Solid-phase oligonucleotide synthesis (SPOS) is now the universal method of choice to create exact DNA and RNA sequences. Custom oligonucleotide synthesis provides a high-fidelity and high-throughput platform for the synthesis of both natural and chemically modified oligonucleotides. With the application of phosphoramidite chemistry on automated synthesizers, one can readily prepare oligonucleotides with a range of different structural modifications, such as 2'-O-substituted nucleosides, fluorescent labels and backbone modifications.
Alfa Chemistry provides a diverse library of oligonucleotide synthesis building blocks to enable researchers to design and synthesize their own custom oligonucleotides. The key intermediate building blocks with their tightly controlled purities and physicochemical properties, such as solubility, for seamless implementation in automated SPOS workflows. The use of these pre-functionalized synthetic units allows for a dramatic reduction in synthetic complexity and synthesis yields and also allows for modular design of complex oligonucleotides.
Solid-phase oligonucleotide synthesis is a step-growth chemical synthesis in which nucleotides are added sequentially to a growing chain bound to an insoluble solid-phase carrier. The solid-phase support is often controlled pore glass (CPG). The solid-phase method allows easy reagent and by-product removal between each chemical step. Automation of solid-phase synthesis is straightforward. Building blocks are phosphoramidite monomers of the four usual nucleosides (adenosine, cytidine, guanosine, and uridine, or thymidine for DNA). Optionally chemically modified nucleotides are doped into the synthesis.
Each cycle of a synthesis involves four primary steps: uncapping, coupling, capping, and oxidation. Uncapping removes the 5'-DMT protecting group, coupling adds the next nucleotide, capping terminates the unreacted site, and oxidation converts the phosphite to a phosphate ester. The synthesis cycle is controlled by a software program and conducted under anhydrous conditions to avoid premature hydrolysis.
Figure 1. Overview of the normal synthetic cycle for solid‐phase oligonucleotide synthesis combined with the concept of making phosphoramidate modifications directly during SPOS[1].
The SPOS platform by Alfa Chemistry is a powerful and versatile solution for the efficient assembly of standard and chemically modified DNA or RNA oligonucleotides. The technology platform is fully compatible with modern automated synthesizers and supports a wide range of applications, from molecular biology research and therapeutic development to diagnostic probe construction.
Chemical modification of oligonucleotides can significantly enhance their function for targeted delivery, enhanced nuclease resistance, or enhanced hybridization specificity. In this scenario, 2-phenylthiomethyl-modified ribonucleotides are incorporated into RNA by standard phosphoramidite chemistry. These monomers were weighed into dry amber vials, dissolved in 0.1 M anhydrous acetonitrile, and stored under argon protection.
An automated synthesizer drew each solution from a sealed vial, dispensing 0.15 mL of solution per coupling step and setting aside 0.15 mL for line initiation. As long as the coupling efficiency is consistent, the modified bases can be seamlessly integrated into the oligonucleotide sequence.
Figure 2. Chemical modifications of oligonucleotides[2].
After chain extension, the oligonucleotide remains bound to the solid phase carrier and must be cleaved and deprotected. The standard treatment is to remove the bases and phosphate protecting groups by treatment with AMA (1:1 methylamine/ammonia) for 1.5 hours at 60 °C. The second deprotection step uses triethylamine/ammonia. The second deprotection step uses triethylamine, N-methylpyrrolidone, and triethylamine-trihydrofluoroacetate to remove the 2'- protecting group of the RNA.
| Step | Reagent/System | Conditions |
| Cleavage from support | AMA (40% methylamine:ammonia, 1:1) | 60 °C, 1.5 h |
| 2'-O deprotection (RNA only) | Et₃N/NMP/Et3N•3HF (2:2:3) | 60 °C, 1.5 h |
| Gel purification | 20% Urea-PAGE | Bromophenol marker at 2/3 gel |
| Desalting | C18 Sep-Pak cartridge | Elution with 60% aqueous methanol |
Purification was performed using 20% denaturing polyacrylamide gel electrophoresis (PAGE) followed by reverse-phase C18 chromatography. Gel bands were visualized under 254 nm UV light, cut off and eluted into NaCl/EDTA buffer. Final desalting by a Sep-Pak column (pretreated with acetonitrile and aqueous NH4Cl) yields highly pure oligonucleotides suitable for downstream applications.
UV-Vis spectroscopy is employed to determine oligonucleotide concentration using Beer-Lambert Law:
A = ε·c·l,
where A is absorbance, ε is the molar extinction coefficient (calculated using tools like DINAMelt), c is concentration, and l is path length (typically 1 mm).
MALDI-TOF mass spectrometry is utilized to confirm molecular weight and verify incorporation of modifications. Prior to loading, samples are desalted via C18 tips and mixed with THAP/ammonium fluoride matrix. Spotting on MALDI plates and analysis in positive-reflector mode yields precise mass spectra.
CD spectroscopy is used to study RNA secondary structure and thermal stability. Modified and unmodified RNA strands (3 µM) are dissolved in sodium phosphate buffer (pH 7.2) with NaCl and MgCl2. Duplex formation can be induced by annealing with complementary strands. Spectra are recorded from 200–350 nm, and thermal melts are performed to calculate melting temperature (Tm), revealing conformational stability.
Data is processed via subtraction of blank signals and exported for plotting and first derivative analysis to obtain Tm. Triplicate experiments ensure statistical relevance.
Figure 3. CD spectra of Single- and Double-stranded RNA, Canonical and Modified. CD spectra of samples containing zero (1, A), one (2, B), or two (3, C) modifications before (blue) and after (red) hybridization[3].
Q1: What is the average yield per synthesis cycle for RNA oligonucleotides?
It depends on the length and modification density, but typical yields range between 1 and 5 OD (optical density units) for 20-mer sequences synthesized at 1 µmol scale.
Q2: Can I synthesize oligonucleotides longer than 100 bases?
Yes, though efficiency drops with length. Special protocols and longer coupling times are required. Enzymatic ligation may be considered for extremely long constructs.
Q3: How stable are 2'-modified oligonucleotides?
2'-modifications like 2'-OMe or 2'-thiophenylmethyl significantly enhance nuclease resistance, improving shelf life and in vivo stability.
Q4: What solvents are critical during phosphoramidite synthesis?
Anhydrous acetonitrile is essential; all reagents must be moisture-free to avoid phosphoramidite hydrolysis.
Q5: How should synthesized oligonucleotides be stored?
Lyophilized oligonucleotides should be stored at –20 °C in RNase-free conditions. For RNA, aliquoting in RNase-free water and avoiding freeze-thaw cycles is recommended.
Q6: Is MALDI-TOF analysis sufficient to detect single-base substitutions?
MALDI-TOF can detect mass shifts corresponding to base modifications, but sequencing or tandem MS is recommended for base-resolution validation.
References
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