A new enzymatic method for truncation of aptamers from 5'-end has been developed and demonstrated on a newly selected indole-modified β-conglutin aptamer. This method relies on extension of a primer containing ribonucleotides, which can be specifically hydrolyzed by RNase, thereby removing the whole or 5'-part of the primer. This approach enables flexible synthesis of modified aptamers truncated from the 5' end, 3' end, or both ends-a capability previously achievable only through chemical oligonucleotide synthesis. Furthermore, by employing doubly labeled primers, diverse labeled or modified nucleotides are introduced to prepare 5'-labeled aptamers suitable for fluorescence- or immobilization-based assays. The different variations of this method also enable the synthesis of mutated sequences to study the effect of modifications and their positions. Overall, this enzymatic method provides a valuable alternative for the truncation or labeling of (hyper)modified and functional oligonucleotides avoiding the need for chemical synthesis.

Department of Organic Chemistry Faculty of Science Charles University Prague Hlavova 8 12843 Prague 2 Czech Republic

Institució Catalana de Recerca i Estudis Avancats 08010 Barcelona Spain

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam 542 2 160 00 Prague 6 Czech Republic

Interfibio Consolidated Research Group Departament d'Enginyeria Química Universitat Rovira i Virgili 43007 Tarragona Spain

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