Synthetic DNA for Cell Surface Engineering: Experimental Comparison between Click Conjugation and Lipid Insertion in Terms of Cell Viability, Engineering Efficiency, and Displaying Stability.

The cell surface can be engineered with synthetic DNA for various applications ranging from cancer immunotherapy to tissue engineering. However, while elegant methods such as click conjugation and lipid insertion have been developed to engineer the cell surface with DNA, little effort has been made to systematically evaluate and compare these methods. Resultantly, it is often challenging to choose a right method for a certain application or to interpret data from different studies. In this study, we systematically evaluated click conjugation and lipid insertion in terms of cell viability, engineering efficiency, and displaying stability. Cells engineered with both methods can maintain high viability when the concentration of modified DNA is less than 25-50 µM. However, lipid insertion is faster and more efficient in displaying DNA on the cell surface than click conjugation. The efficiency of displaying DNA with lipid insertion is 10-40 times higher than that with click conjugation for a large range of DNA concentration. However, the half-life of physically inserted DNA on the cell surface is 3-4 times lower than that of covalently conjugated DNA, which depends on the working temperature. While the half-life of physically inserted DNA molecules on the cell surface is shorter than that of DNA molecules clicked onto the cell surface, lipid insertion is more effective than click conjugation in the promotion of cell-cell interactions under the two different experimental settings. The data acquired in this work are expected to act as a guideline for choosing an approximate method for engineering the cell surface with synthetic DNA or even other biomolecules.

Pharmacological Activation of cGAS for Cancer Immunotherapy.

When compartmentally mislocalized within cells, nucleic acids can be exceptionally immunostimulatory and can even trigger the immune-mediated elimination of cancer. Specifically, the accumulation of double-stranded DNA in the cytosol can efficiently promote antitumor immunity by activating the cGAMP synthase (cGAS) / stimulator of interferon genes (STING) cellular signaling pathway. Targeting this cytosolic DNA sensing pathway with interferon stimulatory DNA (ISD) is therefore an attractive immunotherapeutic strategy for the treatment of cancer. However, the therapeutic activity of ISD is limited by several drug delivery barriers, including susceptibility to deoxyribonuclease degradation, poor cellular uptake, and inefficient cytosolic delivery. Here, we describe the development of a nucleic acid immunotherapeutic, NanoISD, which overcomes critical delivery barriers that limit the activity of ISD and thereby promotes antitumor immunity through the pharmacological activation of cGAS at the forefront of the STING pathway. NanoISD is a nanoparticle formulation that has been engineered to confer deoxyribonuclease resistance, enhance cellular uptake, and promote endosomal escape of ISD into the cytosol, resulting in potent activation of the STING pathway via cGAS. NanoISD mediates the local production of proinflammatory cytokines via STING signaling. Accordingly, the intratumoral administration of NanoISD induces the infiltration of natural killer cells and T lymphocytes into murine tumors. The therapeutic efficacy of NanoISD is demonstrated in preclinical tumor models by attenuated tumor growth, prolonged survival, and an improved response to immune checkpoint blockade therapy.

The combined action of cations and anions of ionic liquids modulates the formation and stability of G-quadruplex DNA.

G-Quadruplex (Gq) formation and stabilization by any molecule is an essential requirement for its application in therapy, especially in oncology. Metal cations have shown higher propensity of the formation of the Gq structure and its stabilization. In this study, the role of both cations and anions of ionic liquids (ILs) on the Gq formation of human telomere (hTeloG) and its stability was investigated using spectroscopic and molecular dynamics simulation techniques. Irrespective of the nature of anions of ILs, tetramethylguanidinium (TMG) cations associated with different anions can form an antiparallel Gq structure in hTeloG. However, the propensity of the formation of an antiparallel Gq structure and its stability depend on the chain length of anions of ILs. Gq is significantly less stable in ILs having longer hydrocarbon chain anions compared to the short chain anions suggesting that the hydrophobicity of the anion plays a critical role in the stability and formation of the Gq structure by ILs. The data indicate that longer hydrocarbon chain anions of ILs preferably interact in the loop region of Gq through hydrophobic interaction which enhances the overall binding of the cation of ILs with Gq causing a decrease in the stacking energy between the G-quartets as well as Hoogsteen hydrogen bonds between the guanine bases leading to the destabilization of the antiparallel Gq structure.

Optochemical control of transcription by the use of 7-deaza-adenosine-based diarylethenes.

Out of nine different 7-deaza-adenosine diarylethenes, we identified a high-performance photoswitch, suitable for the synthesis of photochromic DNA. By using solid phase synthesis, a photoresponsive T7 promotor was generated which allowed reversibly modulating the rate of enzymatic RNA synthesis in vitro.

DQAsomes as the Prototype of Mitochondria-Targeted Pharmaceutical Nanocarriers : An Update.

DQAsomes (dequalinium-based liposome-like vesicles) are the prototype for all mitochondria-targeted vesicular pharmaceutical nanocarrier systems. First described in 1998 in a paper which has been cited as of May 2020 over 150 times, DQAsomes have been successfully explored for the delivery of DNA and low-molecular weight molecules to mitochondria within living mammalian cells. Moreover, they also appear to have triggered the design and development of a large variety of similar mitochondria-targeted nanocarriers . Potential areas of application of DQAsomes and of related mitochondria-targeted pharmaceutical nanocarriers involve mitochondrial gene therapy , antioxidant and updated therapy as well as apoptosis-based anticancer chemotherapy. Here, detailed protocols for the preparation, characterization, and application of DQAsomes are given and most recent developments involving the design and use of DQAsome-related particles are highlighted and discussed.

1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins.

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

Tetrahedral Framework Nucleic Acids Loaded with Aptamer AS1411 for siRNA Delivery and Gene Silencing in Malignant Melanoma.

siRNA is found to effectively knock down the target gene in cells, which is considered a promising strategy for gene therapy. However, the application of siRNA is limited due to its low efficiency of the cellular uptake. Tetrahedral framework nucleic acids (tFNAs) are synthesized by four single-stranded DNAs and show multiple biological functions in recent studies, especially suitable for drug delivery. More than 60% of malignant melanomas are associated with Braf gene mutation, an attractive therapeutic target for RNA interference. In this study, we modified anti-Braf siRNA (siBraf) with tFNAs to downregulate the target gene. Meanwhile, we directly incorporated AS1411 (a DNA aptamer) to our nanostructure, which assists tFNAs to improve the cellular uptake efficacy of siBraf significantly. The results indicated that tFNAs-AS1411-siBraf exhibited more potent activity to cleave Braf mRNA than free siBraf. This study may provide a new idea for the combination therapy of siRNA and aptamers via DNA nanomaterials to achieve gene silencing.

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA.

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

A DNA-encoded library for the identification of natural product binders that modulate poly (ADP-ribose) polymerase 1, a validated anti-cancer target.

Natural products have been an invaluable source of drug discovery, but their targets remain largely unknown. Natural products enriched DNA-encoded chemical libraries (nDELs) empower the researchers to rapidly and economically screen numerous natural products against various protein targets, and therefore promote the elucidation of the molecular mechanisms. In this work, we used poly (ADP-ribose) polymerase 1 (PARP1), as an example to explore the usage of nDEL for the functional natural products selection. We used late-stage modification approach to label three positive binders with unique DNA barcodes, whose dissociation constants range from sub-micromolar to micromolar. The selection criterion was set up according to the enrichment of these controls. Five natural products selected by this criterion directly bind to PARP1 in SPR, among which luteolin exhibits the highest inhibitory activity against PARP1. Moreover, luteolin selectively induces accumulation of DNA double-strand breaks and G2/M phase arrest in BRCA-deficient cells. All the findings from these investigations on luteolin support that PARP1 inhibition is one of the mechanisms for its anti-cancer activity.

Injectable Drug-Conjugated DNA Hydrogel for Local Chemotherapy to Prevent Tumor Recurrence.

Considering the high rate of postsurgical tumor recurrence due to the possible residual cancer cells and the non-negligible toxicity of postsurgical systemic chemotherapy, we designed an injectable DNA hydrogel assembled by chemodrug-grafted DNA strands for localized chemotherapy. First, a multitude of camptothecin was successfully grafted on backbones of the phosphorothioate DNAs, which could be assembled into two types of Y-shaped building blocks and then hierarchically associated together to form drug-containing hydrogels. The injectable feature of drug-containing DNA hydrogels enables a minimally invasive approach for local drug administration. Owing to the enzymatic degradation, the hydrogel can gradually disassemble into nanosized particles, allowing its good permeation into the residual tumor tissue and efficient uptake by cells. Together with its sustained and responsive drug release behaviors, the drug-containing DNA hydrogel can significantly inhibit the regrowth of tumor cells and prevent cancer recurrence. Compared to the control groups, mice treated with our drug-containing DNA hydrogel show the lowest tumor relapse rate (1/3) and substantial slow tumor progression. Despite the long-term local embedding, negligible systemic toxicity and organ damages are observed after the treatment with our drug-grafted DNA hydrogel. With excellent antitumor efficacy and low side effects in vivo, our DNA-drug conjugate (DDC)-based hydrogel represents a promising candidate for local adjuvant therapy in cancer treatment.

Quantitative Formation of Monomeric G-Quadruplex DNA from Multimeric Structures of c-Myc Promoter Sequence.

G-Quadruplex (G4)-forming DNA sequences have a tendency to form stable multimeric structures. This can be problematic for studies with synthetic oligodeoxynucleotides. Herein, we describe a method that quantitatively converts multimeric intermolecular structures of the Pu27 sequence from the c-myc promoter into the desired monomeric G4 by alkaline treatment and refolding.

Enhancing Antitumor Efficacy of Nucleoside Analog 5-Fluorodeoxyuridine on HER2-Overexpressing Breast Cancer by Affibody-Engineered DNA Nanoparticle.

BACKGROUND: Chemotherapy, as an adjuvant treatment strategy for HER2-positive breast cancer, can effectively improve clinical symptoms and overcome the drug resistance of therapeutic monoclonal antibodies. Nucleoside analogues are a class of traditional chemotherapeutic drugs that are widely applied in adjuvant therapy. However, there are many critical issues that limit their clinical efficiency, including poor selectivity and stability, severe side effects and suboptimal therapeutic efficacy. Hence, this work aims to develop a new DNA nanocarrier for targeted drug delivery to solve the above problems. METHODS: Four 41-mer DNA strands were synthesized and 10 FUdR molecules were attached to 5' end of each DNA strand by DNA solid-phase synthesis. An affibody molecule was connected to the end of polymeric FUdR through a linker in one of the four strands. The affibody-FUdR-tetrahedral DNA nanostructures (affi-F/TDNs) were self-assembled through four DNA strands, in which one vertex was connected to an affibody at the end of a polymeric FUdR tail and three vertices were only polymeric FUdR tails. In vitro cellular uptake of affi-F/TDNs was examined visually with confocal fluorescence microscopy and flow cytometry, and the cytotoxicity of affi-F/TDNs against cancer cells was investigated with MTT assay. Cell apoptosis was detected by Annexin V-FITC/PI double staining method. Using NOD/SCID (Mus Musculus) mice model, the targeted killing efficacy of affi-F/TDNs was also evaluated. RESULTS: The drug-loading of FUdR in affi-TDNs was 19.6% in mole ratio. The in vitro results showed that affi-F/TDNs had high selectivity and inhibition (81.2%) for breast cancer BT474 cells overexpressing HER2 and low toxicity in MCF-7 cells with low HER2 expression. During the in vivo application, affi-F/TDNs displayed good stability in the blood circulation, achieved specific accumulation in tumor region and the best antitumor efficacy (inhibition ratio of 58.1%), and showed excellent biocompatibility. CONCLUSIONS: The affibody-DNA tetrahedrons, as a simple and effective active targeting delivery nanocarrier, provided a new avenue for the transport of nucleoside antitumor drugs.

Homogeneous and Functional Group Tolerant Ring-Closing Metathesis for DNA-Encoded Chemical Libraries.

Reaction heterogeneity, poor pH control, and catalyst decomposition in the ring-closing metathesis (RCM) of DNA-chemical conjugates lead to poor yields of the cyclized products. Herein we address these issues with a RCM reaction system that includes a novel aqueous solvent combination to enable reaction homogeneity, an acidic buffer system which masks traditionally problematic functional groups, and a decomposition-resistant catalyst which maximizes conversion to the cyclized product. Additionally, we provide a systematic study of the substrate scope of the on-DNA RCM reaction, a demonstration of its applicability to a single-substrate DNA-encoded chemical library that includes sequencing analysis, and the first successful stapling of an unprotected on-DNA [i, i+4] peptide.

Design of 2'-O-methyl RNA and DNA double-stranded oligonucleotides: naturally-occurring nucleotide components with strong RNA interference gene expression inhibitory activity.

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.

Synthesis of DNA-Encoded Disulfide- and Thioether-Cyclized Peptides.

DNA-encoded chemical library technologies enable the screening of large combinatorial libraries of chemically and structurally diverse molecules, including short cyclic peptides. A challenge in the combinatorial synthesis of cyclic peptides is the final step, the cyclization of linear peptides that typically suffers from incomplete reactions and large variability between substrates. Several efficient peptide cyclization strategies rely on the modification of thiol groups, such as the formation of disulfide or thioether bonds between cysteines. In this work, we established a strategy and reaction conditions for the efficient chemical synthesis of cyclic peptide-DNA conjugates based on linking the side chains of cysteines. We tested two different thiol-protecting groups and found that tert-butylthio (S-tBu) works best for incorporating a pair of cysteines, and we show that the DNA-linked peptides can be efficiently cyclized through disulfide and thioether bond formation. In combination with established procedures for DNA encoding, the strategy for incorporation of cysteines may be readily applied for the generation and screening of disulfide- and thioether-cyclized peptide libraries.

Construction of a reconfigurable DNA nanocage for encapsulating a TMV disk.

A new reconfigurable DNA nanocage based on a DNA origami method has been constructed to capture a tobacco mosaic virus (TMV) disk. We used a hairpin to control the transformation of the nanocage and a strand of TMV RNA to attract the TMV disk. Our design could inspire new DNA-protein complex designs.

Terminator-free template-independent enzymatic DNA synthesis for digital information storage.

DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA.

Use of synthetic DNA spike-in controls (sequins) for human genome sequencing.

Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of synthetic DNA spike-ins-termed 'sequins' (sequencing spike-ins)-that are directly added to DNA samples before library preparation. Sequins can be used to measure technical biases and to act as internal quantitative and qualitative controls throughout the sequencing workflow. This step-by-step protocol explains the use of sequins for both whole-genome and targeted sequencing of the human genome. This includes instructions regarding the dilution and addition of sequins to human DNA samples, followed by the bioinformatic steps required to separate sequin- and sample-derived sequencing reads and to evaluate the diagnostic performance of the assay. These practical guidelines are accompanied by a broader discussion of the conceptual and statistical principles that underpin the design of sequin standards. This protocol is suitable for users with standard laboratory and bioinformatic experience. The laboratory steps require ~1-4 d and the bioinformatic steps (which can be performed with the provided example data files) take an additional day.

A chemical approach for the synthesis of the DNA-binding domain of the oncoprotein MYC.

We describe the first chemical synthesis of a functional mutant of the DNA binding domain of the oncoprotein MYC, using two alternative strategies which involve either one or two Native Chemical Ligations (NCLs). Both routes allowed the efficient synthesis of a miniprotein which is capable of heterodimerizing with MAX, and replicate the DNA binding of the native protein. The versatility of the reported synthetic approach enabled the straightforward preparation of MYC and Omomyc analogues, as well as fluorescently labeled derivatives.