Structures of the four reaction states of the adenine riboswitch aptamer domain, including a transient intermediate state were solved by serial femtosecond crystallography. The structures not only demonstrate the use of X-ray free-electron lasers for RNA crystallography but have also proven that transient states can be determined in real time by mix-and-inject crystallography. These results illustrate the structural basis for the ligand-induced conformational changes associated with the molecular 'switch'.
In recent years, RNA has been increasingly recognized for its essential roles in biology, functioning not only as a carrier of genetic information but also as a dynamic regulator of gene expression through its interactions with other RNAs, proteins, and itself. Advances in chemical probing techniques have significantly enhanced our ability to identify RNA secondary structures and understand their regulatory roles. These developments, alongside improvements in experimental design and data processing, have greatly increased the resolution and throughput of structural analyses.
2-Aminopurine (2AP) is a fluorescent analog of adenine, and its unique properties make it valuable in various biochemical and biotechnological applications. Its fluorescence property probes local dynamics in DNA and RNA because stacking with the surrounding bases quench its fluorescence. 2AP-labeled DNA or RNA sequences have been used for the detection of genetic mutations, viral RNA, or other nucleic acid-based markers associated with diseases like cancer and infectious diseases.
Lower target concentrations during aptamer selection can lead to aptamers with higher binding affinities, but there may be a lower limit for effective concentration.
In experiments with guanine at concentrations of 5 μM, 500 nM, and 50 nM, successful aptamer enrichment occurred at 5 μM and 500 nM, but not at 50 nM.
The research highlighted the importance of optimal target concentration, showing that a high-affinity aptamer had a K value of 200 nM, and mutation studies identified a key cytosine that affected selectivity between guanine and adenine.
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Straße 7, 60438 Frankfurt am Main, Germany.
Translational riboswitches located in the 5' UTR of the messenger RNA (mRNA) regulate translation through variation of the accessibility of the ribosome binding site (RBS). These are the result of conformational changes in the riboswitch RNA governed by ligand binding. Here, we use a combination of single-molecule colocalization techniques (Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) and Single-Molecule Kinetic Analysis of Ribosome Binding (SiM-KARB)) and microscale thermophoresis (MST) to investigate the adenine-sensing riboswitch in , focusing on the changes of accessibility between the ligand-free and ligand-bound states.
The simultaneous evolution of multiple aptamers can drastically increase the speed of aptamer discovery. Most previous studies used the same concentration for different targets, leading to the dominance of the libraries by one or a few aptamers and a low success rate. To foster the best aptamers to grow independently in the sequence space, it is important to (1) use low target concentrations close to their dissociation constants and (2) stop at an early round before any sequence starts to dominate.
