Aptamer Research Service
According to the different analysis needs of customers, Alpha Lifetech has a variety of aptamer analysis strategies for customers to choose.
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Aptamer Research Service
Introduction to Aptamer
Aptamers are single-stranded nucleic acid ( such as DNA or RNA) molecules that are screened in vitro from a library of synthetic oligonucleotides with randomly assigned sequences by a process called SELEX screening, which involves multiple rounds of iterative selection. The main components of aptamer development services owned by Alpha Lifetech include aptamer library construction, aptamer SELEX screening, aptamer SELEX sequencing, aptamer sequence analysis and other aptamer analysis.
SELEX sequencing combines aptamer SELEX screening and high-throughput sequencing. Through aptamer SELEX sequencing, the specific sequence of the aptamer bound to the target can be efficiently determined, providing basic data for subsequent aptamer sequence analysis and application.
Aptamers have been widely used in scientific research, disease treatment, biosensor construction, medicine discovery and environmental monitoring. However, the oligonucleotide aptamers screened in vitro were easily degraded in vivo and even exhibit toxicity. Therefore, after optimizing the screened aptamers, in vitro analysis is required to evaluate the success of aptamer development. According to the different analysis needs of customers, Alpha Lifetech has a variety of aptamer analysis strategies for customers to choose.
Fig.1 Diagram of aptamer analysis. Reference source: Thevendran R, Citartan M. 2022. Assays to Estimate the Binding Affinity of Aptamers.
Introduction to Aptamer Research Service
Aptamer Stability Analysis
Nuclease degradation experiment
By incubating the aptamer with different types of nucleases (such as DNase, RNase, etc.) under specific conditions, the degradation of the aptamer is observed, so as to evaluate its anti-degradation ability. This is beneficial to determine the stability and durability of the aptamer in practical applications.
Fluorescent labeling method
The stability of the aptamer is evaluated indirectly by labeling the fluorophores on the aptamer and observing the changes of the fluorescence signal before and after binding with the target. For example, when an aptamer binds to a target, its conformation may change, resulting in an enhanced or diminished fluorescence signal.
Thermal stability analysis
The thermal stability of the aptamer is assessed by measuring the change of its melting temperature (Tm value) at different temperatures. The higher the melting temperature of the aptamer, the better its thermal stability.
Dynamic stability analysis
Surface plasmon resonance (SPR) and other technologies were used to monitor the binding and decoupling process between the aptamer and the target in real time, obtain dynamic parameters (such as binding rate constant, dissociation rate constant, etc.), further understand the interaction mechanism between the aptamer and the target, and evaluate its dynamic stability.
Structural stability analysis
X-ray crystallography, nuclear magnetic resonance (NMR) and other structural biology techniques were used to analyze the three-dimensional structure of the aptamer and its structural stability.
Aptamer Specific Analysis
Aptamer binding assay
Affinity is assessed by carring out aptamer binding assay to measure the binding constant between the aptamer and the target molecule, such as the dissociation constant Kd. A lower Kd value indicates a higher affinity. Aptamer binding assay can screen out medicine candidates with high binding ability, and then conduct subsequent pharmacodynamics and pharmacokinetics studies.
Reverse screening experiment
Reverse screening assay is a screening method to quickly exclude non-target molecules from a large number of candidate molecules. Reverse screening is designed to exclude molecules that do not possess this property or function. The key to reverse screening is to select appropriate reverse screening markers or conditions that enable non-target molecules to be identified and removed during the screening process. In this experiment, the selection of non-target molecules is very important. It should be ensured that the non-target molecules selected are representative of substances that may interfere with aptamer screening and that possible interfering factors are covered as fully as possible.
Competitive inhibition experiment
By adding excessive competitors (such as molecules with similar structure to the target molecule) to the binding system of the aptamer and the target molecule, the change of binding ability of the aptamer and the target molecule is observed. If the ability of the aptamer to bind the target molecule is significantly reduced, it indicates that the aptamer has high specificity.
In some cases, competitive inhibition experiments can be used as part of or as an adjunct to reverse screening experiments. For example, in the medicine screening process, the ability of the medicine molecule to bind to the target protein can be evaluated by competitive inhibition experiments, and then reverse screening experiments can be used to remove those compounds that are too strongly bound to normal cells or tissues.
Aptamer Cytotoxicity Analysis
There are a variety of experimental methods for aptamer cytotoxicity analysis, which are designed to evaluate the effects of aptamers on cell survival, proliferation, or function.
| Methods | Detailed Introduction | Advantage | Disadvantage |
|---|---|---|---|
| MTT Detection Method | MTT assay is a method based on enzyme activity in mitochondria of living cells. The succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to the water-insoluble blue-purple crystal Formazan and deposit it in the cell, while dead cells have no such function. Dissolving these crystals by dimethyl sulfoxide (DMSO) and detecting absorbance at specific wavelengths (such as 490nm or 570nm) on an enzymoleter can indirectly reflect the number of living cells and thus assess the cytotoxicity of the aptamer. | High sensitivity, economy and convenience | Heavy workload, organic solvents may cause damage to cells; Only the final experimental results can be obtained, and the complete process of cytotoxicity cannot be viewed |
| CCK-8 Detection Method | Cell Counting Kit-8 (CCK-8) is a high-sensitivity, non-radioactive colorimetric detection method. CCK-8 contains WST-8, which is reduced by dehydrogenase in the mitochondria of living cells to produce highly water-soluble orange methyl zan fuel. The amount of mezan dye produced is linearly related to the number of living cells, and the number of living cells can be indirectly reflected by measuring the light absorption value of Mezan dye at the wavelength of 450nm, so as to evaluate the cytotoxicity of the aptamer. | Simple operation, no need to wash cells, rapid detection, wide linear detection range, high sensitivity, good repeatability, little cytotoxicity | High reagent price; It is sometimes difficult to tell whether the reduced absorbance is due to a decrease in the number of living cells or a decrease in the activity of the cell dehydrogenase itself |
| LDH Detection Method | LDH (lactate dehydrogenase) is an enzyme that is stable in the cytoplasm of cells, and when the cell membrane is damaged, LDH is released outside the cell. LDH can catalyze lactic acid to form pyruvate, and react with INT (tetrazolium salts) to form purple crystalline substance. By measuring its absorbance at a specific wavelength (such as 490nm), the degree of cell damage can be reflected, and then the cytotoxicity of the aptamer can be evaluated. | It directly reflects the death rate of cells, and has less damage to cells, and there is no radioactive isotope contamination | The need to frequently take the cells out of the incubator, the operation is more complicated; Only the final experimental results can be obtained, and the complete process of cytotoxicity cannot be viewed |
| Real-time Live Cell Imaging Analysis | Using real-time live cell imaging analyzer, the instrument was placed in the incubator to observe and record the whole process of cell growth cycle in real time. The cytotoxicity of aptamers can be evaluated by analyzing the morphological changes and growth curves of cells. This method can provide video and quantified results of cytotoxic processes while keeping the cell growth environment stable. | It can monitor the cytotoxic process in real time, non-destructive imaging, reduce the interference and damage to cells; Both video and quantified results are available for in-depth analysis. | The equipment costs are high and requires professional operation skills and data analysis ability |
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Advantage of Aptamer Research Service
Total Quality Control
Optimized Aptamers with higher stability
Economical and efficient. More competitive price, better service
Comprehensive research sites and multiple research methods
Professional pre-sales consultation and after-sales support services
Rich experience in aptamer analysis
High-level R&D team
Customized aptamer related services
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2018-07-16
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