Stabilization of a Bimolecular Triplex by 3′‐S‐Phosphorothiolate Modifications: An NMR and UV Thermal Melting Investigation

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single‐stranded nucleic acids are also relevant in third‐strand binding. Thus, there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes, composed of Watson–Crick (W–C) base‐paired DNA duplexes and a Hoogsteen base‐paired RNA strand, are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently, synthetic efforts have been focused on developing TFOs with RNA‐like structural properties. Here, the structural and stability studies of such a TFO, composed of deoxynucleic acids, but with 3′‐S‐phosphorothiolate (3′‐SP) linkages at two sites is described. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. ¹H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However, the triplex involving the 3′‐SP modifications is more compact. The 3′‐SP modification was previously shown to stabilise G‐quadruplex and i‐motif structures and therefore is now proposed as a generic solution to stabilising multi‐stranded DNA structures.     

3′‐Deoxyribopyranose (4′→2′)‐Oligonucleotide Duplexes (=p‐DNA Duplexes) as Substitutes of RNA Hairpin Structures

By automated synthesis, we prepared hybrid oligonucleotides consisting of covalently linked RNA and p‐DNA sequences (p‐DNA=3′‐deoxyribopyranose (4′→2′)‐oligonucleotides) (see Table 1). The pairing properties of corresponding hybrid duplexes, formed from fully complementary single strands were investigated. An uninterrupted π‐π‐stacking at the p‐DNA/RNA interface and cooperative pairing between the two systems was achieved by connecting them via a 4′‐p‐DNA‐2′→5′‐RNA‐3′ and 5′‐RNA‐2′→4′‐p‐DNA‐2′ phosphodiester linkage, respectively (see Fig. 4). The RNA 2′‐phosphoramidites 9 – 12 , required for the formation of the RNA‐2′→4′‐p‐DNA phosphodiester linkage were synthesized from the corresponding, 3′‐O‐tom‐protected ribonucleosides (tom=[(triisopropylsilyl)oxy]methyl; Scheme 1). Analogues of the flavin mononucleotide (=FMN) binding aptamer 22 and the hammerhead ribozyme 25 were prepared. Each of these analogues consisted of two p‐DNA/RNA hybrid single strands with complementary p‐DNA sequences, designed to substitute stem/loop and stem motifs within the parent compounds. By comparative binding and cleavage studies, it was found that mixing of the two complementary p‐DNA/RNA hybrid sequences resulted in the formation of the fully functional analogues 23 ⋅ 24 and 27 ⋅ 28 of the FMN‐binding aptamer and of the hammerhead ribozyme, respectively.     

Oligonucleotides Containing Novel 4′‐C‐ or 3′‐C‐(Aminoalkyl)‐Branched Thymidines

The synthesis of four novel 3′‐C‐branched and 4′‐C‐branched nucleosides and their transformation into the corresponding 3′‐O‐phosphoramidite building blocks for automated oligonucleotide synthesis is reported. The 4′‐C‐branched key intermediate 11 was synthesized by a convergent strategy and converted to its 2′‐O‐methyl and 2′‐deoxy‐2′‐fluoro derivatives, leading to the preparation of novel oligonucleotide analogues containing 4′‐C‐(aminomethyl)‐2′‐O‐methyl monomer X and 4′‐C‐(aminomethyl)‐2′‐deoxy‐2′‐fluoro monomer Y (Schemes 2 and 3). In general, increased binding affinity towards complementary single‐stranded DNA and RNA was obtained with these analogues compared to the unmodified references (Table 1). The presence of monomer X or monomer Y in a 2′‐O‐methyl‐RNA oligonucleotide had a negative effect on the binding affinity of the 2′‐O‐methyl‐RNA oligonucleotide towards DNA and RNA. Starting from the 3′‐C‐allyl derivative 28 , 3′‐C‐(3‐aminopropyl)‐protected nucleosides and 3′‐O‐phosphoramidite derivatives were synthesized, leading to novel oligonucleotide analogues containing 3′‐C‐(3‐aminopropyl)thymidine monomer Z or the corresponding 3′‐C‐(3‐aminopropyl)‐2′‐O,5‐dimethyluridine monomer W (Schemes 4 and 5). Incorporation of the 2′‐deoxy monomer Z induced no significant changes in the binding affinity towards DNA but decreased binding affinity towards RNA, while the 2′‐O‐methyl monomer Z induced decreased binding affinity towards DNA as well as RNA complements (Table 2).     

DNA with 3′‐5′‐Disulfide Links—Rapid Chemical Ligation through Isosteric Replacement

Efforts to chemically ligate oligonucleotides, without resorting to biochemical enzymes, have led to a multitude of synthetic analogues, and have extended oligomer ligation to reactions of novel oligonucleotides, peptides, and hybrids such as PNA. 1 Key requirements for potential diagnostic tools not based on PCR include a fast templated chemical DNA ligation method that exhibits high pairing selectivity, and a sensitive detection method. Here we report on a solid‐phase synthesis of oligonucleotides containing 5′‐ or 3′‐mercapto‐dideoxynucleotides and their chemical ligations, yielding 3′‐5′‐disulfide bonds as a replacement for 3′‐5′‐phosphodiester units. Employing a system designed for fluorescence monitoring, we demonstrate one of the fastest ligation reactions with half‐lives on the order of seconds. The nontemplated ligation reaction is efficiently suppressed by the choice of DNA modification and the 3′‐5′ orientation of the activation site. The influence of temperature on the templated reaction is shown.     

Synthesis and Hybridization Properties of an Oligonucleotide Consisting of 1′,4′‐Anhydro‐2′,5′‐dideoxy‐2′‐(thymin‐1‐yl)‐D‐altritol

The novel oligonucleotide analogue 7 , consisting of 1′,4′‐anhydro‐2′,5′‐dideoxy‐2′‐(thymin‐1‐yl)‐D ‐altritol ( 4 ), residues was synthesized by the phosphoramidite approach on an automated DNA synthesizer. The phosphoramidite building block 6 was obtained by phosphitylation of the corresponding isonucleoside 5 . Oligoisonucleotide 7 contains an extended phosphodiester linkage with a higher flexibility. Oligoisonucleotide 7 was studied with respect to hybridization properties, enzymatic stability, and CD spectra. It exhibits a high stability towards snake‐venom phosphodiesterase and an acceptable hybridization to complementary single‐stranded DNA and RNA.     

Enzymatic Synthesis of 7′,5′‐Bicyclo‐DNA Oligonucleotides

The selection of artificial genetic polymers with tailor‐made properties for their application in synthetic biology requires the exploration of new nucleosidic scaffolds that can be used in selection experiments. Herein, we describe the synthesis of a bicyclo‐DNA triphosphate (i.e., 7′,5′‐bc‐TTP) and show its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold. 7′,5′‐bc‐TTP is a good substrate for Therminator DNA polymerase, and up to seven modified units can be incorporated into a growing DNA chain. In addition, this scaffold sustains XNA‐dependent DNA synthesis and potentially also XNA‐dependent XNA synthesis. However, DNA‐dependent XNA synthesis on longer templates is hampered by competitive misincorporation of deoxyadenosine triphosphate (dATP) caused by the slow rate of incorporation of 7′,5′‐bc‐TTP.     

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on‐line synthesis of DNA‐3′‐PNA chimeras 1 – 4 and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras 5 – 8 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the N‐terminal part of the PNA via N‐(ω‐hydroxyalkyl)‐N‐[(thymin‐1‐yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA and RNA. All investigated DNA‐3′‐PNA chimeras and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras form more‐stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N‐(3‐hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA‐3′‐PNA chimeras, whereas the (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras showed optimal binding with the homologous N‐(4‐hydroxybutyl)glycine linker. The duplexes of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA‐3′‐PNA chimeras. Surprisingly, we found that the charged (2′‐O‐methyl‐RNA)‐3′‐PNA chimera with a N‐(4‐hydroxybutyl)glycine‐based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras include their use as antisense agents acting by a RNase‐independent mechanism of action, a prerequisite for antisense‐oligonucleotide‐mediated correction of aberrant splicing of pre‐mRNA.     

Synthesis of Photolabile 5′‐O‐Phosphoramidites for the Photolithographic Production of Microarrays of Inversely Oriented Oligonucleotides

The photolabile 3′‐O‐{[2‐(2‐nitrophenyl)propoxy]carbonyl}‐protected 5′‐phosphoramidites ( 16 – 18 ) were synthesized (see Scheme) for an alternative mode of light‐directed production of oligonucleotide arrays. Because of the characteristics of these monomeric building blocks, photolithographic in situ DNA synthesis occurred in 5′→3′ direction, in agreement with the orientation of enzymatic synthesis. Synthesis yields were as good as those of conventional reactions. The resulting oligonucleotides are attached to the surface via their 5′‐termini, while the 3′‐hydroxy groups are available as substrates for enzymatic reactions such as primer extension upon hybridization of a DNA template (see Fig. 2). The production of such oligonucleotide chips adds new procedural avenues to the growing number of applications of DNA microarrays.     

Nucleotides,Part LXV,Synthesis of 2′‐Deoxyribonucleoside 5′‐Phosphoramidites: New Building Blocks for the Inverse (5′‐3′)‐Oligonucleotide Approach

The syntheses of the 3′‐O‐(4,4′‐dimethoxytrityl)‐protected 5′‐phosphoramidites 25 – 28 and 5′‐(hydrogen succinates) 29 – 32 , which can be used as monomeric building blocks for the inverse (5′‐3′)‐oligodeoxyribonucleotide synthesis are described (Scheme). These activated nucleosides and nucleotides were obtained by two slightly different four‐step syntheses starting with the base‐protected nucleosides 13 – 20 . For the protection of the aglycon residues, the well‐established 2‐(4‐nitrophenyl)ethyl (npe) and [2‐(4‐nitrophenyl)ethoxy]carbonyl (npeoc) groups were used. The assembly of the oligonucleotides required a slightly increased coupling time of 3 min in application of the common protocol (see Table 1). The use of pyridinium hydrochloride as an activator (instead of 1H‐tetrazole) resulted in an extremely shorter activation time of 30 seconds. We established the efficiency of this inverse strategy by the synthesis of the oligonucleotide 3′‐conjugates 33 and 34 which carry lipophilic caps derived from cholesterol and vitamin E, respectively, as well as by the formation of (3′‐3′)‐ and (5′‐5′)‐internucleotide linkages (see Table 2).     

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

A fast, high‐yielding and reliable method for the synthesis of DNA‐ and RNA 5′‐triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite was coupled to the 5′‐end. Oxidation of the formed 5′‐phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′‐cycloSal‐oligonucleotides. Reaction of the support‐bonded 5′‐cycloSal‐oligonucleotide with pyrophosphate yielded the corresponding 5′‐triphosphates. The 5′‐triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.     

Spontaneous Aminoacylation of a RNA Sequence Containing a 3′‐Terminal 2′‐Thioadenosine

We report the synthesis of a modified 8mer RNA sequence, (C‐C‐C‐C‐A‐C‐C‐(2′‐thio)A)‐RNA 5′‐(dihydrogen phosphate) ( 9 ) containing a 3′‐terminal 2′‐thioadenosine (Schemes 2 and 3), and its spontaneous and site‐specific aminoacylation with the weakly activated amino acid thioester H Phe SPh ( 12 ). This reaction, designed in analogy to the ‘native chemical ligation’ of oligopeptides, occurs efficiently in buffered aqueous solutions and under a wide range of conditions (Table). At pH values between 5.0 and 7.4, two products, the 3′‐O‐monoacylated and the 3′‐O,2′‐S‐diacylated RNA sequences 10 and 11 are formed fast and quantitatively (Scheme 4). At pH 7.4 and 37°, the 3′‐O‐monoacylated product 10 is formed as major product in situ by selective hydrolysis of the O,S‐diacylated precursor 11 . Additionally, the preparation and isolation of the relevant 3′‐O‐monoacylated product 10 was optimized at pH 5. The here presented concept could be employed for a straightforward aminoacylation of analogously modified tRNAs.     

Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

Functionalization of RNA at the 5′‐terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5′‐nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5′‐phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5′‐end of RNA.     

2′‐Ethynyl‐DNA: Synthesis and Pairing Properties

2‐Ethynyl‐DNA was developed as a potential DNA‐selective oligonucleotide analog. The synthesis of 2′‐arabino‐ethynyl‐modified nucleosides was achieved starting from properly protected 2′‐ketonucleosides by addition of lithium (trimethylsilyl)acetylide followed by reduction of the tertiary alcohol. After a series of protecting‐group manipulations, phosphoramidite building blocks suitable for solid‐phase synthesis were obtained. The synthesis of oligonucleotides from these building blocks was successful when a fast deprotection scheme was used. The pairing properties of 2′‐arabino‐ethynyl‐modified oligonucleotides can be summarized as follows: 1) The 2′‐arabino‐ethynyl modification of pyrimidine nucleosides leads to a strong destabilization in duplexes with DNA as well as with RNA. The likely reason is that the ethynyl group sterically influences the torsional preferences around the glycosidic bond leading to a conformation not suitable for duplex formation. 2) If the modification is introduced in purine nucleosides, no such influence is observed. The pairing properties are not or only slightly changed, and, in some cases (deoxyadenosine homo‐polymers), the desired stabilization of the pairing with a DNA complementary strand and destabilization with an RNA complement is observed. 3) In oligonucleotides of alternating deoxycytidine‐deoxyguanosine sequence, the incorporation of 2′‐arabino‐ethynyl deoxyguanosine surprisingly leads to the formation of a left‐handed double helix, irrespective of salt concentration. The rationalization for this behavior is that the ethynyl group locks such duplexes in a left‐handed conformation through steric blockade.     

Mapping the Landscape of Potentially Primordial Informational Oligomers: (3′→2′)‐D‐Phosphoglyceric Acid Linked Acyclic Oligonucleotides Tagged with 2,4‐Disubstituted 5‐Aminopyrimidines as Recognition Elements

The (3′→2′)‐phosphodiester glyceric acid backbone containing an acyclic oligomer tagged with 2,4‐disubstituted pyrimidines as alternative recognition elements have been synthesized. Strong cross‐pairing of a 2,4‐dioxo‐5‐aminopyrimidine hexamer, rivaling locked nucleic acid (LNA) and peptide nucleic acid (PNA), with complementary adenine‐containing DNA and RNA sequences was observed. The corresponding 2,4‐diamino‐ and 2‐amino‐4‐oxo‐5‐aminopyrimidine‐tagged oligomers were synthesized, but difficulties in deprotection, purification, and isolation thwarted further investigations. The acyclic phosphate backbone structure of the protected oligomer seems to be prone to an eliminative degradation owing to the acidic hydrogen at the 2′‐position—an arrangement that renders the oligomer vulnerable to the conditions used for the removal of the protecting groups on the heterocyclic recognition element. However, the free oligomers seem to be stable under the conditions investigated.     

Synthesis and Pairing Properties of 3′‐Deoxyribopyranose (4′→2′)‐Oligonucleotides (‘p‐DNA')

The preparation and the pairing properties of the new 3′‐deoxyribopyranose (4′→2′)‐oligonucleotide (=p‐DNA) pairing system, based on 3′‐deoxy‐β‐D ‐ribopyranose nucleosides is presented. D ‐Xylose was efficiently converted to the prefunctionalized 3‐deoxyribopyranose derivative 4‐O‐[(tert‐butyl)dimethylsilyl]‐3‐deoxy‐D ‐ribopyranose 1,2‐diacetate 8 (obtained as a 4 : 1 mixture of α‐ and β‐D ‐anomers; Scheme 1). From this sugar building block, the corresponding, appropriately protected thymine, guanine, 5‐methylcytosine, and purine‐2,6‐diamine nucleoside phosphoramidites 29 – 32 were prepared in a minimal number of steps (Schemes 2–4). These building blocks were assembled on a DNA synthesizer, and the corresponding p‐DNA oligonucleotides were obtained in good yields after a one‐step deprotection under standard conditions, followed by HPLC purification (Scheme 5 and Table 1). Qualitatively, p‐DNA shows the same pairing behavior as p‐RNA, forming antiparallel, exclusively Watson‐Crick‐paired duplexes that are much stronger than corresponding DNA duplexes. Duplex stabilities within the three related (i.e., based on ribopyranose nucleosides) oligonucleotide systems p‐RNA, p‐DNA, and 3′‐O‐Me‐p‐RNA were compared with each other (Table 2). Intrinsically, p‐RNA forms the strongest duplexes, followed by p‐DNA, and 3′‐O‐Me‐p‐RNA. However, by introducing the nucleobases purine‐2,6‐diamine (D) and 5‐methylcytosine (M) instead of adenine and cytosine, a substantial increase in stability of corresponding p‐DNA duplexes was observed.     

Synthesis of a Thymidine Dimer Containing a Tetrazole‐2,5‐diyl Internucleosidic Linkage and Its Insertion into Oligodeoxynucleotides

Thymidine dimers in which the natural phosphodiester linkage has been replaced by a 2,5‐disubstituted tetrazole ring are synthesized and incorporated into oligodeoxynucleotides (ODNs). The synthesis is accomplished by two strategies based on an alkylation of 5′‐O‐trityl‐on and 5′‐O‐trityl‐off 3′‐deoxy‐3′‐(1H‐tetrazol‐5‐yl)thymidines with 5′‐iodo‐5′‐deoxythymidine in the presence of Et₃N, and the formation of only 2‐substituted tetrazol‐5‐yl linkages is observed in 89 and 46% yields, respectively. The nucleoside dimer formed is reacted with 4,4′‐dimethoxytrityl chloride (DMTCl), followed by treatment with 2‐cyanoethyl tetraisopropylphosphordiamidite in the presence of N,N‐diisopropylammonium tetrazolide, to afford the 5′‐O‐DMT‐protected dinucleoside phosphoramidite that is used for incorporation into ODNs on an automated DNA synthesizer. The modified ODNs with one and up to five tetrazole internucleosidic linkages are obtained in good yields. The thermal stability of DNA/DNA and DNA/RNA duplexes is studied by UV experiments and reported also.     

Expanding the Scope of 2′‐SCF3 Modified RNA

The 2′‐trifluoromethylthio (2′‐SCF₃) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2′‐SCF₃ adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc‐protecting group strategy, are reported. RNA solid‐phase synthesis provided site‐specifically 2′‐SCF₃‐modified oligoribonucleotides that were investigated intensively. Their excellent behavior in ¹⁹F NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule–RNA interaction. Moreover, comparably to the 2′‐SCF₃ pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off‐target effects and their performance in silencing of the BASP1 gene was demonstrated.     

Reliable Chemical Synthesis of Oligoribonucleotides (RNA) with 2′‐O‐[(Triisopropylsilyl)oxy]methyl(2′‐O‐tom)‐Protected Phosphoramidites

A method for the introduction of the 2′‐O‐[(triisopropylsilyl)oxy]methyl (=tom) group into N‐acetylated, 5′‐O‐dimethoxytritylated ribonucleosides is presented. The corresponding 2′‐O‐tom‐protected phosphoramidite building blocks were obtained in pure form and were successfully employed for the routine synthesis of oligoribonucleotides on DNA synthesizers. Under DNA coupling conditions (2.5 min coupling time for a 1.5‐μmol synthesis scale) and with 5‐(benzylthio)‐1H‐tetrazole (BTT) as activator, 2′‐O‐tom‐protected phosphoramidites exhibited average coupling yields >99.4%. The combination of N‐acetyl and 2′‐O‐tom protecting groups allowed a reliable and complete two‐step deprotection, first with MeNH₂ in EtOH/H₂O and then with Bu₄NF in THF, without concomitant destruction of the product RNA sequences.     

The 2‐Cyano‐2,2‐dimethylethanimine‐N‐oxymethyl Group for the 2′‐Hydroxyl Protection of Ribonucleosides in the Solid‐Phase Synthesis of RNA Sequences

The reaction of 2‐cyano‐2‐methyl propanal with 2′‐O‐aminooxymethylribonucleosides leads to stable and yet reversible 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl)ribonucleosides. Following N‐protection of the nucleobases, 5′‐dimethoxytritylation and 3′‐phosphitylation, the resulting 2′‐protected ribonucleoside phosphoramidite monomers are employed in the solid‐phase synthesis of three chimeric RNA sequences, each differing in their ratios of purine/pyrimidine. When the activation of phosphoramidite monomers is performed in the presence of 5‐benzylthio‐1H‐tetrazole, coupling efficiencies averaging 99 % are obtained within 180 s. Upon completion of the RNA‐chain assemblies, removal of the nucleobase and phosphate protecting groups and release of the sequences from the solid support are carried out under standard basic conditions, whereas the cleavage of 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl) protective groups is effected (without releasing RNA alkylating side‐products) by treatment with tetra‐n‐butylammonium fluoride (0.5 m) in dry DMSO over a period of 24–48 h at 55 °C. Characterization of the fully deprotected RNA sequences by polyacrylamide gel electrophoresis (PAGE), enzymatic hydrolysis, and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry confirmed the identity and quality of these sequences. Thus, the use of 2′‐O‐aminooxymethylribonucleosides in the design of new 2′‐hydroxyl protecting groups is a powerful approach to the development of a straightforward, efficient, and cost‐effective method for the chemical synthesis of high‐quality RNA sequences in the framework of RNA interference applications.