Chemical modifications are fundamental to improving the physicochemical and biological performance of oligonucleotide (ON) therapeutics by enhancing their hybridization affinity, nuclease resistance, and metabolic stability. Among the various sugar modifications developed, 4′-carbon (4′- C ) substitutions have attracted significant attention for their ability to reinforce the sugar-phosphate backbone while preserving the natural C 3′- endo conformation and Watson-Crick base pairing. The 4′- C -α-aminoethoxy (4′AEo) framework, in particular, provides steric protection against nuclease degradation without impairing RNase H activity, making it a valuable scaffold for next-generation antisense oligonucleotides (ASOs). This article presents optimized and reproducible synthetic protocols for two novel 4′- C -modified uridine phosphoramidite building blocks, 4′AEo m U (4′- C -α-aminoethoxy-2′- O -methyl-5-methyluridine) and 4′AEo p U (4′- C -α-aminoethoxy-2′- O -methyl-5-(1-propynyl)uridine), both fully compatible with automated solid-phase oligonucleotide synthesis. The synthesis of 4′AEo m U begins with commercially available 2′- O -methyl-5-methyluridine, which undergoes iodination of the 5′-hydroxyl group, 4′,5′-reductive dehalogenation, and 3′- O -silyl protection, followed by 4′,5′-epoxidation and ZnCl 2 -mediated nucleophilic epoxide opening with N -trifluoroacetylaminoethanol to afford the α-configured product as the major isomer, as confirmed by NOESY NMR analysis. In the 4′AEo p U synthetic route, regioselective O -silyl deprotection of the 4′-C-α-aminoethoxy-2′-O-methyluridine derivative (compound 10 ) is followed by 5-iodination and subsequent Sonogashira-Hagihara coupling to introduce the 5-propynyl substituent. The resulting intermediate then undergoes 5′- O -DMTr protection and phosphitylation to yield the desired phosphoramidite. Both protocols exhibit high stereochemical control, reproducibility, and efficiency, yielding analytically pure intermediates validated by TLC, NMR, and mass spectrometry. Compared with previously reported synthetic approaches relying on glycal or radical intermediates, these methods minimize reaction complexity, improve yields, and maintain compatibility with standard phosphoramidite chemistry. Collectively, the described synthetic routes provide a reliable and scalable platform for generating structurally defined 4′- C -modified uridine derivatives. The resulting phosphoramidites enable the construction of antisense oligonucleotides with enhanced duplex stability, nuclease resistance, and favorable pharmacological properties, thereby advancing the rational design of next-generation ON therapeutics with improved safety and efficacy. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Synthesis of 4′-C-α-aminoethoxy-2′- O -methyl-5-methyluridine phosphoramidite (4AEo m U) (9) Basic Protocol 2 : Synthesis of 4′-C-α-aminoethoxy-2′- O -methyl-5-(1-propynyl) uridine phosphoramidite (4AEo p U) (15)
This article provides a comprehensive set of protocols for the in vitro selection of threose nucleic acid (TNA) enzymes capable of catalyzing RNA cleavage or ligation reactions. The article first outlines the generation of random-sequence TNA libraries (typically comprising ∼1014 unique molecules) covalently attached to their RNA substrates via engineered DNA polymerases. It then describes the procedure for isolating catalytically active TNA sequences through affinity chromatography or gel electrophoresis. These sequences are subsequently reverse-transcribed to cDNA for amplification or sequencing. This set of protocols is primarily designed for selecting RNA-cleaving and RNA ligase TNA enzymes but can be adapted for the discovery of TNA enzymes that act on DNA substrates or enzymes derived from other xeno-nucleic acids, provided that requisite nucleoside triphosphates and compatible tool enzymes are available.© 2025 Wiley Periodicals LLC. Basic Protocol 1: TNA library synthesis Basic Protocol 2: Selection of RNA-cleaving TNA enzyme Alternate Protocol: Selection of RNA ligase TNA enzyme Basic Protocol 3: Reverse transcription of enriched TNA library Basic Protocol 4: Preparation of template DNA for the next round.
Genetic alphabet expansion by creating unnatural base pairs (UBPs) could renovate biological systems and next-generation biotechnologies. Among the expanded genetic letters, TPT3-NaM is one of the most advanced UBPs. It has been utilized in semi-synthetic organisms (SSOs) to recode therapeutic proteins. This article describes the synthesis of isoTAT and demonstrates that isoTAT can convert NaM to G, simultaneously pairing with both NaM and G as a bridging base. Additionally, the inherent base' preference for NaM leads to its transformation into T. Consequently, TPT3-NaM can be converted to C-G or A-T base pairs through simple PCR assays, enabling the first method to locate multiple sites of TPT3-NaM pairs dually. Taken together, this work presents the first general and convenient approach capable of locating, tracing, and sequencing site- and number-unlimited TPT3-NaM pairs. The data presented in this article are based on our previously published reports. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of isoTAT triphosphate Basic Protocol 2: Incorporation and extension reaction of isoTAT for a template containing NaM Basic Protocol 3: Monitor DNA containing multiple UBPs using simple PCR assays with isoTAT through Sanger sequencing.
Aptamer-drug conjugates (ApDCs) represent a powerful platform for targeted drug delivery, offering the potential to enhance therapeutic efficacy while minimizing systemic toxicity. However, conventional methods for ApDC preparation often involve complex, multistep procedures with limited control over drug loading and site specificity. This protocol describes a modular and automated strategy for the synthesis and structural optimization of ApDCs using custom-designed phosphoramidites that incorporate anticancer drugs and functional moieties. The customized phosphoramidite is compatible with standard solid-phase oligonucleotide synthesis, enabling the automated and efficient construction of programmable aptamer-drug conjugates (PApDCs) for targeted drug delivery. We also highlight the programmable optimization of aptamer structures to meet clinical needs. The protocol offers a streamlined and versatile platform for the construction of functional ApDCs, facilitating their application in receptor-targeted chemotherapy and nucleic acid-based therapeutics. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and purification of phosphoramidites Basic Protocol 2: Automated solid-phase synthesis and characterization of ApDCs.
We present here a new reagent enabling the supported synthesis of oligodeoxynucleotides (ODNs) and oligoribonucleotides (ORNs) containing a phosphate group at the 5'-terminal position after complete deprotection. This reagent, derived from a dihydroxyacetone core, contains a dimethoxytrityl (DMTr) group. The procedure for final deprotection is very similar to that routinely used in the synthesis of unmodified ODNs or ORNs. In particular, it preserves the advantages of the "trityl-on" method, namely facile purification by reverse-phase high-performance liquid chromatography (RP-HPLC), short treatment with an acetic acid solution in water, the possibility of on-resin monitoring by trityl cation assay, and the use of equipment commonly founded in chemical laboratories. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Phosphorylation of oligodeoxyribonucleotides (ODNs) Alternate Protocol: Phosphorylation of oligoribonucleotides (ORNs) Support Protocol: Preparation of phosphorylation reagent 1.
