## 3 Results and Discussion
### Extraction of OGNs with Different Lengths and Modifications  
Performed studies on the influence of parameters of adsorption and desorption of OGN on MNP allow for the determination of conditions for extraction to obtain the possible high recovery. The final MDSPE procedure is presented in Table 4. The MNP-Ac gives the highest recoveries, so this MNP was chosen for the final extraction procedure. The IE exchange mode was selected, and the adsorption was performed at pH = 4.5 (Table 4). In the elution step, the 10 mM solution of NH\({}_{4}\)OAc at pH = 9.5 in mixture with MeOH (50/50, \(v/v\)) was applied. Adsorption time was set at 15 min, and desorption time was set at 30 min (Table 4). Figure 5: The impact of desorption time on DNA-20 oligonucleotide recovery from MNP-Ac (oligonucleotide sequence is provided in Table 1, MNP-Ac adsorbent structure on Figure 2, and experimental conditions in Section 2.4). Abbreviations: OGN—oligonucleotide. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**MNP** & **Conditioning** & **Adsorption** & **Desorption** & **Recovery** \\ \hline MNP-Ac & 50 \(\mu\)L H\({}_{2}\)O/ACN 50/50 \(v/v\) & 50 \(\mu\)L DNA20 5 \(\mu\)M + 50 \(\mu\)L ACN & 50 \(\mu\)L NH\({}_{3(\text{aq})}\) pH = 9.5 & 34.7 \(\pm\) 1.1\% \\ \hline MNP-Sul & 50 \(\mu\)L H\({}_{2}\)O/ACN 50/450 \(v/v\) & 50 \(\mu\)L DNA20 5 \(\mu\)M + 450 \(\mu\)L ACN & 50 \(\mu\)L H\({}_{2}\)O & 13.2 \(\pm\) 0.3\% \\  & 50 \(\mu\)L NH\({}_{3(\text{aq})}\) pH = 9.5 & 16.9 \(\pm\) 0.3\% \\ \hline MNP-Mal & 50 \(\mu\)L H\({}_{2}\)O/ACE 50/450 \(v/v\) & 50 \(\mu\)L DNA20 5 \(\mu\)M + 450 \(\mu\)L ACE & 50 \(\mu\)L H\({}_{2}\)O & 13.7 \(\pm\) 0.5\% \\  & 50 \(\mu\)L NH\({}_{3(\text{aq})}\) pH = 9.5 & 16.8 \(\pm\) 0.2\% \\ \hline \hline \end{tabular}
\end{table} 
Table 3: The conditions used for the extraction of DNA-20 in the HI mode. Abbreviations: MNP—magnetic nanoparticles; ACN—acetonitrile; ACE—acetone. The final procedure (Table 4) was applied for oligonucleotides with a different sequence, length, and modifications. The sequences of 20-mer and 11-mer OGNs are analogical with Alicaforsen, the antisense OGN tested as a potential drug for pouchitis in enema formulation. The modifications investigated in the present studies are chosen from the most popular ones used in antisense drugs. Moreover, a microRNAs are potential diagnostic markers; thus, extraction of their analogs was also investigated. The recovery values obtained for each studied OGN are presented in Figure 6.  
Except for MOE-20 and LNA-11, the recovery values in all cases are higher than 80%. Moreover, these values are higher than 90% for unmodified OGNs (Figure 6). Introducing a chemical modification in the structure of the OGN affects the hydrophobicity of the molecule. The phosphorothioate one, where the nonbinding oxygen atom in the phosphate group was replaced by a less electronegative sulfur atom, is more hydrophobic than an unmodified strand of DNA with the same length and sequence. Similarly, the substituent in the 2\({}^{\prime}\) position of the sugar group influences this property. The longer the substituent, the more hydrophobic the OGN will be. The changes in this property can be a potential cause of changes in the recoveries values. While comparing results obtained for 20-mers,