**4.1 PEG attachment**

PEGylation is one of the most common methods to prevent renal filtration of aptamers. PEG is a flexible, uncharged and a highly hydrophilic polymer that is widely conjugated with therapeutic drugs to reduce reticuloendothelial clearance, extend circulation time and improve drug efficiency [35]. Macugen, which is the only FDA approved aptamer drug in the market is also PEGylated. PEG decreases aggregation and increases the solubility of the conjugates. PEGylation of Macugen increased its half-life to 9.5 –12.5 hours after intravenous and subcutaneous injection, respectively, in the plasma, and up to 94 hours in vitreous humor [36, 37]. Similarly, PEGylation also increased *in vivo* half-life of M7, a DNA aptamer, from <1 to 24–48 hours [38]. This modification did not cause any change in the specificity of M7 aptamer toward its target PD-1 (Programmed death protein 1) and suppressed the growth of PD-L1 (Programmed death-ligand 1) positive colon cancer carcinoma *in vivo* [39].
