Dear friend Grigorii Rudakov

The choice of the number of nucleotides in a sticky end depends on several factors, including the size and complexity of the DNA molecule, the stability of the hybridized DNA, and the specificity of the interaction between the complementary sticky ends.

While it is possible to use three nucleotides for a sticky end, it may not be the most common choice because it can result in a weaker interaction compared to using two or more nucleotides. This is because a longer sticky end with more nucleotides provides a greater number of hydrogen bonds between the complementary base pairs, resulting in a stronger binding affinity.

Additionally, the length of the sticky end can affect the specificity of the interaction. Using a longer sticky end with more nucleotides can increase the likelihood of non-specific interactions between complementary sequences, leading to off-target binding and reduced efficiency in constructing the desired structure.

In your case, using a three-nucleotide sticky end may be sufficient for your specific design, but it is important to consider the potential trade-offs between specificity and binding strength. You may also want to consider using other strategies to increase the stability and specificity of the interaction, such as incorporating modified nucleotides or optimizing the sequence of the sticky ends.