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.
This article describes experimental and analytical procedures for evaluating the efficiency and fidelity of DNA replication containing mirror-image thymidine ( L -T) in E. coli . The procedure involves construction of DNA recombinants containing a restriction enzyme (PstI) recognition site in which the L -T lesion is site-specifically located within the PstI recognition sequence (CTGCAG). The recombinants are transfected into DH5α cells. DNA is extracted, amplified, and cleaved into relatively short fragments using different combinations of restriction enzymes to facilitate electrophoretic analysis. Detailed explanations for the restriction enzyme–mediated assay for detection of mutagenic properties of mirror-image thymidine at a predetermined site are also presented. Advantages and limitations of the assay are discussed by comparing it to other techniques used for detecting lesion-induced mutation efficiency, and a troubleshooting guide is provided. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Synthesis of oligonucleotides containing L -T Basic Protocol 2 : Construction of DNA recombinants Basic Protocol 3 : Mutation analysis of L -T-induced replication products using a restriction enzyme–mediated assay
This article contains the detailed synthesis and characterization protocols of azobenzene containing siRNAs, which have photoswitchable properties effectively controlled with light. First, the azobenzene scaffolds are synthesized via reduction of nitrophenyl alcohols in the presence of zinc. Next, the hydroxyl group of azobenzene derivatives are protected with a 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 azobenzene-containing oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxymethyl)-azobenzene phosphoramidite Basic Protocol 2: Synthesis of 4,4'-bis(hydroxyethyl)-azobenzene phosphoramidite Basic Protocol 3: Synthesis, purification and characterization of oligonucleotides containing azobenzene derivatives.
This article contains the detailed biophysical characterization, biological testing, and photo-switching protocols of azobenzene containing siRNAs (siRNAzos), which have photoswitchable properties that can be controlled with light. First, the siRNAzos are characterized by annealing the sense and anti-sense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (Tm ) of the duplexes. Second, the biological testing of the siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to ultraviolet (UV) light in order to inactivate the siRNAzo, and then broadband visible light, which re-activates the siRNAzo. This inactivation/reactivation protocol can be done in real time, and is reversible and robust and can be performed multiple times on the same sample if desired. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Bio-physical characterization of siRNAzo duplexes Basic Protocol 2: Evaluation of azobenzene gene-silencing using Firefly Luciferase Basic Protocol 3: Evaluation of azobenzene gene-silencing using reverse transcriptase-polymerase chain reaction (RT-PCR).
Custom-built DNA nanostructures are now used in applications such as biosensing, molecular computation, biomolecular analysis, and drug delivery. While the functionality and biocompatibility of DNA makes DNA nanostructures useful in such applications, the field faces a challenge in making biostable DNA nanostructures. Being a natural material, DNA is most suited for biological applications, but is also easily degraded by nucleases. Several methods have been employed to study the nuclease degradation rates and enhancement of nuclease resistance. This protocol describes the use of gel electrophoresis to analyze the extent of nuclease degradation of DNA nanostructures and to report degradation times, kinetics of nuclease digestion, and evaluation of biostability enhancement factors. © 2020 Wiley Periodicals LLC. Basic Protocol: Timed analysis of nuclease degradation of DNA nanostructures Support Protocol: Calculating biostability enhancement factors.
This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMadeTM synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMadeTM synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer.
A detailed protocol for preparation 3'-glycoconjugated oligonucleotides is described based on one-pot immobilization of 4,4'-dimethoxytrityl-protected carbohydrates to a solid support followed by on-support peracetylation and automated oligonucleotide assembly. Compared to an appropriate building block approach and post-synthetic manipulation of oligonucleotides, this protocol may simplify the synthesis scheme and increase overall yield of the conjugates. Furthermore, the immobilization to a solid support typically increases the stability of reactants, enabling prolonged storage, and makes subsequent processing convenient. Automated assembly on these carbohydrate-modified supports using conventional phosphoramidite chemistry produces 3'-glycoconjugated oligonucleotides in relatively high yield and purity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 1-O-tert-butyldimethylsilyl-6-O-(4,4'-dimethoxytrityl)-β-D-glucose Basic Protocol 2: Synthesis of 6-O-dimethoxytrityl-2,3,1',3',4',6'-hexa-O-benzoylsucrose Basic Protocol 3: Synthesis of 6″-O-dimethoxytrityl-N-trifluoroacetyl-protected aminoglycosides Basic Protocol 4: Synthesis of 3-O-dimethoxytrityl-propyl β-D-galactopyranoside Basic Protocol 5: Synthesis of trivalent N-acetyl galactosamine cluster Basic Protocol 6: Synthesis of carbohydrate monosuccinates and their immobilization to a solid support Basic Protocol 7: Oligonucleotide synthesis using immobilized carbohydrates.
This article contains detailed synthetic procedures for the implementation of the sulfo-click reaction to nucleoside derivatives. First, 3'-O-TBDMS-protected nucleosides are converted to their corresponding 4'-thioacid derivatives in three steps. Then, various conjugates are synthetized via a biocompatible and chemoselective coupling procedure using sulfonyl azide partners. Finally, to illustrate the potential of the sulfo-click reaction, a nucleoside bearing two orthogonal azido groups is synthesized and engaged in one-pot dual labeling through a sulfo-click/copper-catalyzed azide-alkyne cycloaddition (CuAAC) cascade. The high efficiency of the sulfo-click reaction as applied to nucleosides opens up new possibilities in the context of bioconjugation. © 2020 Wiley Periodicals LLC. Basic Protocol 1: General protocol for the synthesis of 4'-thioacid-nucleoside derivatives Basic Protocol 2: Implementation of the sulfo-click reaction Basic Protocol 3: Synthesis of 3'-azido-4'-(carboxamido)ethane-sulfonyl azide-3'-deoxythymidine Basic Protocol 4: Detailed synthetic procedure for one-pot double-click conjugations.
Tandem catalysis has been at the forefront of synthesis in the past decade due to the reduction in the number of steps and purification needed for the synthesis of commercially relevant molecules. With the right combination of catalyst systems, which could be homometallic or multimetallic, one can construct complex structural motifs in a one-pot procedure without the requirement for the isolation of the intermediates, reducing both reagent waste and time. Over the years, application of tandem catalysis has certainly extended towards arene and heteroarene motifs; nucleoside modification using such a strategy has been rare. In this regard, we would like to report herein the development of numerous homometallic and multimetallic tandem catalytic protocols for the modification of nucleosides, providing efficient access to a diverse range of molecules with promising fluorescent properties, as well as pharmaceutically relevant antiviral drugs such as FV-100. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Double tandem one-pot Sonogashira/cyclization of 5-IdU for the synthesis of FV-100 and analogs Basic Protocol 2: Double tandem one-pot Heck/Suzuki-Miyaura of 5-IdU for the synthesis of fluorescent nucleoside analogs Basic Protocol 3: Double tandem one-pot Suzuki-Miyaura cross-coupling of 5-IdU for the synthesis of fluorescent nucleoside analogs Basic Protocol 4: Double tandem one-pot amination/amidation for the synthesis of Sangivamycin precursor Basic Protocol 5: Triple tandem one-pot chemoselective etherification/Sonogashira coupling/cyclization for synthesis of BCNA analogs Basic Protocol 6: Triple tandem one-pot sequential Heck/borylation/Suzuki-Miyaura reaction.
The protocols presented in this article describe highly detailed synthesis of trifluoromethylated purine nucleotides and nucleosides (G and A). The procedure involves trifluoromethylation of properly protected (acetylated) nucleosides, followed by deprotection leading to key CF3 -containing nucleosides. This gives synthetic access to 8-CF3 -substituted guanosine derivatives and three adenosine derivatives (8-CF3 , 2-CF3 , and 2,8-diCF3 ). In further steps, phosphorylation and phosphate elongation (for selected examples) result in respective trifluoromethylated nucleoside mono-, di-, and triphosphates. Support protocols are included for compound handling, purification procedures, analytical sample preparation, and analytical techniques used throughout the performance of the basic protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of trifluoromethylated guanosine and adenosine derivatives Basic Protocol 2: Synthesis of trifluoromethylated guanosine and adenosine monophosphates Basic Protocol 3: Synthesis of phosphorimidazolides of 8-CF3 GMP and 8-CF3 AMP Basic Protocol 4: Synthesis of trifluoromethylated guanosine and adenosine oligophosphates Support Protocol 1: TLC sample preparation and analysis Support Protocol 2: Purification protocol for Basic Protocol 1 Support Protocol 3: HPLC analysis and preparative HPLC Support Protocol 4: Ion-exchange chromatography.
NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotope‐labeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclear‐detected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotope‐labeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemo‐enzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear‐detected NMR experiments including 13C‐detected experiments for ribose assignment and amino groups, the CN‐spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15N‐detected band‐selective excitation short transient transverse‐relaxation‐optimized spectroscopy (BEST‐TROSY) experiment. © 2020 The Authors.
This article contains detailed synthetic protocols for preparation of 5-cyanomethyluridine (cnm5 U) and 5-cyanouridine (cn5 U) phosphoramidites. The synthesis of the cnm5 U phosphoramidite building block starts with commercially available 5-methyluridine (m5 C), followed by bromination of the 5-methyl group to install the cyano moiety using TMSCN/TBAF. The cn5 U phosphoramidite is obtained by regular Vorbrüggen glycosylation of the protected ribofuranose with silylated 5-cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA-based therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cnm5 U and cn5 U phosphoramidites Basic Protocol 2: Synthesis, purification, and characterization of cnm5 U- and cn5 U-modified RNA oligonucleotides.
This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid-phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed-phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC-MS and LC-MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC-MS is employed first to identify RNA phosphorothioate modifications. An optimized LC-MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2: Digestion of RNA samples extracted from cells Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry.
RNAs with 5' functional groups have been gaining interest as molecular probes and reporter molecules. Copper-catalyzed azide-alkyne cycloaddition is one of the most straightforward methods to access such molecules; however, RNA functionalization with azide group has been posing a synthetic challenge. This article describes a simple and efficient protocol for azide functionalization of oligoribonucleotides 5'-end in solid-phase. An azide moiety is attached directly to the C5'-end in two steps: (i) -OH to -I conversion using methyltriphenoxyphosphonium iodide, and (ii) -I to -N3 substitution using sodium azide. The reactivity of the resulting compounds is exemplified by fluorescent labeling using both copper(I)-catalyzed (CuAAC) and strain-promoted (SPAAC) azide-alkyne cycloaddition reactions, ligation of two RNA fragments, and cyclization of short bifunctionalized oligonucleotides. The protocol makes use of oligoribonucleotides synthesized by standard phosphoramidite approach on solid support, using commercially available 2'-O-PivOM-protected monomers. Such a protection strategy eliminates the interference between the iodination reagent and silyl protecting groups (TBDMS, TOM) commonly used in RNA synthesis by phosphoramidite approach. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of oligoribonucleotide 5'-azides Basic Protocol 2: CuAAC labeling of oligoribonucleotide 5'-azides in solution Alternate Protocol 1: CuAAC labeling of oligoribonucleotide 5'-azides on solid support Basic Protocol 3: SPAAC labeling of oligoribonucleotide 5'-azides Basic Protocol 4: CuAAC ligation of oligoribonucleotide 5'-azides Basic Protocol 5: CuAAC cyclization of oligoribonucleotide 5'-azides Support Protocol: HPLC Purification.
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.
The reaction between N-hydroxy succinimide (NHS) ester-activated carboxylic acids and amino-modified nucleic acids is commonly used for the post-synthetic modification of oligonucleotides. Here, we report a two-step variation of the method in which the NHS ester is replaced by the corresponding parent carboxylic acid. In the first step, the carboxylic acid is activated with a standard peptide coupling reagent like HBTU in an anhydrous water-miscible aprotic organic solvent. In the second step, the solution of the activated carboxylic acid is added to the amino-modified oligonucleotide in water. The method is demonstrated using 40-kDa polyethylene glycol (PEG) carboxylic acid and biotin as examples. Recycling of the carboxylic acid, which is typically used in molar excess over the nucleic acid, is shown for the conjugation with 40-kDa PEG carboxylic acid. This conjugation method is generally applicable to the conjugation of carboxylic acids to amino-modified oligonucleotides, thus enabling the attachment of small to large molecular entities such as dyes, tags, peptides, and other macromolecules. © 2020 Wiley Periodicals LLC. Basic Protocol 1: General protocol for the conjugation of an amino-modified oligonucleotide with a carboxylic acid, exemplified for 40-kDa PEG carboxylic acid Basic Protocol 2: Biotinylation of an amino-modified oligonucleotide using the general conjugation protocol Basic Protocol 3: Recycling of the carboxylic acid component from the conjugation reaction, demonstrated for 40-kDa PEG carboxylic acid using ultrafiltration Support Protocol 1: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with 40-kDa PEG carboxylic acid Support Protocol 2: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with biotin Support Protocol 3: Analytical IP-RP-HPLC method used as in-process control method to monitor the conjugation reaction Alternate Protocol: Separation of 40-kDa PEG carboxylic acid from unreacted and conjugated oligonucleotide by preparative AEX-HPLC.
