Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues

The base‐pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5′‐appended acylamido caps that enhance the stability and base‐pairing fidelity of oligonucleotides with a 5′‐terminal 2′‐deoxyadenosine residue using combinatorial synthesis and MALDI‐monitored nuclease selections. This provided the residue of 4‐(pyren‐1‐yl)butyric acid as a lead. Lead optimization gave (S)‐N‐(pyren‐1‐ylmethyl)pyrrolidine‐3‐phosphate as a cap that increases duplex stability and base‐pairing fidelity. For the duplex of 5′‐AGGTTGAC‐3′ with its fully complementary target, this cap gives an increase in the UV melting point Tm of +10.9°C. The Tm is 6.3–8.3°C lower when a mismatched nucleobase faces the 5′‐terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3′‐terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double‐terminal mismatches that is 5.7–5.9°C greater than that for the unmodified control duplex.