# The Influence of the Capping Step During Solid-Phase Phosphoramidite Synthesis of Oligonucleotides on Synthetic Errors in Oligonucleotides

## TL;DR

This study shows how the capping step in DNA synthesis affects error rates, and suggests better reagents to reduce errors in gene constructs.

## Contribution

The study identifies guanine modification during capping as a source of errors and proposes improved capping reagents to reduce them.

## Key findings

- G to A substitutions in gene constructs result from guanine modification during the capping step.
- Using weaker carboxylic acid anhydrides reduces error rates compared to standard acetic anhydride.
- Phosphoramidite-based capping reagents significantly lower synthetic errors in oligonucleotides.

## Abstract

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry.