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PloS One 2024DNA-functionalized hydrogels are capable of sensing oligonucleotides, proteins, and small molecules, and specific DNA sequences sensed in the hydrogels' environment can...
DNA-functionalized hydrogels are capable of sensing oligonucleotides, proteins, and small molecules, and specific DNA sequences sensed in the hydrogels' environment can induce changes in these hydrogels' shape and fluorescence. Fabricating DNA-functionalized hydrogel architectures with multiple domains could make it possible to sense multiple molecules and undergo more complicated macroscopic changes, such as changing fluorescence or changing the shapes of regions of the hydrogel architecture. However, automatically fabricating multi-domain DNA-functionalized hydrogel architectures, capable of enabling the construction of hydrogel architectures with tens to hundreds of different domains, presents a significant challenge. We describe a platform for fabricating multi-domain DNA-functionalized hydrogels automatically at the micron scale, where reaction and diffusion processes can be coupled to program material behavior. Using this platform, the hydrogels' material properties, such as shape and fluorescence, can be programmed, and the fabricated hydrogels can sense their environment. DNA-functionalized hydrogel architectures with domain sizes as small as 10 microns and with up to 4 different types of domains can be automatically fabricated using ink volumes as low as 50 μL. We also demonstrate that hydrogels fabricated using this platform exhibit responses similar to those of DNA-functionalized hydrogels fabricated using other methods by demonstrating that DNA sequences can hybridize within them and that they can undergo DNA sequence-induced shape change.
Topics: Hydrogels; DNA; Oligonucleotides; Fluorescence
PubMed: 38306330
DOI: 10.1371/journal.pone.0295923 -
BMC Bioinformatics Nov 2023Single-stranded nucleic acids (ssNAs) have important biological roles and a high biotechnological potential linked to their ability to bind to numerous molecular...
BACKGROUND
Single-stranded nucleic acids (ssNAs) have important biological roles and a high biotechnological potential linked to their ability to bind to numerous molecular targets. This depends on the different spatial conformations they can assume. The first level of ssNAs spatial organisation corresponds to their base pairs pattern, i.e. their secondary structure. Many computational tools have been developed to predict the ssNAs secondary structures, making the choice of the appropriate tool difficult, and an up-to-date guide on the limits and applicability of current secondary structure prediction tools is missing. Therefore, we performed a comparative study of the performances of 9 freely available tools (mfold, RNAfold, CentroidFold, CONTRAfold, MC-Fold, LinearFold, UFold, SPOT-RNA, and MXfold2) on a dataset of 538 ssNAs with known experimental secondary structure.
RESULTS
The minimum free energy-based tools, namely mfold and RNAfold, and some tools based on artificial intelligence, namely CONTRAfold and MXfold2, provided the best results, with [Formula: see text] of exact predictions, whilst MC-fold seemed to be the worst performing tool, with only [Formula: see text] of exact predictions. In addition, UFold and SPOT-RNA are the only options for pseudoknots prediction. Including in the analysis of mfold and RNAfold results 5-10 suboptimal solutions further improved the performances of these tools. Nevertheless, we could observe issues in predicting particular motifs, such as multiple-ways junctions and mini-dumbbells, or the ssNAs whose structure has been determined in complex with a protein. In addition, our benchmark shows that some effort has to be paid for ssDNA secondary structure predictions.
CONCLUSIONS
In general, Mfold, RNAfold, and MXfold2 seem to currently be the best choice for the ssNAs secondary structure prediction, although they still show some limits linked to specific structural motifs. Nevertheless, actual trends suggest that artificial intelligence has a high potential to overcome these remaining issues, for example the recently developed UFold and SPOT-RNA have a high success rate in predicting pseudoknots.
Topics: Nucleic Acid Conformation; Oligonucleotides; Artificial Intelligence; RNA; Entropy; Algorithms
PubMed: 37940855
DOI: 10.1186/s12859-023-05532-5 -
Chemistry (Weinheim An Der Bergstrasse,... Oct 2023Fluorine labeling of ribonucleic acids (RNA) in conjunction with F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and...
Fluorine labeling of ribonucleic acids (RNA) in conjunction with F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA-ligand interactions. This study presents the first syntheses of 2'-OCF guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid-phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2'-OCF modification is associated with preferential C2'-endo conformation of the U and G ribose in single-stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2'-OCF label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2'-OCF modification has potential for applications in therapeutic oligonucleotides. To this end, three 2'-OCF modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2'-OCF nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2'-OCF group without any restrictions in placing the modification into the RNA target sequence.
Topics: RNA; RNA, Small Interfering; Oligonucleotides; Molecular Conformation; Magnetic Resonance Spectroscopy; Oligoribonucleotides; Nucleic Acid Conformation
PubMed: 37534701
DOI: 10.1002/chem.202302220 -
Molecular Biotechnology May 2024Aptamers, as a kind of small-molecule nucleic acid, have attracted much attention since their discovery. Compared with biological reagents such as antibodies, aptamers... (Review)
Review
Aptamers, as a kind of small-molecule nucleic acid, have attracted much attention since their discovery. Compared with biological reagents such as antibodies, aptamers have the advantages of small molecular weight, low immunogenicity, low cost, and easy modification. At present, aptamers are mainly used in disease biomarker discovery, disease diagnosis, treatment, and targeted drug delivery vectors. In the process of screening and optimizing aptamers, it is found that there are still many problems need to be solved such as the design of the library, optimization of screening conditions, the truncation of screened aptamer, and the stability and toxicity of the aptamer. In recent years, the incidence of liver-related diseases is increasing year by year and the treatment measures are relatively lacking, which has attracted the people's attention in the application of aptamers in liver diseases. This article mainly summarizes the research status of aptamers in disease diagnosis and treatment, especially focusing on the application of aptamers in liver diseases, showing the crucial significance of aptamers in the diagnosis and treatment of liver diseases, and the use of Discovery Studio software to find the binding target and sequence of aptamers, and explore their possible interaction sites.
Topics: Humans; Aptamers, Nucleotide; Liver Diseases; SELEX Aptamer Technique; Animals; Biomarkers
PubMed: 38305844
DOI: 10.1007/s12033-023-01030-4 -
Nature Structural & Molecular Biology May 2024Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of...
Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.
Topics: Humans; Kinetics; RNA Stability; Poly A; RNA, Messenger; Adenosine; Receptors, CCR4; Exoribonucleases; RNA Nucleotidyltransferases
PubMed: 38374449
DOI: 10.1038/s41594-023-01187-1 -
Advanced Science (Weinheim,... Jan 2024Recruiting endogenous bone marrow mesenchymal stem cells (BMSCs) in vivo to bone defect sites shows great promise in cell therapies for bone tissue engineering, which...
Recruiting endogenous bone marrow mesenchymal stem cells (BMSCs) in vivo to bone defect sites shows great promise in cell therapies for bone tissue engineering, which tackles the shortcomings of delivering exogenous stem cells, including limited sources, low retention, stemness loss, and immunogenicity. However, it remains challenging to efficiently recruit stem cells while simultaneously directing cell differentiation in the dynamic microenvironment and promoting neo-regenerated tissue ingrowth to achieve augmented bone regeneration. Herein, a synthetic macroporous double-network hydrogel presenting nucleic acid aptamer and nano-inducer enhances BMSCs recruitment, and osteogenic differentiation is demonstrated. An air-in-water template enables the rapid construction of highly interconnective macroporous structures, and the physical self-assembly of DNA strands and chemical cross-linking of gelatin chains synergistically generate a resilient double network. The aptamer Apt19S and black phosphorus nanosheets-specific macroporous hydrogel demonstrate highly efficient endogenous BMSCs recruitment, cell differentiation, and extracellular matrix mineralization. Notably, the enhanced calvarial bone healing with promising matrix mineralization and new bone formation is accompanied by adapting this engineered hydrogel to the bone defects. The findings suggest an appealing material approach overcoming the traditional limitations of cell-delivery therapy that can inspire the future design of next-generation hydrogel for enhanced bone tissue regeneration.
Topics: Hydrogels; Osteogenesis; Bone Regeneration; Tissue Engineering; Oligonucleotides; DNA
PubMed: 37949678
DOI: 10.1002/advs.202303637 -
ACS Nano Nov 2023The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that...
The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells and to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.
Topics: Humans; Antiviral Agents; Oligonucleotides; Pandemics; COVID-19; SARS-CoV-2; Extracellular Vesicles; Erythrocytes
PubMed: 37852618
DOI: 10.1021/acsnano.3c06803 -
Molecules (Basel, Switzerland) Jul 2023Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a... (Review)
Review
Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a neutral phosphorodiamidate linkage. PMOs work via a steric blocking mechanism and are stable towards nucleases' inside cells. PMOs are usually synthesized using phosphoramidate P(V) chemistry. In this review, we will discuss the synthesis of PMOs, phosphoroamidate morpholinos (MO), and thiophosphoramidate morpholinos (TMO).
Topics: Morpholinos; Organophosphorus Compounds; Oligonucleotides, Antisense
PubMed: 37513252
DOI: 10.3390/molecules28145380 -
RNA (New York, N.Y.) Oct 2023Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient...
Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.
Topics: Mice; Humans; Animals; Oligonucleotides, Antisense; Tissue Distribution; Liver; In Situ Hybridization; Staining and Labeling
PubMed: 37460153
DOI: 10.1261/rna.079699.123 -
Nature Communications Dec 2023Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities....
Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.
Topics: Male; Humans; Oligonucleotides, Antisense; RNA; Protein Binding; Peptide Nucleic Acids; Nucleic Acid Hybridization; Phosphorothioate Oligonucleotides
PubMed: 38042877
DOI: 10.1038/s41467-023-43714-0