 8
·期 48
·8461-8465
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IF 1.915
Hongyan Wang,Jine Wang,Lijun Xu,Yajie Zhang,Yang Chen,Hongxia Chen,Renjun Pei
Aptamers are single-stranded oligonucleotides which are selected and are capable of binding certain targets tightly and specifically. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) has been used to select aptamers for various targets such as proteins, metal ions, small molecules and so on. For the first time, we have used a modified SELEX technology to obtain DNA aptamers for a fluorogenic dye, thioflavin T (ThT). DNA aptamers with low micromolar dissociation constants were achieved after only 8 rounds of selection and the final minimized 33-mer aptamer showed 191-fold fluorescence enhancement in a saturated concentration. The truncated DNA aptamer showed excellent selectivity and specificity for ThT.
研究领域:
分子生物学
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生物化学
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ANALYTICA CHIMICA ACTA
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2019
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IF 5.256
Huili Tian,Nuo Duan,Shijia Wu,Zhouping Wang
Spermine contamination ranks as one of the food safety issues, it will cause some adverse reactions if the intake of spermine is excessive in human body. So it is of great significance to establish fast and efficient analysis method to detect spermine in foods. In this study, the spermine aptamers with high affinity and specificity were obtained by the capture systematic evolution of ligands by exponential enrichment (Capture-SELEX) technique. Forty-one aptamer sequences were obtained by cloning and sequencing, and were divided into eight families based on homology and secondary structure analysis. The affinity and specificity of candidate aptamers was analyzed by isothermal titration calorimetry (ITC) and fluorescence assay . The aptamers named APJ-6 was picked out as the optimal aptamer that recognizes spermine specifically with the Kd value of 9.648 ± 0.896 nM. In order to verify the practicability of the selected aptamers, the sensitive aptamer-based fluorescene assay was designed. Under optimized conditions, this aptasensor exhibited a low detection limit of 0.052 nM, as well as a linear within the range of 0.1–20 nM. Besides, it has been further applied for the determination of spermine in pork samples and the recoveries ranged from 86.45% to 98.15%, showing its great potential for sensitive analysis in food safety control.
研究领域:
分子生物学
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食品科学
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分析化学
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PubMed
高影响因子
BIOCHEMISTRY
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2026
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IF 2.7
Yuzhe Ding,Qingyu Chen,Yicheng Heng,Emily Hoi Pui Chao,Yunus A. Kaiyum,Philip E. Johnson,Juewen Liu
Kanamycin A, or simply referred to as kanamycin, is an aminoglycoside antibiotic with a narrow therapeutic window. Aptamers are useful recognition molecules for their detection and continuous monitoring. However, a short and high-affinity kanamycin aptamer that works under physiological conditions is still lacking. In this work, we revisited a previous aptamer selection done at pH 8, which had been abandoned due to poor sequence enrichment. Its top sequence, named KAN8–1, shows a Kd of 51 nM at pH 7.5 for kanamycin as measured by isothermal titration calorimetry, and its affinities to kanamycin A and B are similar. Using NMR spectroscopy methods, the KAN8–1 aptamer undergoes ligand-induced folding and likely has a better-defined structure compared to the KAN6–1 aptamer, which was highly enriched in the pH 6 selection. Using the KAN8–1 aptamer, a strand-displacement biosensor was developed, and it has a limit of detection of 0.9 μM with excellent selectivity. This sensor also has a similar performance in serum. The reason for the poor enrichment of KAN8–1 was attributed to its low hybridization efficiency and poor hybridization stability to the capture strand as demonstrated by a fluorescence titration assay and melting analysis, which indicated a limitation of the capture-SELEX method.
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CHINESE CHEMICAL LETTERS
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2021
·
IF 6.779
Yuan Liu,Gaojian Yang,Taotao Li,Yan Deng,Zhu Chen,Nongyue He
An improved ssDNA library immobilized systematic evolution of ligands by enrichment (SELEX) was applied to select aptamers against carbaryl. After nine selection rounds, a highly enriched ssDNA pool was obtained. The Apta3 was demonstrated as the optimal aptamer. In order to facilitate the modification of aptamer, the Apta3 was further truncated with the dissociation constant ( K d ) of 0.364 ± 0.055 μmol/L and a fluorescent aptasensor was developed. The linear range for carbaryl was from 100 nmol/L to 1500 nmol/L, with the limit of detection was as low as 15.23 nmol/L. Besides, the biosensor was validated for the carbaryl spiked real samples, and the recoveries were between 97.7% and 107.3%.
研究领域:
生物化学
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分析化学
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ANALYTICAL BIOCHEMISTRY
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2016
·
IF 2.243
Shijia Wu,Nuo Duan,Weixiao Zhang,Sen Zhao,Zhouping Wang
Patulin (PAT) is a kind of mycotoxin that has serious harmful impacts on both food quality and human health. A high-affinity ssDNA aptamer that specifically binds to patulin was generated using systemic evolution of ligands by exponential enrichment (SELEX) assisted by graphene oxide (GO). After 15 rounds of positive and negative selection, a highly enriched ssDNA pool was sequenced and the representative sequences were subjected to binding assays to evaluate their affinity and specificity. Of the eight aptamer candidates tested, the sequence PAT-11 bound to patulin with high affinity and excellent selectivity with a dissociation constant ( K d ) of 21.83 ± 5.022 nM. The selected aptamer, PAT-11, was subsequently used as a recognition element to develop a detection method for patulin based on an enzyme-chromogenic substrate system. The colorimetric aptasensor exhibited a linear range from 50 to 2500 pg mL −1 , and the limit of detection was found to be 48 pg mL −1 . The results indicated that GO-SELEX technology was appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.
研究领域:
毒理学
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分析化学
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食品科学
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PubMed
高影响因子
RSC Advances
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2015
·Vol. 6
·期 2
·1484-1490
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IF 3.84
Hui-Fang Cui,Ya-Jun Li,Jia Wang,Xiao-Jia Li,Qiong-Lin Wang,Yan-Feng Bai
Pro-gastrin-releasing peptide (31–98) (ProGRP31–98) is a highly reliable, sensitive, and specific tumor marker for small cell lung cancer (SCLC). DNA aptamers for ProGRP31–98 were selected in this study from an 89-mer DNA library with a random region of 48 nucleotides (nt) flanked with two primer hybridization sites, by using the systematic evolution of ligands by the exponential enrichment (SELEX) method. The DNA sequences binding to ProGRP31–98 immobilized magnetic beads were selected by running 12 SELEX cycles and 3 negative selection cycles. The selected DNA sequences were separated by cell based DNA cloning, and then sequence analyzed. The DNA sequences obtained were screened and investigated for binding specificity to ProGRP31–98 by using electrochemiluminescence (ECL) measurement with [Ru(bpy)2dppz]2+, a molecular light-switch, as the ECL probe. One DNA sequence (89 nt) and its random region (48 nt) were identified as specific aptamers for ProGRP31–98. Based on the secondary structures of the obtained DNA aptamers, two other truncated DNA aptamers, 40 nt and 15 nt long, respectively, were identified. The obtained aptamers have strong affinities to ProGRP31–98, with a Kd value of 16 nM, and can detect ProGRP31–98 with a detection limit of 17 nM using the label-free ECL measurement.
研究领域:
分子生物学
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肿瘤学
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分析化学
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2021
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IF 7.46
Hui Yue,Jinri Chen,Xu Chen,Xiaoli Wang,Yuting Zhang,Nandi Zhou
Sialic acids (SAs), particularly its most common derivative N-acetylneuraminic acid (Neu5Ac) are involved in many biological processes in vivo . Thus it is of great significance to detect Neu5Ac and trace the sialylation of glycoproteins and other bio-active molecules. Aptamers are molecular elements for bio-recognition with high affinity, which have been widely used in bioanalysis and imaging. However, the ideal aptamers for saccharides have seldom been reported. Magnetic beads (MB)-based SELEX is a commonly used method for screening of aptamers. Herein, three types of MB-based SELEX with different immobilization manners were carried out to systematically study and compare the screening efficiency, which may provide reference for screening of aptamers specific for low-molecular targets. After screening and sequencing, the obtained aptamer sequences were aligned and analyzed, among which three aptamers were selected as candidates. Then, the aptamer ap 3-1 with the highest affinity was optimized according to the results of molecular simulation docking. An optimal aptamer ap 3-1-a with 21 nucleotides (nt) was eventually obtained, with the K d value of 55.71 ± 12.29 nM. To verify the practicability of the aptamer, a fluorescent biosensor for Neu5Ac was constructed employing graphene oxide (GO)-based fluorescence quenching and Neu5Ac-induced fluorescence restoration. The biosensor showed a linear response range within 20−1000 nM. The specificity was verified and the interferents-resistance was validated by detecting Neu5Ac in actual samples. This study may inspire the efficient and universal strategy for screening and application of robust and practicable aptamers for low-molecular targets.
研究领域:
生物化学
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分子生物学
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分析化学
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高影响因子
ANALYST
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2021
·Vol. 146
·期 6
·1986-1995
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IF 4.616
Yu Luo,Ziying Jin,Jine Wang,Pi Ding,Renjun Pei
Aptamers, which are called chemical antibodies for their high affinity and specificity to targets, have great potential as analytical tools to detect pesticides. In this work, a DNA aptamer for thiamethoxam was isolated by an improved SELEX (systematic evolution of ligands by exponential enrichment) strategy, in which the ssDNA library was fixed on streptavidin-agarose beads through a short biotin labeled complementary strand. After 13 rounds of selection, the random ssDNA pool was successfully enriched. Three sequences were chosen as aptamer candidates through sequencing and analysis and were transformed into fluorescent probes to evaluate their interactions with thiamethoxam. A fluorescent turn-on aptasensor for thiamethoxam based on the best aptamer (FAM-Thi13) and a short quenching strand were further designed and showed a quantitative linear range from 10 to 1000 nM with a detection limit of 1.23 nM for thiamethoxam. Molecular docking and molecular dynamics were used to investigate the binding site of the main probe of the aptasensor (FAM-Thi13) and thiamethoxam. Satisfactory results were also obtained in quantifying thiamethoxam in environmental water samples by the developed fluorescent aptasensor.
研究领域:
分析化学
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环境科学
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分子生物学
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
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2020
·Vol. 21
·期 6
·2211
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IF 4.556
Wei Zhang,Danyang Li,Jianguang Zhang,Lingli Jiang,Zhaofa Li,Jun Sheng Lin
The compound 1,1,1-trichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethane (o,p’-DDT) has been identified as one of the endocrine-disrupting chemicals causing adverse effects on wildlife and even humans through bioaccumulation. Its detection has become increasingly important. We have obtained candidate aptamers binding too,p’-DDT by a systematic evolution of ligands by exponential enrichment (SELEX) protocol. Five out of seventeen candidate sequences were selected for preliminary characterization by SYBR Green I assay. One sequence with highest fluorescence response witho,p’-DDT, designated DDT_13, was chosen for further characterization. Its dissociation constant (Kd) was determined to be 412.3 ± 124.6 nM. DDT_13 exhibited low cross-binding activities on other tested small molecules. The good bioactivities of DDT_13 were demonstrated for the analysis of spiked lake water and tap water samples. This study provides a novelo,p’-DDT-specific probe for its future applications.Keywords:o,p’-DDT;aptamer;SELEX;AuNPs;biosensor;real water sample
研究领域:
环境科学
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分析化学
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生物化学
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SENSORS AND ACTUATORS B-CHEMICAL
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2024
·
IF 8
Wanqi Zhang,Zhicong Sun,Yuhang Tian,Yaoting Mou,Yemin Guo,Xia Sun,Falan Li
Streptomycin (STR) is an antibiotic fungicide that is easily enriched in the environment and threatens human health. In this paper, a ratiometric fluorescence aptasensor with silver nanoclusters-skimmed milk powder@zeolitic imidazolate framework-8 (AgNCs-SMP@ZIF-8) fluorescent material as the response signal and carbon quantum dots (CQDs) functionalized with aptamer as the reference signal was prepared. Magnetic graphene oxide selection technology was used to screen high-affinity STR-specific aptamer. The truncation strategy for removing non-binding site bases was based on molecular simulation docking, and it was found that the stem-loop structure formed by the truncated aptamer 52 (Apt52) was used for STR-specific recognition and had a higher STR affinity ( K d = 14.52 nM) compared to the initial 79-nucleotide aptamer. AgNCs was then covered in a protein coating and encapsulated in ZIF-8 to obtain AgNCs-SMP@ZIF-8 with enhanced red fluorescence intensity . The added Cu 2+ rapidly penetrated into ZIF-8 to induce red fluorescence quenching , while the CQDs modified by Apt52 maintained green fluorescence. Hence, the constructed ratiometric fluorescent sensor with Apt52 as the recognizing element can detect STR within 90 s with a detection limit of 0.98 nM. The ratiometric probe proposed has considerable potential for STR residue detection in milk.
研究领域:
分析化学
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生物技术
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纳米技术
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环境科学
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高影响因子
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