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PloS One 2024Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS...
Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS events, such as expressed sequence tags (EST), microarrays and RNA-seq. However, EST has limitations in identifying low-abundance genes, while microarray and RNA-seq are high-throughput technologies, and PCR-based technology is needed for validation. To overcome the limitations of EST and shortcomings of high-throughput technologies, we established a method to identify AS events, especially for low-abundance genes, by reverse transcription (RT) PCR with gene-specific primers (GSPs) followed by nested PCR. This process includes two major steps: 1) the use of GSPs to amplify as long as the specific gene segment and 2) multiple rounds of nested PCR to screen the AS and confirm the unknown splicing variants. With this method, we successfully identified three new splicing variants, namely, GenBank Accession No. HM623886 for the bdnf gene (GenBank GeneID: 12064), GenBank Accession No. JF417977 for the trkc gene (GenBank GeneID: 18213) and GenBank Accession No. HM623888 for the glb-18 gene (GenBank GeneID: 172485). In addition to its reliability and simplicity, the method is also cost-effective and labor-intensive. In conclusion, we developed an RT-nested PCR method using gene-specific primers to efficiently identify known and novel AS variants. This approach overcomes the limitations of existing methods for detecting rare transcripts. By enabling the discovery of new isoforms, especially for low-abundance genes, this technique can aid research into aberrant splicing in disease. Future studies can apply this method to uncover AS variants involved in cancer, neurodegeneration, and other splicing-related disorders.
Topics: Alternative Splicing; Humans; Brain-Derived Neurotrophic Factor; Reverse Transcriptase Polymerase Chain Reaction; DNA Primers
PubMed: 38935635
DOI: 10.1371/journal.pone.0305201 -
International Journal of Molecular... Jun 2024Affinity reagents, or target-binding molecules, are quite versatile and are major workhorses in molecular biology and medicine. Antibodies are the most famous and... (Review)
Review
Affinity reagents, or target-binding molecules, are quite versatile and are major workhorses in molecular biology and medicine. Antibodies are the most famous and frequently used type and they have been used for a wide range of applications, including laboratory techniques, diagnostics, and therapeutics. However, antibodies are not the only available affinity reagents and they do have significant drawbacks, including laborious and costly production. Aptamers are one potential alternative that have a variety of unique advantages. They are single stranded DNA or RNA molecules that can be selected for binding to many targets including proteins, carbohydrates, and small molecules-for which antibodies typically have low affinity. There are also a variety of cost-effective methods for producing and modifying nucleic acids in vitro without cells, whereas antibodies typically require cells or even whole animals. While there are also significant drawbacks to using aptamers in therapeutic applications, including low in vivo stability, aptamers have had success in clinical trials for treating a variety of diseases and two aptamer-based drugs have gained FDA approval. Aptamer development is still ongoing, which could lead to additional applications of aptamer therapeutics, including antitoxins, and combinatorial approaches with nanoparticles and other nucleic acid therapeutics that could improve efficacy.
Topics: Aptamers, Nucleotide; Humans; Animals; SELEX Aptamer Technique
PubMed: 38928448
DOI: 10.3390/ijms25126742 -
International Journal of Molecular... Jun 2024Selenium is an essential inorganic compound in human and animal nutrition, involved in the proper functioning of the body. As a micronutrient, it actively contributes to...
Selenium is an essential inorganic compound in human and animal nutrition, involved in the proper functioning of the body. As a micronutrient, it actively contributes to the regulation of various metabolic activities, i.e., thyroid hormone, and protection against oxidative stress. However, Se exhibits a narrow concentration window between having a positive effect and exerting a toxic effect. In higher doses, it negatively affects living organisms and causes DNA damage through the formation of free radicals. Increased reactivity of Se anions can also disrupt the integrity and function of DNA-repairing proteins. As the permissible concentration of Se in drinking water is 10 µg/L, it is vital to develop sensitive and robust methods of Se detection in aqueous samples. In this study, for the first time, we proposed a selective aptamer for selenate ion detection, chosen following the SELEX process, and its application in the construction of an electrochemical aptasensor towards SeO ions. Measurement conditions such as the used redox marker and pH value of the measurement solution were chosen. The proposed aptasensor is characterized by good selectivity and an LOD of 1 nM. Conditions for biosensor regeneration and storage were also investigated in this research.
Topics: Aptamers, Nucleotide; Selenic Acid; Biosensing Techniques; Electrochemical Techniques; Ions; SELEX Aptamer Technique; Humans; Limit of Detection
PubMed: 38928366
DOI: 10.3390/ijms25126660 -
International Journal of Molecular... Jun 2024Polymerase Chain Reaction (PCR) amplification is widely used for retrieving information from DNA storage. During the PCR amplification process, nonspecific pairing...
Polymerase Chain Reaction (PCR) amplification is widely used for retrieving information from DNA storage. During the PCR amplification process, nonspecific pairing between the 3' end of the primer and the DNA sequence can cause cross-talk in the amplification reaction, leading to the generation of interfering sequences and reduced amplification accuracy. To address this issue, we propose an efficient coding algorithm for PCR amplification information retrieval (ECA-PCRAIR). This algorithm employs variable-length scanning and pruning optimization to construct a codebook that maximizes storage density while satisfying traditional biological constraints. Subsequently, a codeword search tree is constructed based on the primer library to optimize the codebook, and a variable-length interleaver is used for constraint detection and correction, thereby minimizing the likelihood of nonspecific pairing. Experimental results demonstrate that ECA-PCRAIR can reduce the probability of nonspecific pairing between the 3' end of the primer and the DNA sequence to 2-25%, enhancing the robustness of the DNA sequences. Additionally, ECA-PCRAIR achieves a storage density of 2.14-3.67 bits per nucleotide (bits/nt), significantly improving storage capacity.
Topics: Algorithms; Polymerase Chain Reaction; DNA; Information Storage and Retrieval; DNA Primers; Base Sequence
PubMed: 38928155
DOI: 10.3390/ijms25126449 -
Genes Jun 2024Despite remarkable advances in the diagnosis of invasive fungal infections (IFIs), rapid, specific, sensitive, and cost-effective detection methods remain elusive. Due... (Review)
Review
Despite remarkable advances in the diagnosis of invasive fungal infections (IFIs), rapid, specific, sensitive, and cost-effective detection methods remain elusive. Due to their stability, ease of production, and specificity to signature molecules of fungal pathogens, short single-stranded sequences of DNA, RNA, and XNA, collectively called aptamers, have emerged as promising diagnostic markers. In this perspective, we summarize recent progress in aptamer-based diagnostic tools for IFIs and discuss how these tools could potentially meet the needs and provide economical and simple solutions for point-of-care for better management of IFIs.
Topics: Humans; Aptamers, Nucleotide; Invasive Fungal Infections; Fungi; SELEX Aptamer Technique
PubMed: 38927669
DOI: 10.3390/genes15060733 -
Biomolecules Jun 2024De-differentiation and subsequent increased proliferation and inflammation of vascular smooth muscle cells (VSMCs) is one of the mechanisms of atherogenesis. Maintaining...
De-differentiation and subsequent increased proliferation and inflammation of vascular smooth muscle cells (VSMCs) is one of the mechanisms of atherogenesis. Maintaining VSMCs in a contractile differentiated state is therefore a promising therapeutic strategy for atherosclerosis. We have reported the 18-base myogenetic oligodeoxynucleotide, iSN04, which serves as an anti-nucleolin aptamer and promotes skeletal and myocardial differentiation. The present study investigated the effect of iSN04 on VSMCs because nucleolin has been reported to contribute to VSMC de-differentiation under pathophysiological conditions. Nucleolin is localized in the nucleoplasm and nucleoli of both rat and human VSMCs. iSN04 without a carrier was spontaneously incorporated into VSMCs, indicating that iSN04 would serve as an anti-nucleolin aptamer. iSN04 treatment decreased the ratio of 5-ethynyl-2'-deoxyuridine (EdU)-positive proliferating VSMCs and increased the expression of α-smooth muscle actin, a contractile marker of VSMCs. iSN04 also suppressed angiogenesis of mouse aortic rings ex vivo, which is a model of pathological angiogenesis involved in plaque formation, growth, and rupture. These results demonstrate that antagonizing nucleolin with iSN04 preserves VSMC differentiation, providing a nucleic acid drug candidate for the treatment of vascular disease.
Topics: Nucleolin; Animals; RNA-Binding Proteins; Muscle, Smooth, Vascular; Aptamers, Nucleotide; Cell Proliferation; Phosphoproteins; Cell Differentiation; Humans; Rats; Myocytes, Smooth Muscle; Mice; Cells, Cultured; Oligodeoxyribonucleotides; Male; Rats, Sprague-Dawley; Mice, Inbred C57BL
PubMed: 38927112
DOI: 10.3390/biom14060709 -
Biomolecules Jun 2024MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal...
Downregulation of miR-1388 Regulates the Expression of Antiviral Genes via Tumor Necrosis Factor Receptor ()-Associated Factor 3 Targeting Following poly(I:C) Stimulation in Silver Carp ().
MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal physiological functions in fish. Nevertheless, the specific physiological role of miRNAs in lower vertebrates, particularly in comparison to mammals, remains elusive. Additionally, the mechanisms underlying the control of antiviral responses triggered by viral stimulation in fish are still not fully understood. In this study, we investigated the regulatory impact of miR-1388 on the signaling pathway mediated by IFN regulatory factor 3 (). Our findings revealed that following stimulation with the viral analog poly(I:C), the expression of miR-1388 was significantly upregulated in primary immune tissues and macrophages. Through a dual luciferase reporter assay, we corroborated a direct targeting relationship between miR-1388 and tumor necrosis factor receptor ()-associated factor 3 (). Furthermore, our study demonstrated a distinct negative post-transcriptional correlation between miR-1388 and . We observed a significant negative post-transcriptional regulatory association between miR-1388 and the levels of antiviral genes following poly(I:C) stimulation. Utilizing reporter plasmids, we elucidated the role of miR-1388 in the antiviral signaling pathway activated by . By intervening with siRNA-, we validated that miR-1388 regulates the expression of antiviral genes and the production of type I interferons () through its interaction with . Collectively, our experiments highlight the regulatory influence of miR-1388 on the -mediated signaling pathway by targeting post poly(I:C) stimulation. These findings provide compelling evidence for enhancing our understanding of the mechanisms through which fish miRNAs participate in immune responses.
Topics: Animals; MicroRNAs; Poly I-C; Carps; TNF Receptor-Associated Factor 3; Down-Regulation; Interferon Regulatory Factor-3; Gene Expression Regulation; Fish Proteins; Signal Transduction
PubMed: 38927097
DOI: 10.3390/biom14060694 -
Biomolecules May 2024Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need...
Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.
Topics: Humans; Ovarian Neoplasms; Female; RNA, Long Noncoding; Peptide Nucleic Acids; Cyclopentanes; Cell Line, Tumor; Biomarkers, Tumor
PubMed: 38927013
DOI: 10.3390/biom14060609 -
Biosensors May 2024A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living...
A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging.
Topics: Aptamers, Nucleotide; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Biosensing Techniques; Nucleic Acid Hybridization; Fluorescent Dyes
PubMed: 38920578
DOI: 10.3390/bios14060274 -
Biosensors May 2024Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding...
Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.
Topics: Biosensing Techniques; Thrombin; Lipid Bilayers; Quartz Crystal Microbalance Techniques; Aptamers, Nucleotide; Humans; Phosphatidylcholines
PubMed: 38920574
DOI: 10.3390/bios14060270