Owing to their enhanced functional properties, 2 ',4 '-bridged nucleic acids/locked nucleic acids (2 ',4 '-BNAs/LNAs) are considered promising candidates for antisense oligonucleotide therapeutics. The spatial arrangement of LNA residues within the oligonucleotide sequence is a key determinant of binding affinity to the target RNA. In this study, a series of antisense oligonucleotides containing LNA at different positions was synthesized, and their binding affinities toward the target RNA were systematically evaluated. Oligonucleotides with LNA modifications at the terminal regions exhibited approximately 100-fold lower affinity than those modified near the central region. These results offer valuable insights for the rational design of LNA-based antisense oligonucleotides.
Autism spectrum disorder is a complex neurological and developmental disorder that is characterized by altered brain structures and interconnectivity, which results in a vast array of psychosocial and physiological irregularities. This is due to the complex genetic topography of autism and to the intersectionality of genetic and environmental factors that contribute to the development of this disorder. To better understand the genetic factors that cause autism, the gene linc00896, which encodes a long noncoding intergenic RNA, was biochemically and biologically analyzed primarily through circular dichroism, liquid chromatography, and mass spectrometry. From this analysis, it was determined that LINC00896 RNA has a vast interactome and that through this interactome, LINC00896 RNA influences numerous cellular processes that contribute to the symptoms of autistic patients. Additionally, the structural analysis of LINC00896 RNA indicated stable but flexible secondary and tertiary structures that support the numerous binding interactions identified in the interactome. Through these empirical findings, the linc00896 gene was identified as being an important genetic factor that contributes to the development of autism.
The I-R3 DNA enzyme, in its trans -acting form, is capable of cleaving single-stranded DNA (ssDNA) molecules. We have collected all published information on the activity levels of the original I-R3 DNA enzyme and its known variants and embedded that information into a program (we called IR3 ). The program was applied to the sequences of a set of ssDNA viruses and identified all potential catalytic core substrates (targets) and output optimal I-R3 DNA enzyme sequences for all the targets, along with expected activity levels of the enzymes at those targets. Upon experimentally measuring the in vitro cleavage activities of the I-R3 variants, we found marked differences between the program-predicted and experimentally measured values. This demonstrated the incompleteness of the I-R3 model: The sequence of the nucleotides of the catalytic core is not sufficient to fully determine its activity level. A set of experiments was carried out in which the effect of all possible combinations of Watson–Crick base pairs at two positions near the catalytic core, termed S I and S II , was tested. To confirm a newly formed hypothesis, the nucleotide at the S II position of the enzyme strand was mutated to a G and a T, with the substrate strand mutated accordingly. In every case, this led to an increase in relative activity when changed to a G and a decrease, when changed to a T, of the variant I-R3 DNA enzyme. Clearly, the discovered base pair peripheral to the catalytic core has a substantial effect on cleavage activity. This improves the current model of essential nucleotides, and the IR3 software outputs I-R3 enzyme-sequence recommendations that make them more likely to cleave their targets. The software is available for download at https://github.com/XinxinTree/IR3.
The CCAAT/enhancer-binding protein beta (CEBPB or C/EBPβ) is a transcription factor that plays a critical role in cellular differentiation, metabolism, and immune response. Emerging evidence has highlighted its complex involvement in both solid and hematological cancers, such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC), where it can act as an oncogene or a tumor suppressor, depending on the context. In this study, we describe the design and evaluation of a conjugate formed by a small interfering RNA (siRNA) for CEBPB and a transferrin receptor targeting aptamer (TfR-siCEBPB). The designed conjugate is active in human and mouse cells, by transfection and by passive uptake, demonstrating target engagement with strong downregulation of CEBPB mRNA transcript. In murine models of metastatic PDAC and cirrhotic HCC, treatment with TfR-siCEBPB was associated with reduction in tumor burden and improvement in liver function biomarkers. This novel aptamer conjugate allows delivery of targeted oligonucleotide therapy and is a promising therapeutic tool to take forward to human trials.
