Synthesizing Long Oligos

Why this matters

Long oligo synthesis is fundamentally less forgiving than short oligo synthesis. As length increases, small inefficiencies compound rapidly, leading to:

lower full-length yield
higher deletion/truncation levels
more variability run-to-run
more purification difficulty

This article outlines the most important factors that control long-oligo success and how to troubleshoot when results drift.

1) Key Principle: Small Efficiency Losses Become Big Problems

Every cycle (detritylation, coupling, capping, oxidation) must stay consistent efficiently.
A small drop in performance early or mid-run can dramatically reduce the final full-length fraction.

Bottom line: long-oligo success depends on stable, repeatable performance across all cycles.

2)Steric Hindrance (Additional Steps May Be Needed)

As the oligonucleotide grows in length, steric hindrance increases, which can reduce reagent access and slow reaction completion across the synthesis cycle. To maintain full-length yield and purity on long sequences, it may be necessary to increase reaction time or add additional repetitions of one or more steps, including deblock (detritylation), coupling, capping, and oxidation, especially when performance declines later in the run or long-oligo impurity levels increase.

3) Support Selection

Why the solid support matters more on long sequences

As an oligo grows, the support surface becomes crowded. This can slow diffusion of reagents into the support and make reactions less consistent over time.

Common impacts include:

slower reagent penetration
reduced coupling uniformity later in the run
increased truncation and mixed impurities

General guidance for long oligos

For longer targets (commonly >75–100 nt), supports with larger pore structures often provide better access for reagents as strand length increases.

Tradeoffs to consider
Supports optimized for long sequences may introduce different practical constraints such as:

lower loading capacity
increased fragility
more sensitivity to handling and column packing

Standard (Functionalized) vs Universal

Standard support includes the first 3’ base, so you get one less coupling step.
It often allows simpler / less harsh cleavage conditions.
Universal support is more flexible, but typically requires an extra coupling and can require harsher cleavage to fully release the oligo

Best practice

Choose support type and loading level based on what you care about most:

maximum yield
maximum purity
maximum repeatability

For long oligos, repeatability is usually the best starting target.

4) Coupling Step (Highest Impact Chemical Variable)

Why coupling is the primary yield driver

Coupling is the step where the next base is added to the growing chain. For long oligos, coupling must remain extremely consistent across dozens or hundreds of cycles.

Even small coupling losses are amplified with length.

The #1 coupling risk: moisture (water contamination)

Moisture lowers coupling efficiency in two main ways:

A) Water competes with the intended reaction
Activated monomer should react with the growing strand. Water can consume activated species instead, lowering coupling yield.

B) Water gradually reduces monomer effectiveness
Moisture exposure over time can degrade monomers while they sit on the instrument, causing coupling to worsen later in the run.

Common causes of moisture exposure

humid environments
solvent bottles not sealed tightly
older acetonitrile that absorbed moisture
gas supply not sufficiently dry
instruments sitting unused (internal plumbing equilibrates to ambient moisture)

Best practices to protect coupling efficiency

Use dry acetonitrile (instrument + monomer dilution)
Use trap packs or molecular sieeves to further ensure dryness
Prepare fresh monomer solutions and minimize open-air exposure
Keep activator clean, fresh, and properly stored
Ensure inert gas (argon/helium) is dry and leak-free
After downtime, consider running a conditioning/purge routine before critical long-oligo runs

5) Capping Step

What capping is supposed to do

Capping blocks strands that failed to couple in that cycle, preventing them from reacting in later steps.

Why capping issues hurt long oligos more

If capping is incomplete:

failure strands stay reactive
they continue growing
they form deletion impurities (n–1 family)

These deletion impurities are especially problematic because they can be very difficult to remove during purification.

Signs of weak capping performance

high levels of deletion impurities
purity that does not improve despite “okay” coupling metrics
inconsistent final product purity even when yield looks decent

Best practices for capping on long oligos

Keep capping conditions consistent across the full run
If your system allows it, increase:
- capping volume
- contact time
  (especially when long-oligo purity is the priority)
- Confirm capping reagent integrity and line function (no partial delivery

Quick Troubleshooting Guide

If full-length yield is low across the run:

focus on coupling and dryness control
verify monomer freshness and solvent quality
check inert gas dryness and leaks

If deletion impurities are high:

focus on capping strength and delivery consistency
confirm detritylation completion (missed detritylation can also increase deletions)

If results drift after downtime:

suspect moisture uptake in lines/solvent
run purge/conditioning steps
replace or refresh critical reagents

Best Practices Checklist (Long Oligos)

Support selected for long-length diffusion/access
Dry acetonitrile installed and sealed
Fresh monomers prepared with low moisture exposure
Activator is fresh and handled correctly
Gas supply is dry / conditioned
Capping delivery is strong and consistent
Conditioning/purge performed after downtime when needed