Recent progress in DNA data storage based on high-throughput DNA synthesis.

DNA data storage has emerged as a solution for storing massive volumes of data by utilizing nucleic acids as a digital information medium. DNA offers exceptionally high storage density, long durability, and low maintenance costs compared to conventional storage media such as flash memory and hard disk drives. DNA data storage consists of the following steps: encoding, DNA synthesis (i.e., writing), preservation, retrieval, DNA sequencing (i.e., reading), and decoding. Out of these steps, DNA synthesis presents a bottleneck due to imperfect coupling efficiency, low throughput, and excessive use of organic solvents. Overcoming these challenges is essential to establish DNA as a viable data storage medium. In this review, we provide the overall process of DNA data storage, presenting the recent progress of each step. Next, we examine a detailed overview of DNA synthesis methods with an emphasis on their limitations. Lastly, we discuss the efforts to overcome the constraints of each method and their prospects.

Semisynthetic Glycoconjugates as Potential Vaccine Candidates Against Haemophilus influenzae Type a.

Glycoconjugate vaccines are based on chemical conjugation of pathogen-associated carbohydrates with immunogenic carrier proteins and are considered a very cost-effective way to prevent infections. Most of the licensed glycoconjugate vaccines are composed of saccharide antigens extracted from bacterial sources. However, synthetic oligosaccharide antigens have become a promising alternative to natural polysaccharides with the advantage of being well-defined structures providing homogeneous conjugates. Haemophilus influenzae (Hi) is responsible for a number of severe diseases. In recent years, an increasing rate of invasive infections caused by Hi serotype a (Hia) raised some concern, because no vaccine targeting Hia is currently available. The capsular polysaccharide (CPS) of Hia is constituted by phosphodiester-linked 4-ß-d-glucose-(1→4)-d-ribitol-5-(PO4→) repeating units and is the antigen for protein-conjugated polysaccharide vaccines. To investigate the antigenic potential of the CPS from Hia, we synthesized related saccharide fragments containing up to five repeating units. Following the synthetic optimization of the needed disaccharide building blocks, they were assembled using the phosphoramidite approach for the installation of the phosphodiester linkages. The resulting CPS-based Hia oligomers were conjugated to CRM197 carrier protein and evaluated in vivo for their immunogenic potential, showing that all glycoconjugates were capable of raising antibodies recognizing Hia synthetic fragments.

Phosphamide-Based Washing-Durable Flame Retardant for Cotton Fabrics.

A formaldehyde-free reactive flame retardant, an ammonium salt of triethylenetetramine phosphoryl dimethyl ester phosphamide phosphoric acid (ATPEPDPA), was synthesized and characterized using nuclear magnetic resonance (NMR). Fourier transform infrared spectroscopy test (FT-IR), durability test and scanning electron microscopy (SEM) results suggested that ATPEPDPA was successfully grafted on cotton fabrics through a -N-P(=O)-O-C covalent bond. Moreover, the limiting oxygen index (LOI) value of 20 wt% ATPEPDPA-treated cotton was 44.6%, which met stringent washing standard after 50 laundering cycles (LCs). The high washing resistance of the ATPEPDPA-treated cotton was due to the p-π conjugation between the N atom and the P(=O) group in the flame-retardant molecule, which strengthened the stability of the -N-P(=O)-O-C bonds between ATPEPDPA and cellulose, and the -N-P(=O)-(O-CH3)2 groups in the ATPEPDPA. The cone calorimetric test showed that the treated cotton had excellent flame retardance. In addition, the TG and TG-IR tests suggested that ATPEPDPA performed a condensed flame retardance mechanism. Furthermore, the physical properties and hand feel of the treated cotton were well maintained. These results suggested that introducing -N-P(=O)-(O-CH3)2 and -N-P(=O)-(ONH4)2 groups into ATPEPDPA could significantly increase the fire resistance and durability of cotton fabrics.

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.

"Difficult" deoxyribonucleotide sequences in the solid-phase synthesis by the phosphoramidite chemistry.

This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite chemistry-based solid phase synthesis. In total, 76 sequences with different dinucleotide and trinucleotide repeats were synthesized, and the fully-deprotected products were analyzed by denaturing anion exchange HPLC. Overall, sequences containing more 2'-deoxyadenosine residues were obtained in relatively lower yields, likely due to the relative ease of 2'-deoxyadenosine to undergo depurination during the detritylation reaction. Furthermore, dinucleotide steps, such as d(CG)/d(GC) and d(AG)/d(GA), likely contribute the overall lower yields of full-length products as well.

Synthesis of an Ortho-Functionalized Tetrafluorinated Azobenzene Phosphoramidite for Incorporation into a Tetrafluorinated Azobenzene-Containing siRNA for Photocontrolled Gene Silencing.

This article presents the detailed synthesis and characterization protocols for an ortho-functionalized tetrafluorinated azobenzene containing siRNA, which has photoswitchable properties. To design this tetrafluorinated azobenzene scaffold, several synthetic steps are performed to generate a symmetrical tetrafluorinated azobenzene diol. This diol is treated with dimethoxytrityl chloride (DMT-Cl) to protect one of the alcohols. Next, the DMT-protected tetrafluorinated monoalcohol is phosphitylated to afford the DMT-phosphoramidite building block used for solid-phase synthesis. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of an ortho-functionalized fluorinated azobenzene containing siRNA (F-siRNA), which has photoswitchable properties that can be controlled with visible light. First, the F-siRNA was characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile and melting temperature (Tm ) of the duplexes. Second, biological testing of the F-siRNA is performed in cell culture to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to green light to inactivate the F-siRNA, followed by blue light, which reactivates the F-siRNA. The F-siRNA can be kept inactive for up to 72 hr and reactivated at any time within this 72-hr window. © 2023 Wiley Periodicals LLC.

Fluorination Influences the Bioisostery of Myo-Inositol Pyrophosphate Analogs.

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.

From CPG to hybrid support: Review on the approaches in nucleic acids synthesis in various media.

Solid-phase synthesis is, to date, the preferred method for the manufacture of oligonucleotides, in quantities ranging from a few micrograms for research purposes to several kilograms for therapeutic or commercial use. But for large-scale oligonucleotide manufacture, scaling up and hazardous waste production pose challenges that necessitate the investigation of alternate synthetic techniques. Despite the disadvantages of glass supports, using soluble supports as a substitute presents difficulties because of their high overall yield and complex purification steps. To address these challenges, various independent approaches have been developed; however, other problems such as insufficient cycle efficiency and synthesis of oligonucleotide chains of desired length continue to exist. In this study, we present a review of the current developments, advantages, and difficulties of recently reported alternatives to supports based on controlled pore glass, and discuss the importance of a support choice to resolve issues arising during oligonucleotide synthesis.

Preparation of a 4'-Thiouridine Building-Block for Solid-Phase Oligonucleotide Synthesis.

Starting from a commercially available thioether, we report a nine-step synthesis of a 4'-thiouridine phosphoramidite building-block. We install the uracil nucleobase using Pummerer-type glycosylation of a sulfoxide intermediate followed by a series of protecting group manipulations to deliver the desired phosphite. Notably, we introduce a 3',5'-O-di-tert-butylsilylene protecting group within a 4'-thiosugar framework, harnessing this to ensure regiospecific installation of the 2'-O-silyl protecting group. We envisage this methodology will be generally applicable to other 4'-thionucleosides and duly support the exploration of their inclusion within related nucleic acid syntheses. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: (2R,3S,4R)-2,3-O-Isopopropylidene-5-O-tert-butyldiphenylsilyl-1-(4-sulfinyl)cyclopentane: Sulfoxidation Basic Protocol 2: 2',3'-O-Isopropylidene-5'-O-tert-butyldiphenylsilyl-4'-thiouridine: Pummerer glycosylation Basic Protocol 3: 4'-Thiouridine: Deprotection Basic Protocol 4: 2'-O-tert-Butyldimethylsilyl-3',5'-di-tert-butylsiloxy-4'-thiouridine: 2',3',5'-O-silylation Basic Protocol 5: 2'-O-tert-Butyldimethylsilyl-4'-thiouridine: Selective 3'-5'-desilylation Basic Protocol 6: 2'-O-tert-Butyldimethylsilyl-5'-O-dimethoxytrityl-4'-thiouridine: 5'-O-dimethoxytritylation Basic Protocol 7: 2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethoxy)(N,N-diisopropylamino)phosphino]-5'-O-dimethoxytrityl-4'-thiouridine: 3'-O-phosphitylation.

Synthesis of 4'-Thiomodified c-di-AMP Analogs.

Cyclic diadenosine monophosphate (c-di-AMP) is a bacterial cyclic dinucleotide (CDN) comprising two adenosine monophosphates covalently linked by two 3',5'-phosphodiester bonds. c-di-AMP works as a second messenger, regulating many biological processes in bacteria such as cell wall homeostasis, DNA integrity, and sporulation via specific protein and/or RNA receptors. Moreover, c-di-AMP can function as an immunomodulatory agent in eukaryote cells via the stimulator of interferon genes (STING) signaling pathway. This protocol describes the chemical synthesis of two c-di-AMP analogs with a sulfur atom at the 4'-position of the furanose ring instead of an oxygen atom: c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2). Analogs 1 and 2 have resistance to phosphodiesterase-mediated degradation and are therefore useful for understanding the diverse biological phenomena regulated by c-di-AMP. In this protocol, two 4'-thioadenosine monomers are initially prepared via a Pummerer-like reaction assisted by hypervalent iodine. The CDN skeleton is then constructed through two key reactions based on phosphoramidite chemistry: dimerization of two appropriately protected nucleoside monomers to produce a linear dinucleotide, followed by macrocyclization of the resulting linear dinucleotide to form the CDN skeleton. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4'-thioadenosine monomers 13 and 14 Basic Protocol 2: Preparation of c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2).

A DNA Cleavage Assay Using Synthetic Oligonucleotide Containing a Single Site-Directed Lesion for In Vitro Base Excision Repair Study.

Many chemicals cause mutation or cancer in animals and humans by forming DNA lesions, including base adducts, which play a critical role in mutagenesis and carcinogenesis. A large number of such adducts are repaired by the DNA glycosylase-mediated base excision repair (BER) pathway, and some are processed by nucleotide excision repair (NER) and nucleotide incision repair (NIR). To understand what structural features determine repair enzyme specificity and mechanism in chemically modified DNA in vitro, we developed and optimized a DNA cleavage assay using defined oligonucleotides containing a single, site specifically placed lesion. This assay can be used to investigate novel activities against any newly identified derivatives from chemical compounds, substrate specificity and cleavage efficiency of repair enzymes, and quantitative structure-function relationships. Overall, the methodology is highly sensitive and can also be modified to explore whether a lesion is processed by NER or NIR activity, as well as to study its miscoding properties in translesion DNA synthesis (TLS).

A general and efficient approach to synthesize the phosphoramidites of 5'-18O labeled purine nucleosides.

5'-18O labeled RNA oligos are important probes to investigate the mechanism of 2'-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5'-18O labeled nucleosides starting from the corresponding commercially available 5'-O-DMT protected nucleosides. Using this method, we prepared 5'-18O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5'-18O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5'-18O-2'-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5'-18O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2'-O-transphosphorylation reactions.

Chemical Synthesis of Oligonucelotide Sequences: Phosphoramidite Chemistry.

Oligonucleotides are used in a variety of molecular biology techniques, from next-generation sequencing to genetic testing. Maintaining the sequence fidelity of synthetic oligonucleotides is critical to their use. This chapter describes the steps of solid phase oligonucleotide synthesis and purification, which enables the synthesis of oligonucleotides with specific sequences and high purity.

An Alternate Process for the Solid-Phase Synthesis and Solid-Phase Purification of Synthetic Nucleic Acid Sequences.

The chemical synthesis of a riboside phosphoramidite has been achieved to provide a 5-O-capture linker and a 2-O-silyl ether protecting group with the intent of enabling an efficient solid-phase purification of synthetic DNA sequences. The riboside phosphoramidite has been incorporated into a DNA sequence while performing the penultimate automated solid-phase synthesis cycle of the sequence. The terminal 5-O-riboside moiety of the resulting DNA sequence is then conjugated to a capture linker to create an anchor for the solid-phase purification of the DNA sequence conjugate. Release of all DNA sequences from the synthesis support is achieved under standard basic conditions to yield a mixture of the desired DNA sequence conjugate along with unconjugated, shorter-than-full-length sequence contaminants. Upon exposure of all DNA sequences to a capture solid support, only the DNA sequence conjugate is chemoselectively captured, thereby allowing the unconjugated shorter-than-full-length DNA sequences to be efficiently washed away from the capture support. After 2-O-cleavage of the silyl ether protecting group from the terminal riboside ethylphosphate triester conjugate, the solid-phase-purified DNA sequence is efficiently released from the capture support through an innovative intramolecular cyclodeesterification of the ethylphosphate triester, prompted by the riboside's rigid cis-diol conformer, to provide a highly pure DNA sequence. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-1,4-anhydro-D-ribitol (3) Basic Protocol 2: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-3-O-[(N,N-diisopropylamino)ethyloxyphosphinyl]-1,4-anhydro-D-ribitol (6). Basic Protocol 3: Automated synthesis of the chimeric solid-phase-linked DNA sequence 8. Support Protocol: Preparation of 2-cyanoethyl-(5-oxohexyl)-N,N-diisopropylphosphoramidite (9). Basic Protocol 4: Solid-phase purification of the chimeric DNA sequence 10.

The construction of oligonucleotide-cycloastragenol and the renoprotective effect study.

Traditional Chinese Medicine (TCM) provides unique therapeutic effects for many diseases with identified efficacy during long practice. Astragalus Membranaceus (AM) is the Chinese herbal applied for kidney injury in the clinic, but it remains challenging to further enhance the efficacy. Cycloastragenol (CAG) is the ingredient isolated from AM with poor water solubility, which has shown a renoprotective effect. Herein we designed and synthesized the corresponding solid-phase module of CAG, from which CAG as a pharmaceutical element was incorporated into oligonucleotides (ON) as an ON-CAG conjugate in a programmable way by a DNA synthesizer. Cell viability study demonstrated that ON-CAG conjugate remains similar renoprotective effect as that of CAG, which efficiently recovers the activity of HK-2 cells pretreated with cisplatin. Similarly, in the renal cells treated with the conjugate, the biomarkers of kidney injury such as KIM-1 and IL-18 are downregulated, and cytokines are reduced as treated with anti-inflammatory agents. Overall, we have managed to incorporate a hydrophobic ingredient of TCM into ON and demonstrate the oligonucleotide synthesis technology as a unique approach for the mechanism study of TCM, which may facilitate the discovery of new therapeutics based on TCM.

Site-Specific Radioiodination of Oligonucleotides with a Phenolic Element in a Programmable Approach.

Radioiodination of oligonucleotides provides an extra modality for nucleic acid-based theranostics with potential applications. Herein, we report the design and synthesis of a phosphoramidite embedded with a phenolic moiety and demonstrate that oligonucleotides can be readily functionalized with phenol as a precursor by general DNA synthesis. It was identified that the introduction of the precursor does not block the specificity of an aptamer, and the radioiodination is applicable to both DNA and RNA oligonucleotides in a site-specific approach with a commercial kit.

Synthesis of 4'-C-(Aminoethyl)thymidine and 4'-C-[(N-Methyl)aminoethyl] Thymidine Nucleosides to Enhance DNA Stability.

Antisense oligonucleotide (ASO) therapeutics target the pathogenic mRNA directly and modulate protein expression. Novel chemical modifications help to improve the action of ASOs with better thermal stability and resistance against nucleases. Oligodeoxynucleotides (ODNs) containing 4'-C-(aminoethyl)thymidine modifications exhibit efficient and stable hybridization with complementary DNA as well as RNA strands showing remarkably improved resistance against nucleolytic hydrolysis, which makes them promising candidates for antisense therapeutics. This article describes the synthesis of a novel nucleoside analog, 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T), 3, and previously reported 4'-C-aminoethyl-thymidine (4'-AE-T), 2, through a newly designed synthetic route to obtain a high overall yield. This has been established by changing the starting material from thymidine to diacetone-D-glucofuranose and synthesizing the known 4-C-hydroxyethyl pentofuranose. Conversion of the hydroxy group to an azide functional group through Mitsunobu azidation and performing acetolysis, provide the common intermediate 4-C-(2-azidoethyl)-ribofuranose. Subsequent coupling of the thymine nucleobase with the common intermediate under Vorbrüggen glycosylation conditions provides the corresponding modified nucleoside in high yield. It was subjected for conversion of the azide to an amine by Staudinger reaction and 2'-deoxygenation using Barton-McCombie conditions. Debenzylation with Lewis acid and mono-dimethoxytritylation of the 5'-OH afforded a fully protected 3'-OH intermediate for phosphitylation to give the corresponding phosphoramidites. In the case of 4'-MAE-T, benzyloxymethyl protection of the N3 -position and methylation were carried out prior to debenzylation. These phosphoramidite monomers were suitable with conventional oligonucleotide synthesis, and imparted ameliorated nuclease resistance, and competent RNase H activity, suggesting its potential utilization in ASO drugs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4-C-(2-azidoethyl)-ribofuranose (6) Basic Protocol 2: Synthesis of 4'-C-aminoethyl thymidine phosphoramidite (15) Basic Protocol 3: Synthesis of 4'-C-(N-methyl)aminoethyl thymidine phosphoramidite (20).

Synthesis of Ortho-Functionalized Tetrachlorinated Azobenzene Phosphoramidites for Incorporation Into siRNAzos for Photocontrolled Gene Silencing.

This paper contains the detailed synthesis and characterization protocols of ortho-functionalized tetrachlorinated azobenzene-containing small interfering RNAs (siRNAs), which have photoswitchable properties effectively controlled with visible light. To design this tetrachlorinated azobenzene scaffold, a late-stage chlorination with N-chlorosuccinimide and palladium is used. Next, a single hydroxyl group from the tetrachlorinated azobenzene is protected with a 4,4'-dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate tetrachlorinated azobenzene-containing oligonucleotides. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of ortho-functionalized chlorinated azobenzene-containing siRNAs (Cl-siRNAzos), which have photoswitchable properties that can be controlled with visible light. First, the Cl-siRNAzos are characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (Tm ) of the duplexes. Secondly, the biological testing of the Cl-siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to red light in order to inactivate the Cl-siRNAzo, and then either violet light or infrared thermal relaxation is deployed, which re-activates the Cl-siRNAzo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxyethyl) ortho-functionalized tetrachlorinated azobenzene phosphoramidite (5) Basic Protocol 2: Synthesis, purification, and characterization of siRNAs containing ortho-functionalized tetrachlorinated azobenzene Basic Protocol 3: Gene-silencing evaluation of ortho-functionalized tetrachlorinated azobenzene using firefly luciferase.

Low-scale syringe-made synthesis of 15 N-labeled oligonucleotides.

Fast and reasonable low-scale (200 nmol) syringe-made synthesis of 15 N-labeled oligonucleotides representing DNA trinucleotide codons is communicated. All codons were prepared by solid-phase controlled pore glass synthesis column technique via the phosphoramidite method. Twenty-four labeled oligonucleotides covering the DNA genetic code alphabet were prepared using commercially available reagents and affordable equipment in a reasonably short period of time, with acceptable yields and purity for direct applications in mass spectrometry.

Enantioselective 1,3-Dipolar Cycloaddition Using (Z)-α-Amidonitroalkenes as a Key Step to the Access to Chiral cis-3,4-Diaminopyrrolidines.

The enantioselective 1,3-dipolar cycloaddition between imino esters and (Z)-nitroalkenes bearing a masked amino group in the ß-position was studied using several chiral ligands and silver salts. The optimized reaction conditions were directly applied to the study of the scope of the reaction. The determination of the absolute configuration was evaluated using NMR experiments and electronic circular dichroism (ECD). The reduction and hydrolysis of both groups was performed to generate in an excellent enantiomeric ratio the corresponding cis-2,3-diaminoprolinate.