Design and Synthesis of DNA Origami Nanostructures to Control TNF Receptor Activation.

Clustering of type II tumor necrosis factor (TNF) receptors (TNFRs) is essential for their activation, yet currently available drugs fail to activate signaling. Some strategies aim to cluster TNFR by using multivalent streptavidin or scaffolds based on dextran or graphene. However, these strategies do not allow for control of the valency or spatial organization of the ligands, and consequently control of the TNFR activation is not optimal. DNA origami nanostructures allow nanometer-precise control of the spatial organization of molecules and complexes, with defined spacing, number and valency. Here, we demonstrate the design and characterization of a DNA origami nanostructure that can be decorated with engineered single-chain TNF-related apoptosis-inducing ligand (SC-TRAIL) complexes, which show increased cell killing compared to SC-TRAIL alone on Jurkat cells. The information in this chapter can be used as a basis to decorate DNA origami nanostructures with various proteins, complexes, or other biomolecules.

Distinctive Nucleic Acid Recognition by Lysine-Embedded Phenanthridine Peptides.

Three new phenanthridine peptide derivatives (19, 22, and 23) were synthesized to explore their potential as spectrophotometric probes for DNA and RNA. UV/Vis and circular dichroism (CD) spectra, mass spectroscopy, and computational analysis confirmed the presence of intramolecular interactions in all three compounds. Computational analysis revealed that compounds alternate between bent and open conformations, highlighting the latter's crucial influence on successful polynucleotide recognition. Substituting one glycine with lysine in two regioisomers (22, 23) resulted in stronger binding interactions with DNA and RNA than for a compound containing two glycines (19), thus emphasizing the importance of lysine. The regioisomer with lysine closer to the phenanthridine ring (23) exhibited a dual and selective fluorimetric response with non-alternating AT and ATT polynucleotides and induction of triplex formation from the AT duplex. The best binding constant (K) with a value of 2.5 × 107 M-1 was obtained for the interaction with AT and ATT polynucleotides. Furthermore, apart from distinguishing between different types of ds-DNA and ds-RNA, the same compound could recognize GC-rich DNA through distinct induced CD signals.

Interaction of dinuclear Co(III) cylinders with higher-order DNA structures.

Alternative DNA structures play critical roles in fundamental biological processes linked to human diseases. Thus, targeting and stabilizing these structures by specific ligands could affect the progression of cancer and other diseases. Here, we describe, using methods of molecular biophysics, the interactions of two oxidatively locked [Co2L3]6+ cylinders, rac-2 and meso-1, with diverse alternative DNA structures, such as junctions, G quadruplexes, and bulges. This study was motivated by earlier results demonstrating that both Co(III) cylinders exhibit potent and selective activity against cancer cells, accumulate in the nucleus of cancer cells, and prove to be efficient DNA binders. The results show that the bigger cylinder rac-2 stabilizes all DNA structures, while the smaller cylinder meso-1 stabilizes just the Y-shaped three-way junctions. Collectively, the results of this study suggest that the stabilization of alternative DNA structures by Co(III) cylinders investigated in this work might contribute to the mechanism of their biological activity.

Alternative DNA structures in hematopoiesis and adaptive immunity.

Besides the canonical B-form, DNA also adopts alternative non-B form conformations which are highly conserved in all domains of life. While extensive research over decades has centered on the genomic functions of B-form DNA, understanding how non-B-form conformations influence functional genomic states remains a fundamental and open question. Recent studies have ascribed alternative DNA conformations such as G-quadruplexes and R-loops as important functional features in eukaryotic genomes. This review delves into the biological importance of alternative DNA structures, with a specific focus on hematopoiesis and adaptive immunity. We discuss the emerging roles of G-quadruplex and R-loop structures, the two most well-studied alternative DNA conformations, in the hematopoietic compartment and present evidence for their functional roles in normal cellular physiology and associated pathologies.

Exploring the mechanism of interaction of glipizide with DNA: Combined in vitro and bioinformatics approach.

DNA, vital for biological processes, encodes hereditary data for protein synthesis, shaping cell structure and function. Since revealing its structure, DNA has become a target for various therapeutically vital molecules, spanning antidiabetic to anticancer drugs. These agents engage with DNA-associated proteins, DNA-RNA hybrids, or bind directly to the DNA helix, triggering diverse downstream effects. These interactions disrupt vital enzymes and proteins essential for maintaining cell structure and function. Analysing drug-DNA interactions has significantly advanced our understanding of drug mechanisms. Glipizide, an antidiabetic drug, is known to cause DNA damage in adipocytes. However, its extract mechanism of DNA interaction is unknown. This study delves into the interaction between glipizide and DNA utilizing various biophysical tools and computational technique to gain insights into the interaction mechanism. Analysis of UV-visible and fluorescence data reveals the formation of complex between DNA and glipizide. The binding affinity of glipizide to DNA was of moderate strength. Examination of thermodynamic parameters at different temperatures suggests that the binding was entropically spontaneous and energetically favourable. Various experiments such as thermal melting assays, viscosity measurement, and dye displacement assays confirmed the minor grove nature of binding of glipizide with DNA. Molecular dynamics studies confirmed the glipizide forms stable complex with DNA when simulated by mimicking the physiological conditions. The binding was mainly favoured by hydrogen bonds and glipizide slightly reduced nucleotide fluctuations of DNA. The study deciphers the mechanism of interaction of glipizide with DNA at molecular levels.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

A New Strategy to Investigate RNA:DNA Triplex Using Atomic Force Microscopy.

Over the past decade, long non-coding RNAs (lncRNAs) have been recognized as key players in gene regulation, influencing genome organization and expression. The locus-specific binding of these non-coding RNAs (ncRNAs) to DNA involves either a non-covalent interaction with DNA-bound proteins or a direct sequence-specific interaction through the formation of RNA:DNA triplexes. In an effort to develop a novel strategy for characterizing a triple-helix formation, we employed atomic force microscopy (AFM) to visualize and study a regulatory RNA:DNA triplex formed between the Khps1 lncRNA and the enhancer of the proto-oncogene SPHK1. The analysis demonstrates the successful formation of RNA:DNA triplexes under various conditions of pH and temperature, indicating the effectiveness of the AFM strategy. Despite challenges in discriminating between the triple-helix and R-loop configurations, this approach opens new perspectives for investigating the role of lncRNAs in gene regulation at the single-molecule level.

Determinant of m6A regional preference by transcriptional dynamics.

N6-Methyladenosine (m6A) is the most abundant chemical modification occurring on eukaryotic mRNAs, and has been reported to be involved in almost all stages of mRNA metabolism. The distribution of m6A sites is notably asymmetric along mRNAs, with a strong preference toward the 3' terminus of the transcript. How m6A regional preference is shaped remains incompletely understood. In this study, by performing m6A-seq on chromatin-associated RNAs, we found that m6A regional preference arises during transcription. Nucleosome occupancy is remarkedly increased in the region downstream of m6A sites, suggesting an intricate interplay between m6A methylation and nucleosome-mediated transcriptional dynamics. Notably, we found a remarkable slowdown of Pol-II movement around m6A sites. In addition, inhibiting Pol-II movement increases nearby m6A methylation levels. By analyzing massively parallel assays for m6A, we found that RNA secondary structures inhibit m6A methylation. Remarkably, the m6A sites associated with Pol-II pausing tend to be embedded within RNA secondary structures. These results suggest that Pol-II pausing could affect the accessibility of m6A motifs to the methyltransferase complex and subsequent m6A methylation by mediating RNA secondary structure. Overall, our study reveals a crucial role of transcriptional dynamics in the formation of m6A regional preference.

Zα Domain of ADAR1 Binds to an A-Form-like Nucleic Acid Duplex with Low Micromolar Affinity.

The left-handed Z-conformation of nucleic acids can be adopted by both DNA and RNA when bound by Zα domains found within a variety of viral and innate immune response proteins. While Z-form adoption is preferred by certain sequences, such as the commonly studied (CpG)n repeats, Zα has been reported to bind to a wide range of sequence contexts. Studying how Zα interacts with B-/A-form helices prior to their conversion to the Z-conformation is challenging as binding coincides with Z-form adoption. Here, we studied the binding of Zα fromHomo sapiens ADAR1 to a locked "A-type" version of the (CpG)3 construct (LNA (CpG)3) where the sugar pucker is locked into the C3'-endo/C2'-exo conformation, which prevents the duplex from adopting the alternating C2'/C3'-endo sugar puckers found in the Z-conformation. Using NMR and other biophysical techniques, we find that ZαADAR1 binds to the LNA (CpG)3 using a similar interface as for Z-form binding, with a dissociation constant (KD) of ∼4 µM. In contrast to Z-DNA/Z-RNA, where two ZαADAR1 bind to every 6 bp stretch, our data suggests that ZαADAR1 binds to multiple LNA molecules, indicating a completely different binding mode. Because ZαADAR1 binds relatively tightly to a non-Z-form model, its binding to B/A-form helices may need to be considered when experiments are carried out which attempt to identify the Z-form targets of Zα domains. The use of LNA constructs may be beneficial in experiments where negative controls for Z-form adoption are needed.

Nuclear morphology is shaped by loop-extrusion programs.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-ß (MENß) are two long noncoding RNAs upregulated in multiple cancers, marking these RNAs as therapeutic targets. While traditional small-molecule and antisense-based approaches are effective, we report a locked nucleic acid (LNA)-based approach that targets the MALAT1 and MENß triple helices, structures comprised of a U-rich internal stem-loop and an A-rich tract. Two LNA oligonucleotides resembling the A-rich tract (i.e., A9GCA4) were examined: an LNA (L15) and a phosphorothioate LNA (PS-L15). L15 binds tighter than PS-L15 to the MALAT1 and MENß stem loops, although both L15 and PS-L15 enable RNA•LNA-RNA triple-helix formation. Based on UV thermal denaturation assays, both LNAs selectively stabilize the Hoogsteen interface by 5-13 °C more than the Watson-Crick interface. Furthermore, we show that L15 and PS-L15 displace the A-rich tract from the MALAT1 and MENß stem loop and methyltransferase-like protein 16 (METTL16) from the METTL16-MALAT1 triple-helix complex. Human colorectal carcinoma (HCT116) cells transfected with LNAs have 2-fold less MALAT1 and MENß. This LNA-based approach represents a potential therapeutic strategy for the dual targeting of MALAT1 and MENß.

Structural insights into branch site proofreading by human spliceosome.

Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2-BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.

Unexpectedly stable homopurine parallel triplex of SNA:RNA*SNA and L-aTNA:RNA*L-aTNA.

Homopurine strands are known to form antiparallel triplexes stabilized by G*G and A*A Hoogsteen pairs, which have two hydrogen bonds. But there has been no report on the parallel triplex formation of homopurine involving both adenosine and guanosine to the duplex. In this paper, we first report parallel triplex formation between a homopurine serinol nucleic acid (SNA) strand and an RNA/SNA duplex. Melting profiles revealed that the parallel SNA:RNA*SNA triplex was remarkably stable, even though the A*A pair has a single hydrogen bond. An L-acyclic threoninol nucleic acid (L-aTNA) homopurine strand also formed a stable parallel triplex with an L-aTNA/RNA duplex.

Intramolecular aptamer switches.

Aptamer switches as effective biosensing tools have become a focal point of research in engineered aptasensors. Intramolecular aptamer switches are more versatile, affordable, and simpler than classical "open-close" and strand displacement-based aptamer switches. Recently, many new aptamers with an overall hairpin structure have been reported. In this study, intramolecular aptamer switches were developed by adding new base pairs to the end of aptamers. The additional nucleotides can pair with the internal domains of the aptamer, causing a change in its conformation from the original secondary structure without a target. When a target binds to an aptamer, a marked change in the structure of the aptamer is expected. As models for testing this intramolecular aptamer switch idea, aptamers of oxytetracycline (OTC), 17ß-estradiol (E2), and adenosine were employed. When the additional base pairs are too long, binding the target to the aptamer becomes more challenging. This research offers valuable insights into the development of intramolecular aptamer switches and their potential applications in biosensor design.

RMBase v3.0: decode the landscape, mechanisms and functions of RNA modifications.

Although over 170 chemical modifications have been identified, their prevalence, mechanism and function remain largely unknown. To enable integrated analysis of diverse RNA modification profiles, we have developed RMBase v3.0 (http://bioinformaticsscience.cn/rmbase/), a comprehensive platform consisting of eight modules. These modules facilitate the exploration of transcriptome-wide landscape, biogenesis, interactome and functions of RNA modifications. By mining thousands of epitranscriptome datasets with novel pipelines, the 'RNA Modifications' module reveals the map of 73 RNA modifications of 62 species. the 'Genes' module allows to retrieve RNA modification profiles and clusters by gene and transcript. The 'Mechanisms' module explores 23 382 enzyme-catalyzed or snoRNA-guided modified sites to elucidate their biogenesis mechanisms. The 'Co-localization' module systematically formulates potential correlations between 14 histone modifications and 6 RNA modifications in various cell-lines. The 'RMP' module investigates the differential expression profiles of 146 RNA-modifying proteins (RMPs) in 18 types of cancers. The 'Interactome' integrates the interactional relationships between 73 RNA modifications with RBP binding events, miRNA targets and SNPs. The 'Motif' illuminates the enriched motifs for 11 types of RNA modifications identified from epitranscriptome datasets. The 'Tools' introduces a novel web-based 'modGeneTool' for annotating modifications. Overall, RMBase v3.0 provides various resources and tools for studying RNA modifications.

Structural basis of branching during RNA splicing.

Branching is a critical step in RNA splicing that is essential for 5' splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5' splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2'-OH of the adenosine for nucleophilic attack on the 5' scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome.

Dissection of integrated readout reveals the structural thermodynamics of DNA selection by transcription factors.

Nucleobases such as inosine have been extensively utilized to map direct contacts by proteins in the DNA groove. Their deployment as targeted probes of dynamics and hydration, which are dominant thermodynamic drivers of affinity and specificity, has been limited by a paucity of suitable experimental models. We report a joint crystallographic, thermodynamic, and computational study of the bidentate complex of the arginine side chain with a Watson-Crick guanine (Arg×GC), a highly specific configuration adopted by major transcription factors throughout the eukaryotic branches in the Tree of Life. Using the ETS-family factor PU.1 as a high-resolution structural framework, inosine substitution for guanine resulted in a sharp dissection of conformational dynamics and hydration and elucidated their role in the DNA specificity of PU.1. Our work suggests an under-exploited utility of modified nucleobases in untangling the structural thermodynamics of interactions, such as the Arg×GC motif, where direct and indirect readout are tightly integrated.

Exploring the interaction of tepotinib with calf thymus DNA using molecular dynamics simulation and multispectroscopic techniques.

In recent times, there has been a surge in the discovery of drugs that directly interact with DNA, influencing gene expression. As a result, understanding how biomolecules interact with DNA has become a major area of research. One such drug is Tepotinib (TPT), an FDA-approved anti-cancer medication known as a MET tyrosine kinase inhibitor, used in chemotherapy for metastatic non-small cell lung cancer (NSCLC) with MET exon 14 skipping alterations. In our study, we adopted both biophysical and in-silico methods to investigate the binding relationship of TPT and ctDNA. The absorption spectra of ctDNA exhibited a hypochromic effect when titrated with TPT and the binding constant of TPT-ctDNA complex was calculated, Ka = 9.91 × 104 M-1. By computing bimolecular enhancement constant (KB) and thermodynamic enhancement constant (KD) in fluorometric investigations, it was found that the fluorescence enhancement is a result of a static process involving the ctDNA-TPT complex formation in the ground state, as opposed to a dynamic process. The displacement assay results further supported this finding, showing that TPT exhibits a binding preference for minor groove of ct-DNA and was also demonstrated by KI quenching and CD spectroscopy. The molecular docking and molecular dynamic simulations validated TPT's groove binding nature and binding pattern with ctDNA, respectively. Thus, the results of our present investigation offer valuable insights into the interaction between TPT and ctDNA. It is evident that TPT, as an anti-cancer medication, binds to the minor groove of ctDNA.

NMR analysis of a loop-bulge structure of UUCGA pentaloop.

Although structures of many RNA loops, such as GNRA and UNCG tetraloops, were well known, it is still possible to find more RNA structures. In the present study, solution structure of an RNA fragment having UUCGA pentaloop was analyzed by NMR spectroscopy. It was found that the UUCG tetraloop is formed and the adenosine residue at the 3' side of the tetraloop is bulged out. The characteristic motif of the loop-bulge structure has also been found in other RNAs including CUUGU and CUGGC pentaloops. Along with the recently found T-hairpin structure with a UUUGAUU loop, in which UUUGA pentaloop and UU bulge are formed, the loop-bulge structures can be categorized as an RNA motif and it may be called as the integrated structure loop, I-loop.

Reversible Protection and Targeted Delivery of DNA Origami with a Disulfide-Containing Cationic Polymer.

DNA nanostructures have considerable biomedical potential as intracellular delivery vehicles as they are highly homogeneous and can be functionalized with high spatial resolution. However, challenges like instability under physiological conditions, limited cellular uptake, and lysosomal degradation limit their use. This paper presents a bio-reducible, cationic polymer poly(cystaminebisacrylamide-1,6-diaminohexane) (PCD) as a reversible DNA origami protector. PCD displays a stronger DNA affinity than other cationic polymers. DNA nanostructures with PCD protection are shielded from low salt conditions and DNase I degradation and show a 40-fold increase in cell-association when linked to targeting antibodies. Confocal microscopy reveals a potential secondary cell uptake mechanism, directly delivering the nanostructures to the cytoplasm. Additionally, PCD can be removed by cleaving its backbone disulfides using the intracellular reductant, glutathione. Finally, the application of these constructs is demonstrated for targeted delivery of a cytotoxic agent to cancer cells, which efficiently decreases their viability. The PCD protective agent that is reported here is a simple and efficient method for the stabilization of DNA origami structures. With the ability to deprotect the DNA nanostructures upon entry of the intracellular space, the possibility for the use of DNA origami in pharmaceutical applications is enhanced.