SHORT COMMUNICATION Monitoring the progression of the in vitro selection of nucleic acid aptamers by denaturing high-performance liquid chromatography Jens Müller & Osman El-Maarri & Johannes Oldenburg & Bernd Pötzsch & Günter Mayer Received: 25 July 2007 / Revised: 10 October 2007 / Accepted: 11 October 2007 / Published online: 10 November 2007 # Springer-Verlag 2007 Abstract Monitoring of in-vitro selection experiments is crucial in evaluation of the success and outcome of such approaches. Furthermore, monitoring running in parallel with the selection procedure enables early intervention and adjustment of stringency to achieve the desired activities of the selected nucleic acid species. Here we describe the use of a non-radioactive method that enables monitoring of a SELEX procedure on the basis of sequence diversity. We employ denaturing HPLC and describe for the first time an experimental set-up that is useful both for analysis of the progression of in-vitro selection experiments and for separation of distinct aptamer sequences. Keywords Aptamer . SELEX . dHPLC WAVE Aptamers are short, single stranded nucleic acids which fold into well-defined 3D shapes [1, 2]. They bind to cognate target molecules with high affinity and specificity and can be obtained by an in vitro selection procedure, also termed as SELEX (systematic evolution of ligands by exponential enrichment), utilizing divers nucleic acid libraries as starting molecule libraries [3–8]. Critical for the success of an in-vitro selection experiment, and thus the characteristics of the selected aptamers, is efficient moni- toring of the progression of the selection process. This allows early intervention and adjustment of the selection pressure and stringency to achieve the desired activities of the selected nucleic acid species. Commonly, the progression of a selection experiment is monitored by use of radioac- tively labelled nucleic acids. However, use of radioactivity is not possible in every laboratory and needs strict safety regulations to be addressed. Alternative methods that enable monitoring of aptamer selection include capillary electro- phoresis and affinity chromatography [9, 10]. Here, we set out to develop an easy alternative method for monitoring the progression of the in vitro selection process. Given the fact that during successful selection the sequence diversity of a nucleic acid library is dramatically reduced, we hypothesized that this reduction might be detectable using denaturing high-performance liquid chro- matography (dHPLC). Materials and methods SELEX The in vitro selection will be described in detail elsewhere. In brief, recombinant human APC (Xigris) was biotinylated and coupled to streptavidin-labelled magnetic beads (Dyna- beads M-280 Streptavidin; Invitrogen) to allow the removal of unbound ssDNA molecules during the SELEX process. Initially, 500 pmol of a random oligonucleotide pool (D1, 5′-GCCTGTTGTGAGCCTCCTAAC-N49-CATGCTTAT- TCTTGTCTCCC-3′) was incubated with the immobilised APC. After washing, bound aptamers were recovered by thermal elution and introduced to amplification. Reverse Anal Bioanal Chem (2008) 390:1033–1037 DOI 10.1007/s00216-007-1699-8 J. Müller : O. El-Maarri : J. Oldenburg : B. Pötzsch Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany G. Mayer (*) Life and Medical Sciences Bonn, c/o Kekulé-Institute for Org. Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany e-mail: gmayer@uni-bonn.de