Enzymatic ligation strategies for the preparation of purine riboswitches with site-specific chemical modifications.
One of the most versatile riboswitch classes refers to purine nucleoside metabolism. In the cell, purine riboswitches of the respective mRNAs either act at the transcriptional or translational level and off- or on-regulate genes upon binding to their dedicated ligands. Biophysical studies on ligand-induced folding of these RNA domains in vitro contribute to understanding their regulation mechanisms in vivo. For such studies, in particular, for approaches using fluorescence spectroscopy, the preparation of large RNAs with site-specific chemical modifications is required. Here, we describe a strategy for the preparation of riboswitch aptamers and aptamers adjoined to their expression platforms by chemical synthesis and enzymatic ligation. The modular design enables fast access to a large number of purine riboswitch derivatives with the modification of interest at any strand position. We exemplarily provide a detailed protocol for the preparation of adenosine deaminase (add) A-riboswitch variants with 2-aminopurine (AP) modifications at the 40-nmol scale.
Rieder
Renate
R
Institute of Organic Chemistry, Leopold Franzens University, Innrain 52a, A-6020, Innsbruck, Austria.
Höbartner
Claudia
C
Micura
Ronald
R
D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D011088:DNA Ligases / Q000378:metabolism*; D013058:Mass Spectrometry; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011687:Purines / Q000737:chemistry / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D020506:Untranslated Regions / Q000737:chemistry* / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D011687:Purines; D038621:Regulatory Sequences, Ribonucleic Acid; D020506:Untranslated Regions; D011088:DNA Ligases; D011130:RNA Ligase (ATP)