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NMR spectroscopy is a powerful tool for research on protein dynamics. In the past decade, there has been significant progress in the development of NMR methods for studying charged side chains. In particular, NMR methods for lysine side-chain NH3+ groups have been proven to be powerful for investigating the dynamics of hydrogen bonds or ion pairs that play important roles in biological processes. However, relatively low sensitivity has been a major practical issue in NMR experiments on NH3+ groups. In this paper, we present a unique and simple approach to improve sensitivity in 15N relaxation measurements for NH3+ groups. In this approach, the efficiency of coherence transfers for the desired components are maximized, whereas undesired anti-phase or multi-spin order components are purged through pulse schemes and rapid relaxation. For lysine side-chain NH3+ groups of a protein-DNA complex, we compared the data obtained with the previous and new pulse sequences under the same conditions and confirmed that the 15N relaxation parameters were consistent for these datasets. While retaining accuracy in measuring 15N relaxation, our new pulse sequences for NH3+ groups allowed an 82% increase in detection sensitivity of 15N longitudinal and transverse relaxation measurements.

Nucleic acid aptamers are short RNA- or DNA-based affinity reagents typically selected from combinatorial libraries to bind to a specific target such as a protein, a small molecule, whole cells or even animals. Aptamers have utility in the development of diagnostic, imaging and therapeutic applications due to their size, physico-chemical nature and ease of synthesis and modification to suit the application. A variety of oligonucleotide modifications have been used to enhance the stability of aptamers from nuclease degradation in vivo. The non-bridging oxygen atoms of the phosphodiester backbones of RNA and DNA aptamers can be substituted with one or two sulfur atoms, resulting in thioaptamers with phosphorothioate or phosphorodithioate linkages, respectively. Such thioaptamers are known to have increased binding affinity towards their target, as well as enhanced resistance to nuclease degradation. In this review, we discuss the development of phosphorothioate chemistry and thioaptamers, with a brief review of selection methods.

High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5′dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes.