PEGylation is an attractive approach to modify oligonucleotides intended for therapeutic purposes. The PEG conjugation reduces protein interaction with the oligonucleotide, and helps to overcome its intrinsic biopharmaceutical shortcomings, such as poor enzymatic stability, rapid body clearance, and unwanted immunostimulation. While it is known that the molecular weight and architecture of the PEG play an important role in its effectiveness, the manner in which the PEG component interferes with the hybridization of the nucleic acid remains poorly understood. In this study, we systematically compare the hybridization thermodynamics as well as protein accessibility of several DNA conjugates involving linear, Y-shaped, and brush-type PEG, and conduct PEGylation on different sequence positions for brush-type PEGs to obtain a protein shielding depth profile. It is found that the PEGylated DNA experiences two opposing effects: local excluded volume effect and chemical interaction, the strengths of which are architecture-dependent. Notably, the brush architecture is able to offer significantly greater protein shielding capacity than its linear or Y-shaped counterparts, while maintain nearly identical free energy for DNA hybridization compared with free DNA.