Patterning of biomolecules on graphene layers could provide new avenues to modulate their electrical properties for novel electronic devices. Single-stranded deoxyribonucleic acids (ssDNAs) are found to act as negative-potential gating agents that increase the hole density in single-layer graphene. Current–voltage measurements of the hybrid ssDNA/graphene system indicate a shift in the Dirac point and “intrinsic” conductance after ssDNA is patterned. The effect of ssDNA is to increase the hole density in the graphene layer, which is calculated to be on the order of 1.8 × 1012 cm−2. This increased density is consistent with the Raman frequency shifts in the G-peak and 2D band positions and the corresponding changes in the G-peak full width at half maximum. Ab initio calculations using density functional theory rule out significant charge transfer or modification of the graphene band structure in the presence of ssDNA fragments.