Potency of Nucleic Acid
Nucleic acid therapy technology plays an important role in modern biomedicine. They can be divided into three major categories: nucleic acids used for gene knockdowns, such as small interfering RNA (siRNA), antisense oligonucleotide (ASO), single guide RNA (sgRNA) for CRISPR/Cas9, nucleic acids used for gene overexpression (such as mRNA and circRNA) and nucleic acids used for diagnostics, such as probes. The potency of nucleic acid is pivotal for its effectiveness, encompassing functional activity in various settings. The methods for detecting the potency of these types of nucleic acids are different. Evaluating their potency in vitro and in vivo requires tailored approaches based on their specific functions.
Gene Knockdown Nucleic Acids
Nucleic acids such as small interfering RNA (siRNA), antisense oligonucleotides (ASOs), and single guide RNA for CRISPR/Cas9 (sgRNA) are used for gene knockdown, which involves reducing target gene or protein levels.
- In Vitro Potency Assessment
Evaluating the potency of gene knockdown nucleic acids in vitro is crucial for understanding their effectiveness in silencing target genes or proteins. Tab.1 shows various methods used to measure the potency of gene knockdown nucleic acids in vitro.
Tab.1 Methods for determining the potency of gene knockdown nucleic acids in vitro.
| Items | Description | Methods |
|---|---|---|
| Structural Integrity and Stability | Including secondary structures and duplex formation. | Gel Electrophoresis, UV Spectrophotometry, Capillary Electrophoresis |
| Off-Target Analysis | To predict potential off-target sites using Bioinformatics Tools and evaluate potential off-target effects. | TargetScan, Microarray, RNA-seq, Co-Immunoprecipitation (Co-IP), and Mass Spectrometry (MS). |
| mRNA levels | Measures reduction in target mRNA levels. | qRT-PCR |
| Protein levels | Assesses a decrease in target protein levels. | Western blotting, Flow Cytometry |
| Efficiency | Knockdown effect for siRNA | Luciferase Reporter Assay |
| ASO-induced mRNA degradation | Luciferase Reporter Assay | |
| Cleavage efficiency for sgRNA | T7E1 Assay |
- In vivo Biological Activity
The methods for detecting in vivo potency of nucleic acid drugs vary depending on the disease type. For cancer, the focus is on tumor growth inhibition, imaging, and molecular and cellular changes. In neurological diseases, key assessments include behavioral testing, neuroimaging, and electrophysiological testing. For cardiovascular diseases, the evaluation centers on changes in cardiac function, hemodynamics, imaging techniques, and biomarkers. By employing these diverse methods, the potency and therapeutic effects of nucleic acid drugs can be comprehensively evaluated.
Gene Overexpression Nucleic Acids
Gene overexpression nucleic acids, including mRNA, circRNA, and probes, have diverse applications in therapy and research. They are used in vaccines, direct protein replacement therapies, and as essential tools for genetic studies. Evaluating their potency both in vitro and in vivo is crucial for ensuring their efficacy and safety.
- In Vitro Potency Assessment
For overexpression use, such as mRNA and circRNA, potency analysis includes qPCR to measure RNA levels, confirming successful transfection and expression, and Western blotting to detect the presence and quantity of the target protein. Functional assays assess the biological activity of the expressed protein, such as enzyme activity or cell viability.
- In vivo Biological Activity
For mRNA vaccines and therapeutics, immunogenicity assays measure the immune response elicited by mRNA vaccines in animal models or clinical trials.
For circRNA therapeutics, in vivo, expression analysis includes qPCR and RNA-seq to quantify circRNA levels and examine expression profiles in tissues, as well as protein assays to measure the levels and activity of proteins encoded by circRNA. Functional and toxicity studies evaluate the therapeutic effects and potential toxicities in animal models.
Diagnostics Nucleic Acids
Nucleic acids used for diagnostics, such as probes undergo distinct evaluation approaches for both in vitro potency assessment and in vivo imaging and diagnostics. In vitro, hybridization techniques such as Fluorescence in Situ Hybridization (FISH) detect and localize specific nucleic acids within cells, ELISA quantifies nucleic acids captured by probes, and microscopy visualizes probe binding in cellular contexts. For in vivo applications, biodistribution studies analyze the distribution of labeled probes within organisms, adapted FISH techniques detect nucleic acids in live tissues, and advanced imaging modalities like PET and MRI provide real-time visualization of probe localization, crucial for diagnostic and therapeutic purposes.
SERVICES
By assessing the potency of nucleic acids across these multifaceted dimensions, researchers can gain comprehensive insights into their functional activity and suitability for various applications, including therapeutic development, research, and diagnostic purposes. Utilizing our extensive proficiency and state-of-the-art technology, Creative Biolabs offers comprehensive characterization programs dedicated to evaluating nucleic acid potency, ensuring the delivery of top-quality products tailored to your specifications. Contact us for additional information on nucleic acid potency testing, and we're here to offer personalized assistance to meet your specific needs.
