Q: I notice that longer oligos are more expensive. Why does the length of my oligo affect its synthesis yield and purity?

A: Think of oligonucleotide synthesis like building a chain of LEGO® blocks, where each block is a nucleotide. Every time we add a new block (or nucleotide), there’s a small chance the connection isn’t perfect. Even if our success rate for each connection, or „coupling efficiency,“ is very high—like 99-99.5%—these small imperfections accumulate as the chain gets longer.

This efficiency compounds exponentially as a function of length, following the formula (coupling efficiency)^n, where n is the number of couplings.

For example, at a 99% coupling efficiency, a 20-mer oligo would have about 82% yield (0.99^20) of correct full-lenth oligonucleotide, while a 100-mer would only have about 37% yield (0.99^100).

As the oligo gets longer, the cumulative effect of these inefficiencies means fewer full-length sequences are produced, which lowers the overall yield after purification. This is why for longer oligos simply, we have to perform a higher number of syntheses to achieve the same yield as a shorter oligo.

On top of that, longer oligos are harder to purify. They tend to include more incomplete or truncated sequences that need to be separated from the full-length product to achieve the high purity required for reliable results. At Ella Biotech, we recommend using RP-HPLC purification for all the oligos longer than 25 nucleotide to ensure you get a high-quality product suitable for your experiments.

So, in short, the length of your oligo affects yield and purity because of the stepwise nature of synthesis and the challenges in ensuring all the sequences are complete and free from impurities. While longer oligos are more challenging and expensive to produce, careful synthesis and purification ensure you get a high-quality product suitable for your experiments.

Q: What’s considered a „long“ oligo, and why are they challenging to synthesize?

A: We consider sequences longer than 80 bases to be „long“ oligos, with those exceeding 100 bases presenting synthesis challenges. The main challenge lies in the cumulative effect of coupling efficiency during synthesis – even with highly optimized protocols and excellent coupling efficiency for each base addition, the yield of full-length product inevitably decreases as the sequence gets longer.

Despite these challenges, we at Ella Biotech, have successfully synthesized oligos up to 200 bases in length through careful optimization of our synthesis protocols. If your research project requires long oligonucleotides, please get in touch! We can work together to ensure you get the high-quality, long oligos your project needs.

Q: Are there any sequence-specific challenges in oligo synthesis I should be aware of?

A: Yes, certain sequences can be trickier to synthesize and purify due to their specific characteristics. For example:

• High G content: Oligos with a high percentage of guanine (G) residues, especially consecutive G’s, can be challenging. These sequences often form secondary structures or aggregates, which can affect both synthesis and purification. They may also have lower solubility, making handling more difficult. In these cases, ion-exchange HPLC (IEX HPLC) is our recommended purification method to ensure a high-quality product.
• Self-complementary sequences: Sequences that are self-complementary can form duplexes or hairpins during synthesis and purification. These structures complicate the purification process and can reduce overall yield.
• High number of modifications: multiple different modifications in one oligo may require lots of complex reaction steps and intermediate purification steps and can demand special deprotection and cleavage conditions, reducing the overall yield.

If your sequence has any of these characteristics, we recommend discussing it with us. We can suggest strategies, modifications, or alternative designs to overcome these challenges and ensure the best possible outcome for your oligo synthesis.

Q: Why is purification necessary for oligonucleotides?

A: Purification is a critical step in ensuring your oligonucleotides are ready for use in experiments. During synthesis, incomplete products and chemical impurities, like protecting groups, can accumulate. If these impurities aren’t removed, they can cause several issues in your applications.

For example, impurities can interfere with hybridization, leading to non-specific binding or amplification. This means your oligos might bind where they shouldn’t, reducing the specificity of your experiments. Additionally, impure oligos can result in lower efficiency for techniques like qPCR or sequencing, as truncated or incomplete sequences don’t perform as expected. Worst of all, contaminants can introduce background noise, making your data unreliable or inaccurate.

By using an appropriate purification method, these problems can be avoided. Purification removes unwanted by-products and ensures your oligos are clean and functional, so you get the specificity, efficiency, and accuracy needed for dependable experimental results. It’s an essential step to ensure your research runs smoothly!