2'‐O‐Acetal Levulinic Ester Ribonucleoside 3'‐Phosphoramidites for the Solid‐Phase Synthesis of Long RNA

Abstract

Solid‐phase RNA synthesis is challenging because the 2'‐hydroxyl requires a protecting group that is stable during chain assembly yet easily removable without degrading the product. Driven by the demand for long RNA therapeutics, we report a synthesis method using 2'‐O‐acetal levulinic ester (ALE) ribonucleoside 3'‐O‐phosphoramidites. This approach utilizes a rapid, base‐labile on‐column deprotection strategy that preserves the 5'‐O‐dimethoxytrityl (DMTr) group, facilitating DMTr‐ON reverse‐phase (RP) purification. The resulting protocols enable the efficient production of long, functional RNAs, such as single‐guide RNAs with superior yield and purity compared to conventional silyl‐based methods, while remaining compatible with diverse chemical modifications. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Automated solid‐phase synthesis of 2'‐ALE RNA

Basic Protocol 2: Fast on‐column deprotection of 2'‐ALE RNA

Basic Protocol 3: DMTr‐ON reverse‐phase high‐performance liquid chromatography (RP‐HPLC) purification of long RNA

Support Protocol 1: Automated deprotection setup of 2'‐ALE RNA

Support Protocol 2: Denaturing polyacrylamide gel electrophoresis (PAGE) purification of RNA

INTRODUCTION

The success of phosphoramidite‐based oligonucleotide synthesis relies heavily on the strategic selection of protecting groups. RNA synthesis is inherently more complex than DNA due to the necessity of an orthogonal protecting group at the 2'‐hydroxyl position. Ideally, this group must remain stable throughout the repetitive acidic and oxidative conditions of the synthesis cycle (Flemmich et al., 2024), while still allowing for removal without degrading the RNA product. Meeting this dual requirement has been the primary challenge in the field and a major catalyst for methodological innovation.

Over several decades, various 2'‐protecting groups have been developed to balance synthetic stability, coupling efficiency, and deprotection compatibility. While the 2'‐O‐tert‐butyldimethylsilyl (2'‐TBDMS) method pioneered by Ogilvie et al. (1988) remains a benchmark for automated synthesis, its steric bulk limits routine production <100 nt. These constraints often lead to significant truncated failure sequences. Additionally, fluoride‐based deprotection [e.g., tetra‐n‐butylammonium fluoride (TBAF) or triethylamine trihydrofluoride (TREAT‐HF)] can trigger premature 5'‐O‐dimethoxytrityl (DMTr) loss, complicating “trityl‐on” purification (Wincott et al., 1995). To address these hurdles, alternatives like the 2'‐[tris(isopropylsilyl)oxy]methyl (2'‐TOM) group offer reduced steric hindrance for longer RNA strands but remain fluoride‐dependent. The 2'‐bis(acetoxyethoxy)methyl (2'‐ACE) group achieves stepwise yields >99%, yet its acid‐lability is incompatible with standard 5'‐O‐DMTr protection, requiring specialized 5'‐silyl ethers and modified synthesizers (Scaringe et al., 1998). Conversely, the 2'‐tert‐butyldithiomethyl (DTM) group offers a bio‐labile alternative; it is cleaved under mild reducing conditions, making it a promising candidate for RNA prodrug applications (Biscans et al., 2016; Ochi et al., 2013). However, its widespread use is hindered by the inherent instability and reversibility of the disulfide bond and a short shelf life. Similarly, while the 2'‐cyanoethoxymethyl (CEM) group facilitated the synthesis of a 110‐mer RNA (Shiba et al., 2007), its removal requires 0.5 M TBAF, failing to eliminate fluoride from the deprotection workflow. These cases highlight the ongoing trade‐offs in RNA synthesis between coupling efficiency, mild deprotection, and operational simplicity.

Base‐labile 2'‐protecting groups streamline RNA synthesis by mimicking the simplicity of DNA deprotection. For example, 2'‐pivaloyloxymethyl (PivOM) acetal esters are cleaved under standard basic conditions, aligning RNA workflows with DNA protocols (Biscans et al., 2014; Lavergne et al., 2008, 2010). Similarly, 2'‐O‐thionocarbamate (TC) groups allow for single‐step deprotection of both nucleobases and the 2'‐OH using 1,2‐diamines under anhydrous conditions (Dellinger et al., 2011). More recently, the 2'‐O‐imino‐2‐propanoate group introduced a two‐step base‐mediated process involving NaOH‐driven saponification followed by rapid intramolecular cleavage (Takahashi et al., 2021).

Initially developed for microarray‐based RNA synthesis to bypass fluoride treatments, 2'‐O‐ acetal levulinic ester (ALE) protecting groups effectively prevent 2'‐3' acyl migration while maintaining stability during synthesis cycles (Lackey et al., 2009). While early applications focused on 2'‐branched RNA mimics for Dbr1 structural studies (Clark et al., 2016; Katolik et al., 2014, 2017; Montemayor et al., 2014), ALE‐based phosphoramidites have more recently enabled the synthesis of functional RNAs exceeding 200 nt, including single guide RNAs (sgRNAs) and minimal messenger RNAs (mRNAs) (Lyu et al., 2026). The protocols described here provide a practical workflow for routine long‐RNA synthesis. We detail automated assembly (Basic Protocol 1), rapid on‐column deprotection at room temperature (Basic Protocol 2 and Support Protocol 1), and high‐resolution purification via reverse‐phase high‐performance liquid chromatography (RP‐HPLC) or polyacrylamide gel electrophoresis (PAGE) (Basic Protocol 3 and Support Protocol 2). Combined, these methods facilitate the production of long RNAs such as 100 nt sgRNAs through fast coupling and mild, base‐mediated deprotection.

Basic Protocol 1. AUTOMATED SOLID‐PHASE SYNTHESIS OF 2'‐ALE RNA

This protocol details the automated solid‐phase synthesis of RNA at a 1 µmol scale using 2'‐ALE phosphoramidites on Sierra BioSystems K&A (S‐4‐LC or H‐8 SE) synthesizers operated with software version [02.00‐64]. The method can be readily adapted to any synthesizer with comparable reagent delivery and pressure specifications. To enable rapid, base‐mediated deprotection while preserving RNA integrity, the phosphoramidites utilize base‐labile protecting groups (e.g., Lev and dmf; Fig. 1) in combination with succinyl‐LCAA supports. For 100 nt sgRNAs, 2000 Å controlled‐pore glass (CPG) pore sizes are used to improve diffusion and coupling efficiency (Kozlov et al., 2005), though this reduces the overall CPG loading density.

Figure 1.

Structure of 2'‐O‐acetal levulinic ester (2'‐ALE) RNA phosphoramidite building block with the base labile protecting group structures. Abbreviations: DMTr, 4,4'‐dimethoxytrityl; Lev, levulinyl (levulinic acid derived protecting group); Dmf, N,N‑dimethylformamidine.

Materials

• Acetone ACS reagent (Sigma, cat. no. 179124‐4L)

• 5'‐DMTr‐2'‐ALE‐adenosine(N‐Lev) 3'‐CE phosphoramidite (ChemGenes, cat. no. ANP‐7385)

• 5'‐DMTr‐2'‐ALE‐guanosine (N‐Dmf) 3'‐CE phosphoramidite (ChemGenes, cat. no. ANP‐7387)

• 5'‐DMTr‐2'‐ALE‐cytidine (N‐Lev) 3'‐CE phosphoramidite (ChemGenes, cat. no. ANP‐7386)

• 5'‐DMTr‐2'‐ALE‐uridine 3'‐CE phosphoramidite (ChemGenes, cat. no. ANP‐7384)

• Cylinder argon, pre‐purified (Praxair, cat. no. AR 4.8‐T)

• Acetonitrile HPLC reagent (Fisher Scientific, cat. no. A998‐4)

• Succinyl‐3'‐LCAA CPG 2000 or 3000 Å (ChemGenes)

• Methylene chloride (dichloromethane, DCM), ACS reagent (Fisher Chemical, cat. no. D37‐4)

• Detritylation reagent (3% trichloroacetic acid/dichloromethane) (ChemGenes, cat. no. RN‐1462)

• Activation solution (5‐ethylthiotetrazole 0.25 M in acetonitrile) (ChemGenes, cat. no. RN‐1466)

• CAP A (acetic anhydride/pyridine/tetrahydrofuran) (ChemGenes, cat. no. RN‐1458)

• CAP B (16% N‐methylimidazole in tetrahydrofuran) (ChemGenes, cat. no. RN‐7776)

• Oxidation solution (0.1 M iodine/pyridine/H₂O/tetrahydrofuran) (ChemGenes, cat. no. RN‐1456)

• Amber glass bottles compatible with synthesizer reagent ports

• Drying oven

• Vacuum desiccator

• K&A H‐8 or S‐4‐LC synthesizer (Sierra BioSystems)

• Drying trap XL (ChemGenes, cat. no. DMT‐1972)

• Empty synthesis columns, screw type, ABI/Expedite, 1.0 µmol (ChemGenes, cat. no. E‐1007‐ST)

• Air compressor (Panther Silent Air Compressor model P30TC)

• 1A day before synthesis, wash amber bottles with acetone and oven‐dry them overnight at 160°C. Cool to room temperature in a vacuum desiccator before use.
• 2Weigh the 2'‑ALE phosphoramidites into clean, dried amber bottles. Vacuum‐dry for at least 2 hr and release under argon. To ensure optimal results for long RNA synthesis, prepare 75 mM solutions in acetonitrile (ACN) (Lyu et al., 2026). Calculate the required volume based on your synthesizer model (estimated 250 µl/cycle for K&A H‐8; 200 µl/cycle for K&A S‐4‐LC), adding an extra 1000 µl to prime the lines.
• 3Dissolve the monomers in pre‐dried anhydrous ACN to obtain a final concentration of 75 mM.

  Add an XL‐drying trap to the ACN bottle at least 1 day before using it as a diluent.

• 4Weigh the appropriate nucleoside‐derivatized CPG into columns (with top and bottom frits) at a 1 µmol scale, using the manufacturer's loading capacity. Ensure the columns are tightly sealed.

• 5Place all synthesizer reagents (anhydrous ACN, DCM, detritylation reagent, activator, capping reagents, and oxidation reagents) onto their designated ports.
• 6Prime lines individually via manual valve control (e.g., select “TCA” and “WST” until flow is visible) or use the “PRIME” command from the “START” menu to prime all lines simultaneously.
• 7Attach phosphoramidite solutions to the assigned ports (e.g., A, G, C, and U). To minimize reagent loss, prime these lines using the “PULSE” function (0.1 s intervals) until flow is observed in the waste line.
• 8Pressurize all reagents under 0.04 MPa argon. Set the air compressor to ∼0.4 MPa to ensure proper valve closure.
• 9
  Program the cycle according to Table 1, following these subroutines:

  • a.
    Four 3 s deliveries of TCA with 10 s wait times (30 s total).
  • b.
    Coupling, alternating activator and amidite delivery (1 s pulses, 0.4 s soak) for a total of 4 min per monomer.
  • c.
    Capping, sequential delivery of Cap A and Cap B (30 s total).
  • d.
    Oxidation, deliver the iodine solution for 2.5 s and allow an 18 s reaction.
  • e.
    Capping, sequential delivery of Cap A and Cap B (30 s total). This last step removes residual water from the solid support after oxidation.
    For long RNA synthesis, thorough DCM washes must be integrated before and after detritylation. This prevents acetonitrile from complexing with the deblocking acid, which can significantly inhibit detritylation kinetics, particularly beyond 100 cycles (Paul & Royappa, 1996). Pre‐detritylation manually with extended time may be necessary.

Table 1.

Automated Solid‐Phase Synthesis Cycle of Long RNA (>100 nt) Using ALE Chemistry ^a

Step	Command	Function description	Time (s)
Subroutine 1: Detritylation
1	DCM to column	Columns are flushed with anhydrous dichloromethane	9
2	Gas to column	Solid supports are dried by flushing with argon	5
3	TCA to column	Detritylation reagent TCA delivery to the columns	3
4	Delay	Wait time	10
5	Repeat steps 3 and 4 three times
6	Gas to column	TCA is cleared by flushing with argon	5
7	DCM to column	Columns are flushed with anhydrous dichloromethane	9
8	Gas to column	Solid supports are dried by flushing with argon	5
9	ACN to column	Columns are flushed with anhydrous acetonitrile	10
Subroutine 2: Coupling b
1	Gas to column	Solid supports are dried by flushing with argon	4
2	AMD mixed with TET to column	Alternated delivery of phosphoramidite and activator solution to the columns	1
3	Delay	Wait time	6
4	AMD mixed with TET to column	Alternated delivery of phosphoramidite and activator solution to the columns	0.4
5	ACN to M_W	Valve block is washed with ACN to the waste	1
6	Gas to M_W	Valve block is flushed with argon	1
7	Delay	Coupling time	240
8	Gas to column	Columns are cleared by flushing with argon	4
9	ACN to column	Columns are flushed with anhydrous acetonitrile	2.5
10	Gas to column	Columns are dried by flushing with argon	4
11	ACN to M_W	Valve block is washed with ACN to the waste	1
12	Gas to M_W	Valve block is flushed with argon	1
Subroutine 3: Capping
1	CP_A mixed with CP_B to column	Alternated delivery of Cap A and Cap B to the columns	2
2	Delay	Wait time	10
3	CP_A mixed with CP_B to column	Repeat alternated delivery of Cap A and Cap B to the columns	0.8
4	Delay	Wait time	20
5	Gas to column	Columns are cleared by flushing with argon	4
6	ACN to M_W	Valve block is washed with ACN to the waste	1
7	ACN to column	Columns are flushed with anhydrous acetonitrile	2
8	Gas to M_W	Valve block is flushed with argon	1
9	Gas to column	Columns are dried by flushing with argon	4
Subroutine 4: Oxidation
1	OXI to column	Delivery of oxidation solution to the columns	2.5
2	Delay	Wait time	9+9
3	ACN to M_W	Valve block is washed with ACN to the waste	1.5
4	Gas to column	Columns are cleared by flushing with argon	4
5	ACN to column	Columns are flushed with anhydrous acetonitrile	4
6	Repeat steps 3‐5 again
7	Gas to column	Columns are dried by flushing with argon	4
Subroutine 5: Subroutine 3 (Capping) is repeated after oxidation

^a On the K&A H‐8 and S‐4‐LC Synthesizer with Software Version [02.00‐64].

^b Note: Step 2 to 6 are programmed in branch subroutine “1ubrnch” that is dedicated to each base.

• 10Install the columns and check for leaks by manually flushing ACN through each. Run the capping subroutine (Table 1, Subroutine 3) once to acetylate any unreacted amino groups on the support.
• 11In the “CONTROL” menu, select “START” and assign sequences to their respective ports. Set “DMT OFF” to “NO” to retain the 5'‐DMTr group for HPLC purification, then initiate the run.
• 12Once the run is complete, manually flush the ports with argon to dry the columns before deprotection.

Basic Protocol 2. FAST ON‐COLUMN DEPROTECTION OF 2'‐ALE RNA

This protocol describes a rapid, on‐column deprotection workflow for RNA oligonucleotides synthesized with 2'‐ALE phosphoramidites (Fig. 2) (Lyu et al., 2026). The method utilizes nucleophilic amines in non‐polar solvents (e.g., toluene) to ensure that the deprotected, negatively charged RNA remains associated with the solid support throughout the deprotection (Dellinger et al., 2011). We use 1,2‐ethylenediamine (EDA) in toluene to prevent hydroxide‐mediated strand cleavage and preserve RNA integrity. In brief, 10% diethylamine in ACN first removes 2‐cyanoethyl phosphate groups, after which a 1:1 (v/v) EDA:toluene solution deprotects the nucleobases and cleaves the succinyl linker (Fig. 2). The RNA remains bound to the CPG via non‐covalent interactions until final elution with water. This workflow can be carried out manually, as detailed here, or implemented directly on the synthesizer using spare reagent lines (see Support Protocol 1).

Figure 2.

Deprotection scheme of RNA synthesized by 2'‐ALE phosphoramidites.

2'‐O‐methyl (2'‐OMe) modifications are frequently used in functional RNAs, such as sgRNAs, to enhance binding specificity, nuclease stability, and in vivo activity (Finn et al., 2018; Ke et al., 2022). However, commercially available 2'‐OMe‐A phosphoramidites often utilize a benzoyl (Bz) protecting group, given that guanosine is dmf‐protected, the Bz deprotection becomes the rate‐limiting step during deprotection. Time‐course liquid chromatography–mass spectrometry (LC–MS) analysis showed that 2 hr of EDA exposure is required for complete Bz removal under standard conditions (Table 2). Accordingly, we recommend extending the EDA:toluene treatment to 2 hr for any sequence containing Bz‐protected 2'‐OMe‐A monomers, and up to 3 hr when Bz content is high or sequences are particularly modification‐rich.

Table 2.

LC–MS Analysis (ESI‐) of a 26‐mer 2'‐OMe RNA Oligonucleotide Following Benzoyl (Bz) Deprotection by Ethylenediamine (30 min to 4 hr)

EDA deprotection a	Observed product mass (Da)	Observed incomplete deprotection mass (Da) a
30 min	8753.3815	+ 209.0341 (+ two Bz), + 313.0718 (+ three Bz)
1 hr	8754.3955	+ 104.0115 (+ one Bz), + 208.0517 (+ two Bz)
2 hr	8754.3965	None
3 hr	8754.3749	None
4 hr	8754.3534	None

^a Theoretical Exact Mass: 8750.5842 Da.

Materials

• 2'‐ALE RNA (from Basic Protocol 1)

• Diethylamine (DEA) (Thermo Scientific Chemicals, cat. no. 149452500)

• Acetonitrile (ACN) HPLC reagent (Fisher Scientific, cat. no. A998‐4)

• Ethylenediamine (EDA) (Sigma, cat. no. E26266)

• Toluene (Thermo Scientific, cat. no. T324‐4)

• H₂O, RNase‐free (Invitrogen, cat. no. 10977‐015 or equivalent)

• 1‐, 5‐, and 10‐ml Luer‐lock syringe

• House vacuum or vacuum manifold

• 1.5‐ml microcentrifuge tube, RNase‐free

• Sterile 0.22‐µm PES membrane syringe filter, 13‐mm (NEST, cat. no. 331211)

CAUTION: EDA is corrosive, strongly odorous, and irritating to the skin, eyes, and respiratory tract. All EDA handling should be performed in a certified chemical fume hood while wearing appropriate personal protective equipment, including lab coat, nitrile gloves, and eye protection. Because EDA is hygroscopic and volatile, containers should be kept tightly closed when not in use, and any spills should be cleaned immediately according to institutional safety procedures.

• 1Remove the argon‐dried columns once synthesis is complete.
• 2Remove phosphate protecting groups by slowly pulsing 10% DEA in dry ACN through the column (5‐ml Luer‐lock syringe) every 5 min for a total of 20 min at room temperature.

  10% DEA (de‐cyanoethylation) solution: Mix 1 ml DEA with 9 ml dry ACN. Prepare freshly in a pre‐dried bottle immediately before use.

• 3Rinse the column three times with 10 ml ACN using a syringe. Briefly vacuum‐dry.
• 4Apply a 1:1 (v/v) EDA/toluene solution to the column. Incubate at room temperature for 1 to 3 hr to cleave the RNA and remove base‐labile groups.

  EDA solution: Mix equal volumes of EDA and dry toluene (pre‐dried over 3‐Å molecular sieves).

• 5Wash the column twice with 10 ml toluene, then three times with 10 ml ACN.
• 6Keep the column under vacuum for at least 1 hr to remove remaining hemiacetal groups (2'‐O‐CH₂‐OH). Transfer the CPG to a 1.5‐ml RNase‐free microcentrifuge tube.
• 7Elute the RNA from the support by washing the CPG three times with 400 µl RNase‐free water, pooling the eluates. Filter through a 0.22‐µm PES syringe filter to remove CPG particles. The filtered solution contains the fully deprotected RNA and can be taken forward to purification by DMTr‐ON RP‐HPLC or denaturing PAGE.

Support Protocol 1. AUTOMATED DEPROTECTION SETUP OF 2'‐ALE RNA

This protocol describes how to carry out the ALE deprotection scheme (diethylamine de‑cyanoethylation followed by EDA treatment) directly on a K&A synthesizer by using two spare phosphoramidite ports and an auxiliary solvent line. This protocol outlines the automated deprotection process and provides custom scripts (see Table 3) for implementation on the K&A synthesizer S‐4‐LC or H‐8 SE using K&A software version [02.00‐64], to be executed immediately following synthesis. Automating deprotection minimizes manual handling of CPG, reduces variability between runs, and is particularly useful for high‐throughput long RNA synthesis.

Table 3.

Automated ALE‐made RNA Deprotection Cycle ^a

Step	Command	Function description	Time (s)
Subroutine 1: De‐cyanoethylation b
1	Gas to column	Solid supports are dried by flushing with argon	4
2	AMD to column	Delivery of de‐cyanoethylation solution in a specific phosphoramidite port) to the columns	1
3	Delay	Wait time	600
4	AMD to column	Repeat delivery of de‐cyanoethylation solution in a specific phosphoramidite port) to the columns	1
5	ACN to M_W	Valve block is washed with ACN to the waste	1
6	Gas to M_W	Valve block is flushed with argon	1
7	Delay	Wait time	600
8	Gas to column	Columns are cleared by flushing with argon	4
9	ACN to column	Columns are flushed with anhydrous acetonitrile	5
Subroutine 2: EDA exposure c
1	Gas to column	Solid supports are dried by flushing with argon	4
2	S2 to column	Columns are flushed with anhydrous toluene (S2)	5
3	Gas to column	Solid supports are dried by flushing with argon	5
4	AMD to column	Delivery of EDA solution in a specific phosphoramidite port to the columns	2
5	Delay	Wait time	1200
6	AMD to column	Delivery of EDA solution in a specific phosphoramidite port to the columns	2
7	Delay	Wait time	1200
8	AMD to column	Delivery of EDA solution in a specific phosphoramidite port to the columns	2
9	ACN to M_W	Valve block is washed with ACN to the waste	3
10	Gas to M_W	Valve block is flushed with argon	1
11	Delay	Wait time	1200
12	Gas to column	Solid supports are dried by flushing with argon	4
13	S2 to column	Columns are flushed with anhydrous toluene (S2)	8
14	Gas to column	Solid supports are dried by flushing with argon	4
15	ACN to column	Columns are flushed with anhydrous acetonitrile	8
16	Gas to column	Solid supports are dried by flushing with argon	8

^a This streamlined protocol utilizes two subroutines modeled after the standard “coupling” program: a 20‐min de‐cyanoethylation step (Subroutine 1) followed by a 1‐hr EDA treatment (Subroutine 2).

^b Note: Step 2 to 7 are programmed in branch subroutine named “de‐CE” that is dedicated to the amidite port that installed the de‐cyanoethylation solution.

^c Note: Step 3 to 11 are programmed in branch subroutine named “EDA” that is dedicated to the amidite port that installed the EDA solution.

Materials

• Cylinder argon pre‐purified (Praxair, cat. no. AR 4.8‐T)

• Acetone ACS reagent (Sigma, cat. no. 179124‐4L)

• Diethylamine (DEA) (Thermo Scientific Chemicals, cat. no. 149452500)

• Acetonitrile (ACN) HPLC reagent (Fisher Scientific, cat. no. A998‐4)

• Ethylenediamine (EDA) (Sigma, cat. no. E26266)

• Toluene (Thermo Scientific, cat. no. T324‐4)

• H₂O, RNase‐free (Invitrogen, cat. no. 10977‐015 or equivalent)

• Amber glass bottles for synthesizer

• Drying oven

• Vacuum desiccator

• Drying trap XL (ChemGenes, cat. no. DMT‐1972)

• K&A H‐8 or S‐4‐LC synthesizer (Sierra BioSystems)

• Air compressor (Panther Silent Air Compressor model P30TC)

• 1.5‐ml microcentrifuge tube, RNase‐free

• Sterile 0.22‐µm PES membrane syringe filter, 13‐mm (NEST, cat. no. 331211)

CAUTION: EDA is corrosive, strongly odorous, and irritating to the skin, eyes, and respiratory tract. All EDA handling should be performed in a certified chemical fume hood while wearing appropriate personal protective equipment, including lab coat, nitrile gloves, and eye protection. Because EDA is hygroscopic and volatile, containers should be kept tightly closed when not in use, and any spills should be cleaned immediately according to institutional safety procedures.

• 1One day before synthesis, wash two amber reagent bottles with acetone and dry them overnight at 160°C. Allow the bottles to cool to room temperature in a vacuum desiccator before use.
• 2Prepare the de‐cyanoethylation solution by mixing 1 ml DEA with 9 ml dry ACN. Prepare freshly in a pre‐dried bottle immediately before use.
• 3Prepare a 1:1 v/v EDA:toluene solution (EDA solution) by mixing equal volumes (e.g., 5 ml + 5 ml) of EDA and dry toluene pre‑dried over 3‐Å molecular sieves.
• 4Place the de‑cyanoethylation solution and the EDA solution in two spare amidite ports on the synthesizer. Prime the corresponding amidite lines using the manual valve controls until solution is visible in the waste “WST” line.
• 5Install a bottle of dry toluene (pre‑dried over 3‐Å molecular sieves) on an auxiliary solvent line (e.g., port S2). Prime this line by selecting ports “S2” and “WST” in the manual control menu and pressing “OK” until toluene flows steadily to waste.
• 6
  Program the automated deprotection cycle as summarized in Table 3. The cycle comprises two branch subroutines that mimic “coupling” steps but deliver only the deprotection solutions (no activator):

  • a.
    Subroutine 1: De‐cyanoethylation. The de‐cyanoethylation solution is delivered from its dedicated amidite port twice, each followed by a 600 s wait, giving a total reaction time of 20 min. The columns are then washed with ACN and dried with argon.
  • b.
    Subroutine 2: EDA exposure. Columns are pre‐wet with toluene via line S2, then treated with the EDA solution in three cycles (each involving delivery from the amidite port followed by a 1200 s incubation). For RNA containing Bz‐protected residues, total EDA exposure should be increased from 1 hr to 3 hr by extending the wait intervals. Post‐treatment, columns are flushed with argon and washed extensively with toluene and acetonitrile.
• 7Remove the columns and deprotection bottles from the synthesizer. Thoroughly wash the manifold, all ports, and lines that carried EDA using toluene and acetone, followed by ACN to ensure no crystallization in the synthesizer.
• 8Vacuum‐dry the columns for at least 1 hr to scavenge residual hemiacetal intermediates. Then, transfer the CPG from each column into individual 1.5‐ml RNase‐free microcentrifuge tubes.
• 9Elute the RNA by washing the CPG three times with 400 µl RNase‐free water, pooling the eluates. To remove residual CPG particles, clarify the solution by passing it through a sterile 0.22‐µm PES syringe filter. The resulting filtrate contains fully deprotected RNA and is ready for DMTr‐ON RP‐HPLC or denaturing PAGE purification (see Basic Protocol 3 and Support Protocol 2).

Basic Protocol 3. DMTr‐ON RP‐HPLC PURIFICATION OF LONG RNA

Purifying long RNA (>100 nt) is a major bottleneck because even high per‐cycle coupling efficiencies yield substantial amounts of n‐x failure strands that closely resemble the full‐length product in charge and hydrophobicity. This difficulty is further compounded by secondary structures that may create a heterogeneous mix of conformers leading to broad and overlapping peaks. While the hydrophobic 5'‐DMTr group serves as a robust separation handle for DNA purification, its use in conventional 2'‐TBDMS RNA synthesis is limited because high‐temperature fluoride deprotection often causes premature DMTr loss (Wincott et al., 1995). In contrast, ALE chemistry avoids these harsh reagents and temperatures, ensuring the DMTr group remains intact under its mild and room temperature deprotection conditions. This enables robust DMTr‐ON RP‐HPLC purification for RNA of varying lengths made by ALE chemistry, including 100 nt sequences. Below, we outline a semipreparative RP‐HPLC protocol optimized for 1 to 3 µmol scale ALE‐synthesized RNAs. The method is readily adjustable to other column dimensions and loading amounts by tuning injection volume, gradient slope, and flow rate. A final ethanol precipitation step allows efficient recovery and desalting, after which the 5'‐DMTr group can be removed under mild acidic conditions.

Materials

• RP‐Buffer A: 0.1 M triethylammonium acetate (TEAA) buffer, pH 7 (see recipe)

• RP‐Buffer B: Acetonitrile (ACN) HPLC reagent (Fisher Scientific, cat. no. A998‐4)

• H₂O, Milli‐Q (18.2 MΩ·cm at 25°C)

• 3 M sodium acetate buffer, pH 5.5 (see recipe)

• 100% ethanol (Commercial Alcohols, cat. no. P016EAAN)

• H₂O, RNase‐free (Invitrogen, cat. no. 10977‐015 or equivalent)

• Glacial acetic acid (Fisher Scientific, cat. no. A38‐500)

• Agilent Prep 100‐Å C18, 50 × 50–mm, 5‐µm (Agilent, cat. no. 446905‐502)

• Agilent 1260 Infinity II HPLC system with:

  • 1290 Infinity preparative binary pump (Agilent, cat. no. G7161B)

  • 1260 Infinity II diode array detector (Agilent, cat. no. G7115A)

  • 1260 Infinity II multicolumn thermostat (Agilent, cat. no. G7116A)

  • 1290 Infinity II preparative open‐bed sampler/collector (Agilent, cat. no. G7158B)

• Sterile 0.22‐µm PES membrane syringe filter, 13‐mm (NEST, cat. no. 331211)

• 15‐ or 50‐ml conical sterile centrifuge tubes (Falcon, cat. no. 352095 or 352070 or equivalent)

• –20°C freezer

• Refrigerated centrifuge (Eppendorf, cat. no. 5430R), 4°C

• 1.5‐ml microcentrifuge tube, RNase‐free

• Glen Gel‐Pak desalting columns (Glen Research, cat. no. 61‐5025 or 61‐5010)

• 1
  Set up the HPLC method. Configure the HPLC system with the following parameters:

  • a.
    Flow rate: 5 ml/min.
  • b.
    Column temperature: 65°C.
  • c.
    Signal collection: 260 and 280 nm, bandwidth 4 nm, peak width 20 Hz.
  • d.
    Fraction collection mode: Peak collection covering the region 5 to 25 min with desired peak detection mode.
  • e.
    Needle wash: Milli‐Q water. Wash for 15 sec each at post run.
  • f.
    Gradient as follows:

  Time (min)	Buffer A %	Buffer B %	Flow rate (ml/min)
  0	90	10	3
  2	90	10	3
  2.5	90	10	5
  5	90	10	5
  25	75	25	5
  26	10	90	5
  31	10	90	5
  32	90	10	5
  37	90	10	5

• 2Prime the solvent lines. Place Buffer A (0.1 M TEAA) and Buffer B (ACN) on the instrument. Prime each line at high flow rate of 30 ml/min for at least 10 min with the purge valves open and column bypassed, to remove any residual solvent and bubbles.
• 3Equilibrate the column. Direct flow through the C18 column and equilibrate at the starting mobile‐phase composition (e.g., 90% Buffer A/10% Buffer B) and target temperature until the baseline and back pressure stabilize.
• 4Inject the DMTr‑ON crude RNA sample. Filter the crude RNA through a 0.22‐µm filter to remove particulates, then inject onto the column. Collect the DMTr‑ON full‑length product peak, which is typically the second major peak, into appropriately labeled 15‑ or 50‑ml conical tubes depending on the fraction volume.

• 5Add 0.1 volumes of 3 M sodium acetate (pH 5.5) to the collected HPLC fractions (e.g., add 1 ml per 10 ml fraction).
• 6Add 2.5 to 3 volumes of ice‐cold 100% ethanol. Mix thoroughly by inversion.
• 7Incubate at –20°C for at least 1 hr (overnight is recommended to maximize yield).
• 8Centrifuge 15 min at 12,000 × g, 4°C, to pellet the RNA. Carefully remove the supernatant without disturbing the pellet.
• 9Wash the pellet by adding 1 ml of cold 75% ethanol. Centrifuge again 10 min at 12,000 × g, 4°C.
• 10Remove the supernatant. Air‐dry or vacuum‐dry the pellet until no liquid remains.

  Tip: Do not over‐dry, as the pellet may become difficult to resuspend.

• 11To remove the 5'‐DMT group, add 200 µl of 20% (v/v) acetic acid in water to the dried, HPLC‑purified RNA in a microcentrifuge tube.
• 12Incubate at room temperature for 1 hr.
• 13Quench the detritylation by adding 0.1 volume (e.g., 20 µl) of 3 M sodium acetate buffer, pH 5.5.
• 14Repeat the ethanol precipitation to recover the DMTr‐OFF RNA. For LC–MS or high‐sensitivity applications, desalt the sample using Glen Gel‐Pak columns or equivalent gel‐filtration cartridge or ultra centrifugal filter units to ensure the removal of residual salts and by‐products.

Support Protocol 2. DENATURING PAGE PURIFICATION OF RNA

While RP‐HPLC is convenient and scalable, denaturing PAGE remains the method of choice when high resolution by length is required, especially for closely related n‐x species or DMTr‐OFF RNAs. Although it is more labor‐intensive and less amenable to high throughput than chromatography, it provides better resolving power for closely related species and conformers. This protocol covers gel casting, separation, and “crush‐and‐soak” recovery. Expect higher purity but lower yields compared to HPLC due to the hands‐on extraction process.

Materials

• Denaturing PAGE gel stock with 7 M urea (see recipe)

• 10% ammonium persulfate (APS) (see recipe)

• N,N,N′,N′‐tetramethylethylenediamine (TEMED) (Sigma, cat. no. 411019)

• 1× TBE buffer (see recipe)

• H₂O, RNase‐free (Invitrogen, cat. no. 10977‐015 or equivalent)

• Formamide (Sigma, cat. no. F9037)

• Ice

• 2× RNA loading dye (Thermo Scientific, cat. no. R0641)

• Liquid nitrogen

• 3 M sodium acetate buffer, pH 5.5 (see recipe)

• 100% ethanol (Commercial Alcohols, cat. no. P016EAAN)

• Gel casting stand with gaskets (Hoefer, cat. no. SE6015)

• Glass plates, 18 × 16–cm (Sigma, cat. no. GESE6102)

• Spacers, 1.5‐mm thickness, 16‐cm length, 2‐cm width (Hoefer, cat. no. SE6119‐2‐1.5)

• Preparative comb, 1 well, 1.5‐mm thickness (Hoefer, cat. no. SE211A‐R‐1.5)

• SE600 standard dual cooled vertical protein electrophoresis unit (Hoefer, cat. no. SE600)

• Power supply (Bio‐Rad, cat. no. 1645056)

• SpeedVac system (Thermo Scientific, cat. no. SRF110P1‐115)

• Digital heating shaking dry bath (Thermo Scientific, cat. no. 88880028)

• Preparative TLC plates, glass‐backed, silica, 1000‐µm, 20 × 20–cm, F254 (SiliCycle, cat. no. TLG‑R10011B‑341)

• UV lamp for shadowing (Thermo Scientific, cat. no. UVP 95000409)

• Sterile stainless‐steel blades, No. 23 (Fischer Scientific cat. no. 08‐918‐5D)

• 10‐ml transport/culture tube (Sarstedt, cat. no. 62.551.201)

• Nutating mixer

• Sterile 0.22‐µm PES membrane syringe filter, 13‐mm (NEST, cat. no. 331211)

• 50‐ml conical sterile centrifuge tubes (Falcon, cat. no. 352070 or equivalent)

• –20°C freezer

• Refrigerated centrifuge (Eppendorf, cat. no. 5430R), 4°C

• Glen Gel‐Pak desalting columns (Glen Research, cat. no.61‐5025 or 61‐5010)

• 1Assemble two glass plates in the gel casting stand using two 1.5‐mm‐thick spacers to define the gel thickness.
• 2To cast an 18 × 16–cm gel, mix 50 ml of denaturing PAGE stock (7 M urea) with 250 µl of 10% APS and 50 µl TEMED immediately before casting.

  For a 100 nt sgRNA, 10% to 12% acrylamide is recommended.

• 3Pour the gel solution into the casting cassette, insert a 1.5‑mm preparative comb, and allow to polymerize for 20 to 30 min at room temperature.
• 4Mount the gel in the electrophoresis unit and pre‐run in 1× TBE at 150 V for at least 20 min to equilibrate temperature and remove residual unpolymerized acrylamide or urea.
• 5Dry an aliquot of crude, fully deprotected RNA obtained after deprotection (e.g., one‐quarter of a 1 µmol synthesis eluted after Basic Protocol 2, step 7) in a SpeedVac and resuspend in 1:1 (v/v) RNase‐free water/formamide to a final loading volume of up to 200 µl in the 1.5‐mm preparative well.

  Tip: Limit loading to ∼60 OD₂₆₀ units per preparative lane (corresponding to approximately one‐quarter of a 1 µmol synthesis under these conditions) to avoid band broadening. 1 OD₂₆₀ unit is defined as the amount of nucleic acid that gives an absorbance of 1.0 at 260 nm in a 1‐cm pathlength cuvette, in 1 ml of solution.

• 6Denature the RNA at 95°C for 5 min, then immediately chill on ice.
• 7Load the sample into the well. In a separate lane, load RNA loading dye to monitor the run.
• 8Run at 250 V for 2 to 3 hr in 1× TBE, with active cooling with running water to avoid overheating.

• 9Following electrophoresis, remove the gel and place it on an F254 silica‐coated TLC plate. Visualize RNA bands via UV shadowing as dark bands against the fluorescent background.
• 10Using a sterile blade, excise the slowest‐migrating major band (full‐length product) and transfer it to a sterile 10‐ml culture tube. Mince the gel into ∼1‐mm pieces using a pipette tip to maximize surface area for elution.
• 11Add 2 to 3 gel volumes of RNase‐free water. Snap‐freeze the suspension in liquid nitrogen, then incubate at 4°C overnight with gentle agitation (e.g., on a nutating mixer).
• 12The next day, transfer the supernatant and pass it through a sterile 0.22‐µm filter into a clean 50‐ml tube. Perform additional washes of the gel fragments and pool the supernatants to maximize yield.
• 13Supplement the pooled supernatant with 0.1 volume of 3 M sodium acetate (pH 5.5) and 2.5 to 3 volumes of ice‐cold 100% ethanol. Mix gently by inversion.
• 14Precipitate the RNA at –20°C for at least 1 hr (or overnight for optimal recovery).
• 15Centrifuge 15 min at 12,000 × g, 4°C. Carefully decant or aspirate the supernatant without disturbing the RNA pellet, which may be translucent and located at the bottom or side of the tube.
• 16Wash the pellet with 1 ml of cold 75% ethanol. Rinse by gentle inversion.
• 17Centrifuge 10 min at 12,000 × g, 4°C. Remove the supernatant by decanting or aspiration.
• 18Air‐dry or vacuum‐dry the pellet to remove residual ethanol. For high‐sensitivity applications (e.g., LC–MS), desalt the sample using a Glen Gel‐Pak column to remove trace salts.

REAGENTS AND SOLUTIONS

APS, 10%

• For 10 ml:

• Dissolve 1 g of ammonium persulfate (APS) (Sigma, cat. no. A3678) in 10 ml RNase‐free water

• Store the solution up to 1 month at 4°C

Denaturing PAGE gel stock with 7 M urea

• For 250 ml:

• Weigh out 105 g urea (Sigma, cat. no. U5378) into a 250‐ml sterile glass bottle

• Add 6.25 × X ml of 40% acrylamide/bis (19:1) (Bishop, cat. no. ACR011) to make an X% gel

  Example: for a 12% gel, use 75 ml; for a 10% gel, use 62.5 ml.

• Add 25 ml of 10× TBE buffer (see recipe)

• Add DEPC‐treated or RNase‐free water (see recipe) to a total volume of ∼200 ml

• Stir until urea is fully dissolved

• Adjust the final volume to 250 ml with water

• Store up to 2 weeks at room temperature

DEPC‐treated water

• For 1 L:

• Add 1 ml of diethyl pyrocarbonate (DEPC) (Sigma, cat. no. D5758) to ∼900 ml Milli‐Q water in a glass bottle

• Stir to mix and bring final volume to 1 L with Milli‐Q water

• Incubate at 37°C overnight to allow DEPC to inactivate the RNases

• Autoclave to inactivate residual DEPC (liquid cycle, 121°C for 15 to 20 min)

• Let the water cool and store up to 2 months at room temperature

RP‐Buffer A: 0.1 M TEAA buffer, pH 7

• Dilute the 1 M TEAA buffer, pH 7.0 (see recipe) to 0.1 M

• Prepare fresh before use

Sodium acetate, 3 M, pH 5.5

• For 1 L:

• Dissolve 246.1 g of anhydrous sodium acetate (Sigma, cat. no. S8750) in ∼800 ml Milli‐Q water

• Slowly titrate with glacial acetic acid (Fisher Scientific, cat. no. A38‐500) to pH 5.5

• Bring final volume to 1 L with Milli‐Q water

• Filter through sterile 0.22‐µm PES filter (Fisher Scientific, cat. no. 09‐741‐11)

• Store up to 6 months at room temperature

TBE (Tris‐borate‐EDTA) buffer, 10×

• For 1 L:

• Dissolve 108 g of Tris base (Sigma, cat. no. T1503), 55 g of boric acid (Sigma, cat. no. B6768), and 9.3 g of EDTA disodium salt (Sigma, cat. no. E5134) in ∼800 ml Milli‐Q water

• Stir until fully dissolved

• Bring final volume to 1 L with Milli‐Q water

• Verify the pH is 8.3 to 8.5 using a calibrated pH meter

• Store up to 6 months at room temperature

TBE buffer, 1×

• For 1 L:

• Mix 100 ml of 10× TBE buffer (see recipe) with 900 ml DEPC‐treated Milli‐Q water (see recipe)

• Prepare fresh before use

Triethylammonium acetate (TEAA) buffer, 1 M, pH 7.0

• For 1 L:

• 139.4 ml triethylamine (Fisher Scientific, cat. no. O4885‐1)

• 57.2 ml of glacial acetic acid (Fisher Scientific, cat. no. A38‐500)

• Add to ∼800 ml Milli‐Q water (18.2 MΩ·cm at 25°C)

• Adjust the pH to 7.0 ± 0.1 by titrating with acetic acid or triethylamine as needed

• Bring final volume to 1 L with Milli‐Q water

• Filter through sterile 0.22‐µm PES filter (Fisher Scientific, cat. no. 09‐741‐11)

• Store up to 6 months at 4°C

COMMENTARY

Critical Parameters

Solid‐phase synthesis of RNA using 2'‐ALE phosphoramidites

Basic Protocol 1 outlines 2'‑ALE RNA synthesis at a 1 µmol scale on K&A instruments, but the same chemistry can be scaled up to ∼10 µmol on other platforms, provided solvent delivery volumes and reaction times are re‐optimized. All solvents must be rigorously dried and protected from the atmosphere. For long sequences, dichloromethane washes before and after detritylation are essential to prevent incomplete deblocking and column clogging, particularly beyond ∼100 cycles. Coupling with ALE monomers is optimized at 4 min, shorter contact times reduce yield while longer times risk premature acetal ester cleavage. Finally, use succinyl‐linked LCAA CPG supports rather than universal supports to ensure compatibility with ALE's mild deprotection conditions and to avoid incomplete cleavage from the solid support.

Deprotection of RNA made by 2’‐ALE phosphoramidites

For Basic Protocol 2 and Support Protocol 1, freshly prepared, anhydrous 10% DEA in ACN and EDA:toluene solutions are critical. Maintaining a strictly anhydrous environment during these steps prevents strand cleavage and keeps the deprotected, polyanionic RNA bound to the CPG until the final water elution. Sequences containing benzoyl‐protected residues require extended EDA exposure (up to 2 to 3 hr) to achieve complete deprotection; shorter treatments can leave residual protecting groups that compromise downstream function or analytical characterization.

Reverse‐phase HPLC

For DMTr‑ON RP‑HPLC purification (Basic Protocol 3), full equilibration of the system and C18 column at the starting solvent composition and temperature is crucial for reproducible retention times and robust separation of DMTr‑ON full‐length RNA. After each run, thorough washing of the needle and column with a high‐organic mobile phase [10% Buffer A (0.1 M TEAA)/90% Buffer B (ACN)] for at least 5 min prevents carryover of hydrophobic impurities and preserves column performance over multiple preparative injections. The described method is optimized for 1 to 3 µmol scales; for smaller (<0.2 µmol) or larger (>5 µmol) loads, injection volume, gradient slope, and column dimensions should be scaled to avoid overloading and peak distortion.

Ethanol precipitation for RNA

Efficient precipitation from HPLC or PAGE fractions requires ice‐cold 100% ethanol and a monovalent salt such as sodium acetate. Adjusting the sodium acetate solution to pH 5.2 to 5.5 enhances recovery of long RNAs while maintaining chemical stability. When working with low RNA amounts, the pellet may be nearly invisible; adding a carrier such as glycogen (∼50 µg/ml) improves pellet formation and makes it easier to track during washes.

Troubleshooting

Table 4 summarizes common issues that may arise during protocol execution, along with their probable causes and recommended solutions.

Table 4.

Troubleshooting Guide for Long RNA Synthesis and Purification Using 2'‐ALE Phosphoramidites

Problem	Possible cause	Solution
Slow detritylation kinetic as cycle iterates longer; incomplete detritylation	Acetonitrile forms a complex with the deblocking acid TCA, which competes with the oligonucleotide for acid binding	Increase DCM wash time or column drying time before and after detritylation step
Synthesis reagents cannot flow through the column	The frits of the columns are blocked	Replace with clean frits
	The CPG is overloaded or packed too much	Large pore size solid supports like 2000 Å and 3000 Å can have low loading capacity, reduce the CPG to <1 µmol scale and allow for proper reagent diffusion
EDA deprotection solution turns yellow	The EDA is naturally a colorless liquid; it can turn yellow when it is exposed to air over time; it readily reacts with carbon dioxide and water from the air	The ethylenediamine bottle should be septum sealed and stored under argon or nitrogen; prepare fresh EDA solution in toluene before deprotection
Extremely low yield when RNA is purified by DMTr‐ON HPLC	The DMTr group is lost during processing or downstream handling	The DMTr group is typically stable in water; therefore, it is advisable to avoid using mild acidic buffers for RNA elution from the CPG; instead, store the eluted RNA at –20°C if purification is not carried out immediately after deprotection
Extremely low yield regardless of the purification method although trityl monitor suggests successful synthesis	Premature chain cleavage during deprotection	Ensure de‐cyanoethylation step is carried out before EDA step
	RNA degradation by RNase contamination	Maintain an RNase‐free environment for the handling and processing of RNA, particularly long RNA molecules
No RNA pellet is observed after ethanol precipitation	Low RNA yield or concentration that the pellet may be invisible to the naked eye	Carefully decant or aspirate the supernatant from the side opposite to where the RNA pellet is expected, based on the centrifuge orientation
	Incomplete precipitation or centrifugation	Ensure sufficient ethanol, salt ratio, and incubation time; centrifugation should be carried out at 4°C

Understanding Results

The 2'‐O‐acetal levulinic ester (ALE) group was designed to bypass the rapid 2' to 3' acyl migration seen in direct 2'‐levulinyl esters (Lackey et al., 2007, 2009). By bridging the levulinyl unit and the 2'‐hydroxyl with a formaldehyde‐derived acetal, ALE prevents migration while remaining base‐labile. Building on the high coupling efficiencies reported (Lackey et al., 2007), we evaluated ALE‐protected monomers (Fig. 1) as a platform for long RNA synthesis (Lyu et al., 2026). In this setup, exocyclic amines for A and C are protected with levulinyl (Lev) groups, and G with dimethylformamidine (dmf). Syntheses were performed on a K&A synthesizer using the ALE‐optimized cycle (Table 1) and an on‐column deprotection strategy (Fig. 2). This workflow involves treating the support‐bound RNA with 10% DEA in ACN to remove cyanoethyl groups, followed by EDA in toluene to cleave the remaining base‐labile groups and the succinyl linker. This sequence can be performed manually (Basic Protocol 2) or automated via spare amidite lines for high‐throughput applications (Support Protocol 1).

To demonstrate performance, Figure 3 illustrates the synthesis of a chemically modified 100 nt sgRNA (AK100) targeting the Ttr gene (Finn et al., 2018). Due to its extensive 2'‐OMe substitutions and phosphorothioate (PS) linkages, this sequence serves as a stringent test for coupling efficiency and deprotection. Using the ALE protocol (75 mM monomer, 4 min coupling), we extended the EDA deprotection to 2 hr to ensure full benzoyl removal. Compared to the 2'‐TBDMS method (100 mM monomer, 8 min coupling), ALE delivered significantly higher yields: ∼39 nmol (3.9%) vs ∼2.6 nmol (0.3%) after purification (Fig. 3A‐B). LC–MS analysis confirmed the expected mass, indicating complete deprotection (Fig. 3C). These results underscore ALE's superior coupling efficiency and the utility of its DMTr‐ON compatibility.

Figure 3.

Synthesis and characterization of a chemically modified 100 nt sgRNA using 2'‑ALE chemistry. (A) Analytical ion‐exchange HPLC chromatograms comparing crude 100 nt sgRNA synthesized using ALE (75 mM, 4‑min coupling) versus 2'‐TBDMS (100 mM, 8‑min coupling) conditions. (B) Denaturing PAGE analysis of crude and purified sgRNA samples produced with ALE or TBDMS monomers, with isolated % yields indicated above each lane. (C) High‐resolution LC–MS characterization for the HPLC‑purified sgRNA. Reverse‐phase HPLC trace (top), electrospray mass spectrum (middle), and deconvoluted spectrum (bottom and right). Theoretical molecular weight (MW), 32,887.4983 Da; found MW, 32,887.7500 Da.

Figure 4 demonstrates the ALE protocol's application to a 124 nt ydaO‐class riboswitch from T. pseudethanolicus (Gao & Serganov, 2014). Denaturing PAGE of the crude product revealed a prominent full‐length band (Fig. 4A), yielding 18.0 nmol from a 1 µmol synthesis (1.8% yield), consistent with standard solid‐phase methods for this length. LC–MS confirmed the purity and identity of the final RNA (Fig 4B).

Figure 4.

Synthesis and characterization of 124 nt ydaO riboswitch RNA using 2'‑ALE chemistry. (A) 8% denaturing PAGE analysis comparing crude (lanes 1‐3) and HPLC‐purified (lanes 4‐6) 124 nt RNA. (B) High‐resolution LC–MS characterization of the purified RNA showing reverse‐phase HPLC trace (top), electrospray mass spectrum (middle), and deconvoluted spectrum (bottom). Theoretical MW, 40,132.8653 Da; found MW, 40,132.7 Da.

Overall, 2'‐ALE chemistry outperforms conventional 2'‐TBDMS methods by offering higher coupling efficiency with lower monomer consumption and shorter cycle times. The synergy of rapid on‐column deprotection and DMTr‐ON HPLC purification enables the scalable, high‐throughput production of long, chemically modified RNAs, such as sgRNAs, riboswitches, and small mRNAs up to 200 nt in length that are typically challenging to access via traditional silyl‐based chemistry (Lyu et al., 2026).

Time Considerations

ALE chemistry offers significant efficiency gains over TBDMS phosphoramidites by reducing coupling times to 4 min and monomer concentrations to 75 mM. As coupling is the rate‐limiting step, these improvements nearly halve the synthesis time; for a 100 nt sgRNA, ALE coupling takes 6.7 hr compared to over 13 hr with TBDMS. With a full cycle time of 6.4 min, a 100 nt strand is completed in 10.7 hr. Deprotection involves 20 min of de‐cyanoethylation followed by 1 to 3 hr of EDA treatment, depending on the base protection used. After a 1‐hr vacuum step to remove 2'‐acetal groups, RNA is eluted. For purification, automated DMTr‐ON RP‐HPLC (37 min/run) is recommended over labor‐intensive PAGE due to its scalability and minimal hands‐on requirements.

Author Contributions

Zidi Lyu: Conceptualization; data curation; formal analysis; investigation; methodology; validation; visualization; writing—original draft; writing—review and editing. Adam Katolik: Conceptualization; data curation; formal analysis; investigation; methodology; validation; visualization; writing—review and editing. Masad Damha: Conceptualization; funding acquisition; investigation; methodology; project administration; resources; supervision; validation; writing—review and editing.

Conflict of Interest

ALE monomers and processes described here are covered by patents (M.J.D. is an inventor), are licensed for commercial use, and are available through ChemGenes Corporation to any interested party for their own research.

Lyu, Z. , Katolik, A. , & Damha, M. J. (2026). 2'‐O‐Acetal levulinic ester ribonucleoside 3'‐Phosphoramidites for the solid‐phase synthesis of long RNA. Current Protocols, 6, e70426. doi: 10.1002/cpz1.70426

Data Availability Statement

All data generated or analyzed during this study are included in this published article. Additional data that support the findings of this study are available at https://doi.org/10.1093/nar/gkaf1525.