Small interfering RNAs (siRNA) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. siRNA are typically formed by annealing of two complementary single stranded oligoribonucleotides. Compared to purity determination of non-hybridized single strands by denaturing chromatographic methods, characterization of the hybridized duplex is challenging. Here we are reporting a non-denaturing ion pairing-reversed phase (IP-RP) chromatography method capable of separating optimal duplex (full-length single strands only) from non-optimal duplex variants (containing shortmers, longmers and 2',5'-isomers) using ultraviolet- and mass spectrometric detection. The impact of different annealing conditions on siRNA composition was investigated. Optimized annealing conditions lead to a significant increase in optimal duplex, while total duplex content remained constant. The non-denaturing method reported herein showed high mass spectrometric sensitivity and superior separation efficiencies compared to other IP-RP buffer systems. The method is useful for in-process control and release testing of therapeutic double stranded nucleic acids such as siRNA.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1016/j.chroma.2011.06.057 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Aspartic acid unveils as antibiofilm agent and tobramycin adjuvant against mucoid and small colony variants of isolates within cystic fibrosis airway mucus.
Biofilm
June 2025
CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
Antibiotics are central to managing airway infections in cystic fibrosis (CF), yet current treatments often fail due to the presence of biofilms, settling down the need for seeking therapies targeting biofilms. This study aimed to investigate the antibiofilm activity of aspartic acid and its potential as an adjuvant to tobramycin against biofilms formed by mucoid and small colony variant (SCV) tobramycin tolerant strain. We assessed the effect of aspartic acid on both surface-attached and suspended biofilms within CF artificial mucus and investigated the synergistic impact of combining it with non-lethal tobramycin concentrations.
View Article and Find Full Text PDFBlockade of neddylation through targeted inhibition of DCN1 alleviates renal fibrosis.
Clin Sci (Lond)
January 2025
Zhengzhou University First Affiliated Hospital, Zhengzhou, China.
Neddylation is a process of attaching neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) to substrates for the protein function modulation via enzymatic cascades involving NEDD8-activating enzyme (E1), NEDD8-conjugating enzyme (E2), and NEDD8 ligase (E3). Defective in cullin neddylation 1 (DCN1) serves as a co-E3 ligase, that can simultaneously bind E2 UBE2M and cullin proteins to stabilize the catalytic center of the Cullin-Ring E3 ligase (CRL) complex, thereby promoting cullin neddylation. Neddylation is reported to be activated in diverse human diseases, and inhibition of protein neddylation has been regarded as a promising anticancer therapy.
View Article and Find Full Text PDFThe future of siRNA-mediated approaches to treat von Willebrand disease.
Expert Rev Hematol
January 2025
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, the Netherlands.
Introduction: The clinical management of the inherited bleeding disorder von Willebrand disease (VWD) focuses on normalizing circulating levels of von Willebrand factor (VWF) and factor VIII (FVIII) to prevent or control bleeding events. The heterogeneous nature of VWD, however, complicates effective disease management and development of universal treatment guidelines.
Areas Covered: The current treatment modalities of VWD and their limitations are described and why this prompts the development of new treatment approaches.
[Effects of long non-coding RNA KLHL7-AS1 on the proliferation and apoptosis of nucleus pulposus cells under the environment of oxidative stress and its mechanism].
Zhonghua Yi Xue Za Zhi
February 2025
Department of Orthopedics, the First Hospital of Huaian City, Nanjing Medical University, Huaian 223300, China.
To investigate the effects of long non-coding RNA KLHL7-AS1 (LncRNA KLHL7-AS1) on the proliferation and apoptosis of nucleus pulposus cells under oxidative stress and its mechanisms. Human nucleus pulposus cells (HUM-iCell-s012) were divided into 4 groups, and unoxidized nucleus pulposus cells were transfected with an empty pcDNA vector (pcDNA-control) to serve as the blank control group. Based on previous studies on oxidative stress-induced nucleus pulposus cell senescence and preliminary experiments, oxidative stress was induced by treating nucleus pulposus cells with 400 μmol/L HO.
View Article and Find Full Text PDFInhibition of Neutral Sphingomyelinase-2 restrains Enterovirus 71 Infection by Autophagy.
Microb Pathog
January 2025
Department of Laboratory Medicine, Suzhou Mental Health Center, the Affiliated Guangji Hospital of Soochow University, Suzhou215137, Jiangsu, China.
Enterovirus 71 (EV-71) is a major pathogenic factor that causes hand, foot, and mouth disease in young children and infants. Given the limited treatments for EV-71 infection, discovering new host factors and understanding the mechanisms involved will aid in combating this viral infection. Neutral sphingomyelinase-2 (nSMase-2, encoded by SMPD3) is a crucial cellular cofactor in viral infection.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!