Nucleic acid aptamers (NAAs) are short synthetic DNA or RNA molecules that specifically fold into distinct three-dimensional structures able to specifically recognize a target. While NAAs show unprecedented promise in a variety of applications, including sensing, therapeutics and diagnostics, one major limitation involves the lack of stability towards omnipresent nucleases. Therefore, we herein report a systematic truncation and incorporation of 2′-O-methyl bases to a DNA aptamer, which results in increased stability without affecting affinity. One of the newly designed analogues is stable up to 24 hours, demonstrating that 2′-O-methyl RNA is an attractive modification to DNA aptamers, especially when therapeutic applications are intended.