Oligodeoxynucleotide Synthesis Under Non-Nucleophilic Deprotection Conditions.

This protocol describes a method for the incorporation of sensitive functional groups into oligodeoxynucleotides (ODNs). The nucleophile-sensitive epigenetic N4-acetyldeoxycytosine (4acC) DNA modification is used as an example, but other sensitive groups can also be incorporated, e.g., alkyl halide, α-haloamide, alkyl ester, aryl ester, thioester, and chloropurine groups, all of which are unstable under the basic and nucleophilic deprotection and cleavage conditions used in standard ODN synthesis methods. The method uses a 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) group that carries a methyl group at the carbon of the methoxy moiety (meDmoc) for the protection of exo-amines of nucleobases. The growing ODN is anchored to a solid support via a Dmoc linker. With these protecting and linking strategies, ODN deprotection and cleavage are achieved without using any strong bases and nucleophiles. Instead, they can be carried out under nearly neutral non-nucleophilic oxidative conditions. To increase the length of ODNs that can be synthesized using the meDmoc method, the protocol also describes the synthesis of a PEGylated Dmoc (pDmoc) phosphoramidite. With some of the nucleotides being incorporated with pDmoc-CE phosphoramidite, the growing ODN on the solid support carries PEG moieties and becomes more soluble, thus enabling longer ODN synthesis. The ODN synthesis method described in this protocol is expected to make many sensitive ODNs that are difficult to synthesize accessible to researchers in multiple areas, such as epigenetics, nanopore sequencing, nucleic acid-protein interactions, antisense drug development, DNA alkylation carcinogenesis, and DNA nanotechnology. © 2024 Wiley Periodicals LLC. Basic Protocol: Sensitive ODN synthesis Support Protocol 1: Synthesis of meDmoc-CE phosphoramidites Support Protocol 2: Synthesis of a pDmoc-CE phosphoramidite.

Nicked Invader probes: multistranded and sequence-unrestricted recognition of double-stranded DNA.

Major efforts have been devoted to the development of constructs that enable sequence-specific recognition of double-stranded (ds) DNA, fueled by the promise for enabling tools for applications in molecular biology, diagnostics, and medicine. Towards this end, we have previously introduced Invader probes, i.e., short DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. The individual strands of these labile probes display high affinity towards complementary DNA (cDNA), which drives sequence-unrestricted dsDNA-recognition. However, recognition of long targets is challenging due to the high stability of the corresponding probes. To address this, we recently introduced toehold Invader probes, i.e., Invader probes with 5'-single-stranded overhangs. The toehold architecture allows for shorter double-stranded segments to be used, which facilitates probe dissociation and dsDNA-recognition. As an extension thereof, we here report the biophysical and dsDNA-targeting properties of nicked Invader probes. In this probe architecture, the single-stranded overhangs of toehold Invader probes are hybridized to short intercalator-modified auxiliary strands, leading to formation of additional labile segments. The extra binding potential from the auxiliary strands imparts nicked Invader probes with greater dsDNA-affinity than the corresponding toehold or blunt-ended probes. Recognition of chromosomal DNA targets, refractory to recognition by conventional Invader probes, is demonstrated for nicked Invader probes in the context of non-denaturing FISH experiments, which highlights their utility as dsDNA-targeting tools.

Aptamer-PROTAC Conjugates (APCs) for Tumor-Specific Targeting in Breast Cancer.

Development of proteolysis targeting chimeras (PROTACs) is emerging as a promising strategy for targeted protein degradation. However, the drug development using the heterobifunctional PROTAC molecules is generally limited by poor membrane permeability, low in vivo efficacy and indiscriminate distribution. Herein an aptamer-PROTAC conjugation approach was developed as a novel strategy to improve the tumor-specific targeting ability and in vivo antitumor potency of conventional PROTACs. As proof of concept, the first aptamer-PROTAC conjugate (APC) was designed by conjugating a BET-targeting PROTAC to the nucleic acid aptamer AS1411 (AS) via a cleavable linker. Compared with the unmodified BET PROTAC, the designed molecule (APR) showed improved tumor targeting ability in a MCF-7 xenograft model, leading to enhanced in vivo BET degradation and antitumor potency and decreased toxicity. Thus, the APC strategy may pave the way for the design of tumor-specific targeting PROTACs and have broad applications in the development of PROTAC-based drugs.

Synthesis of GalNAc-Oligonucleotide Conjugates Using GalNAc Phosphoramidite and Triple-GalNAc CPG Solid Support.

GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.

Magic Peptide: Unique Properties of the LRR11 Peptide in the Activation of Leukotriene Synthesis in Human Neutrophils.

Neutrophil-mediated innate host defense mechanisms include pathogen elimination through bacterial phagocytosis, which activates the 5-lipoxygenase (5-LOX) product synthesis. Here, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs), which mimic the receptor-recognized sites of bacterial (CpG-ODNs) and genomic (G-rich ODNs) DNAs released from the inflammatory area, on the neutrophil functions after cell stimulation with Salmonella typhimurium. A possible mechanism for ODN recognition by Toll-like receptor 9 (TLR9) and RAGE receptor has been proposed. We found for the first time that the combination of the magic peptide LRR11 from the leucine-rich repeat (LRR) of TLR9 with the CpG-ODNs modulates the uptake and signaling from ODNs, in particular, dramatically stimulates 5-LOX pathway. Using thickness shear mode acoustic method, we confirmed the specific binding of CpG-ODNs, but not G-rich ODN, to LRR11. The RAGE receptor has been shown to play an important role in promoting ODN uptake. Thus, FPS-ZM1, a high-affinity RAGE inhibitor, suppresses the synthesis of 5-LOX products and reduces the uptake of ODNs by neutrophils; the inhibitor effect being abolished by the addition of LRR11. The results obtained revealed that the studied peptide-ODN complexes possess high biological activity and can be promising for the development of effective vaccine adjuvants and antimicrobial therapeutics.

Melting of DNA in confined geometries.

The stability of DNA molecules during viral or biotechnological encapsulation is a topic of active current research. We studied the thermal stability of double-stranded DNA molecules of different lengths in a confined space. Using a statistical model, we evaluate the melting profile of DNA molecules in two geometries: conical and cylindrical. Our results show that not only the confinement, but also the geometry of the confined space plays a prominent role in the stability and opening of the DNA duplex. We find that for more confined spaces, cylindrical confinement stabilizes the DNA, but for less confined spaces conical geometry stabilizes the DNA overall. We also analyse the interaction between DNA sequence and stability, and the evenness with which strand separation occurs. Cylindrical and conical geometries enable a better controlled tuning of the stability of DNA encapsulation and the efficiency of its eventual release, compared to spherical or quasi-spherical geometries.

The Toll-like receptor ligand, CpG oligodeoxynucleotides, regulate proliferation and osteogenic differentiation of osteoblast.

BACKGROUND: This study aimed to investigate the regulation of CpG oligodeoxynucleotides (ODNs) on proliferation and osteogenic differentiation of MC3T3 cells. METHODS: The laser co-focusing and flow cytometry assay were employed to detect cell uptake of CpG ODN 2006. Twelve ODNs were sythesized, and their effects on proliferation and differentiation were detected by MTT and alkaline phosphatase (ALP) activity assay. Flow cytometry assay was used to examine the regulation of CpG ODN on cell cycle. Quantitative real-time PCR (qRT-PCR) and western blot were used to evaluate the regulation of CpG ODN on mRNA and protein expression of osteogenic differentiation genes. RESULTS: The phosphorothioate CpG ODN 2006 could efficiently enter the MC3T3 cells in 1 h and locate in the cytoplasm. The MTT assay demonstrated CpG ODNs could promote MC3T3 cell proliferation and differentiation in the early stage, and gradually attenuated along with the increase of treating time, except for BW001 and FC001. qRT-PCR assay demonstrated that all the 12 CpG ODNs could promote the relative expression level of osteogenic differentiated genes, SP7 and OCN. In addition, western blot analysis suggested the CpG ODNs of BW001 and FC001 could increase the protein expression of P27Kip1 and Runx2 and decrease the protein expression of cyclin D1. CONCLUSION: The selected CpGODNs may be a potential gene therapy for bone regeneration of periodontitis.

Glycoform Differentiation by a Targeted, Self-Assembled, Pattern-Generating Protein Surface Sensor.

A method for generating targeted, pattern-generating, protein surface sensors via the self-assembly of modified oligodeoxynucleotides (ODNs) is described. The simplicity by which these systems can be created enabled the development of a sensor that can straightforwardly discriminate between distinct glycoform populations. By using this sensor to identify glycosylation states of a therapeutic protein, we demonstrate the diagnostic potential of this approach as well as the feasibility of integrating a wealth of supramolecular receptors and sensors into higher-order molecular analytical devices with advanced properties. For example, the facile device integration was used to attach the well-known anthracene-boronic acid (An-BA) probe to a biomimetic DNA scaffold and consequently, to use the unique photophysical properties of An-BA to improve glycoform differentiation. In addition, the noncovalent assembly enabled us to modify the sensor with a trinitrilotriacetic acid (tri-NTA)-Ni2+ complex, which endows it with selectivity toward a hexa-histidine tag (His-tag). The selective responses of the system to diverse His-tag-labeled proteins further demonstrate the potential applicability of such sensors and validate the mechanism underlying their function.

Modulation of cell membrane functionalization with aggregates of oligodeoxynucleotides containing alkyl chain-modified uridines.

In this study, we prepared oligodeoxynucleotides (ODNs) containing the uridine base modified by an alkyl chain at the 5-position (AU) and characterized their aggregate formation, localization, and functions in cells. These experiments revealed that aggregates of these ODNs were readily transported into cells, but their localization was dependent upon the number of hydrophobic units. ODNs with one modified AU were transported in the cytosol, while ODNs with multiple AU modifications resulted in their accumulation at the cell membrane. We also examined the ability of the AU-modified ODNs to capture small molecules at the cell membrane and their cellular uptake. We positioned a thioflavin-T (ThT)-binding aptamer on the cell membrane by means of hybridization with ODNs with three AUs at the strand end. Treatment with ThT resulted in its efficient uptake into cells, due to the capture of the ThT by the aptamers on the cell membrane. Thus, we demonstrated the functionalization of cell membranes with modified ODNs and the efficient delivery of small molecules into the cells.

Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis.

This protocol provides details for the preparation of nucleoside phosphoramidites with 1,3-dithian-2-yl-methyl (Dim) and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) as protecting groups, and a linker with Dmoc as the cleavable function, then using them for solid phase synthesis of sensitive oligodeoxynucleotides (ODNs). Using these Dim-Dmoc phosphoramidites and Dmoc linker, ODN synthesis can be achieved under typical conditions using phosphoramidite chemistry with slight modifications, and ODN deprotection and cleavage can be achieved under mild conditions involving oxidation with sodium periodate at pH 4 followed by aniline at pH 8. Under the mild deprotection and cleavage conditions, many sensitive functional groups including but not limited to esters, thioesters, alkyl halides, N-aryl amides, and α-chloroamides-which cannot survive the basic and nucleophilic deprotection and cleavage conditions such as concentrated ammonium hydroxide and dilute potassium methoxide used in typical ODN synthesis technologies-can survive. Thus, it is expected that the Dim-Dmoc ODN synthesis technology will find applications in the synthesis of ODNs that contain a wide range of sensitive functional groups. © 2020 Wiley Periodicals LLC. Basic Protocol: Synthesis, deprotection, cleavage, and purification of sensitive oligodeoxynucleotides Support Protocol 1: Synthesis of Dim-Dmoc nucleoside phosphoramidites Support Protocol 2: Preparation of CPG with a Dmoc linker Support Protocol 3: Synthesis of a phosphoramidite containing a sensitive alkyl ester group.

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor.

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.

Synthesis of oligodeoxyribonucleotides containing a tricyclic thio analogue of O6-methylguanine and their recognition by MGMT and Atl1.

Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.

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.

The influencing factors and functions of DNA G-quadruplexes.

G-quadruplexes form folded structures because of tandem repeats of guanine sequences in DNA or RNA. They adopt a variety of conformations, depending on many factors, including the type of loops and cations, the nucleotide strand number, and the main strand polarity of the G-quadruplex. Meanwhile, the different conformations of G-quadruplexes have certain influences on their biological functions, such as the inhibition of transcription, translation, and DNA replication. In addition, G-quadruplex binding proteins also affect the structure and function of G-quadruplexes. Some chemically synthesized G-quadruplex sequences have been shown to have biological activities. For example, bimolecular G-quadruplexes of AS1411 act as targets of exogenous drugs that inhibit the proliferation of malignant tumours. G-quadruplexes are also used as vehicles to deliver nanoparticles. Thus, it is important to identify the factors that influence G-quadruplex structures and maintain the stability of G-quadruplexes. Herein, we mainly discuss the factors influencing G-quadruplexes and the synthetic G-quadruplex, AS1411. SIGNIFICANCE OF THE STUDY: This review summarizes the factors that influence G-quadruplexes and the functions of the synthetic G-quadruplex, AS1411. It also discusses the use of G-quadruplexes for drug delivery in tumour therapy.

Solid-phase synthesis of branched oligonucleotides containing a biologically relevant dCyd341 interstrand crosslink DNA lesion.

Branched oligonucleotides containing a biologically relevant DNA lesion, dCyd341, which involves an interstrand crosslink between a cytosine base on one strand and a ribose moiety on the opposite strand, were prepared in a single automated solid-phase synthesis. For this, we first prepared the phosphoramidite analogue of dCyd341 bearing an orthogonal levulinyl protecting group. Then, following the synthesis of the first DNA strand containing dCyd341, the levulinic group was removed and the synthesis was then continued from the free base hydroxyl group at the branching point, using traditional phosphoramidites. The synthesized oligonucleotides were fully characterized by MALDI-TOF/MS and were enzymatically digested, and the presence of the lesion was confirmed by HPLC-MS/MS and the sequence was finally controlled upon exonuclease digestion followed by MALDI-TOF/MS analysis. The developed strategy was successfully employed for the preparation of several short linear and branched oligonucleotides containing the aforementioned lesion.

Sensitive Oligodeoxynucleotide Synthesis Using Dim and Dmoc as Protecting Groups.

In traditional oligodeoxynucleotide (ODN) synthesis, phosphate groups are protected with the 2-cyanoethyl group, and amino groups are protected with acyl groups. At the end of ODN synthesis, deprotection is achieved with strong bases and nucleophiles. Therefore, traditional technologies are not suitable for the synthesis of ODNs containing sensitive functionalities. To address the problem, we report the use of Dim and Dmoc groups, which are based on the 1,3-dithian-2-yl-methyl function, for phosphate and amine protection for the solid phase ODN synthesis. Using the new Dim-Dmoc protection, deprotection was achieved under mild oxidative conditions without using any strong bases and nucleophiles. As a result, the new technology is suitable for the synthesis of ODNs containing sensitive functions. To demonstrate feasibility, seven 20-mer ODNs including four that contain sensitive ester and alkyl chloride groups were synthesized, purified with RP HPLC, and characterized with MALDI-TOF MS and enzyme digestion essays. High purity ODNs were obtained.

User-defined single pot mutagenesis using unamplified oligo pools.

User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.

Polymorphic G:G mismatches act as hotspots for inducing right-handed Z DNA by DNA intercalation.

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.

Thermostability Trends of TNA:DNA Duplexes Reveal Strong Purine Dependence.

The development of high fidelity polymerases and streamlined synthesis of threose nucleic acid (TNA) triphosphates and phosphoramidites has made TNA accessible as a motif for generating nuclease-resistant high-affinity aptamers, antisense oligos, and synthetic genetic biopolymers. Little is known, however, about the thermostability trends of TNA:DNA duplexes. Here we investigate the thermostability of 14 TNA:DNA duplexes with the goal of elucidating the fundamental factors governing TNA:DNA duplex stability. We find that purine content in TNA significantly influences the stability and conformation of TNA:DNA duplexes. Low TNA purine content destabilizes duplexes, with Tm values often 5 °C lower than analogous DNA:DNA and RNA:DNA duplexes. By contrast, TNA:DNA duplexes having high TNA purine content display greater stability than DNA:DNA or RNA:DNA duplexes having the same sequences. High TNA purine content leads TNA:DNA duplexes to adopt conformations similar to RNA:RNA (A-form) configuration, whereas duplexes with low TNA purine content have conformations more similar to DNA:DNA (B-form) configuration. These insights provide a basis for understanding and predicting TNA:DNA duplex stability, which is anticipated to guide the practical use of TNA in biotechnology applications.

Preparation and Purification of Oligodeoxynucleotide Duplexes Containing a Site-Specific, Reduced, Chemically Stable Covalent Interstrand Cross-Link Between a Guanine Residue and an Abasic Site.

Methods for the preparation of DNA duplexes containing interstrand covalent cross-links may facilitate research in the fields of biochemistry, molecular biology, nanotechnology, and materials science. Here we report methods for the synthesis and isolation of DNA duplexes containing a site-specific, chemically stable, reduced covalent interstrand cross-link between a guanine residue and an abasic site. The method uses experimental techniques and equipment that are common in most biochemical laboratories and inexpensive, commercially available oligonucleotides and reagents.