Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5′-GGT TGG TGT GGT TGG-3′ that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent’s aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2′-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.

Pathogenesis-related (PR) proteins of class 10 are abundant in higher plants. Some of these proteins are induced under stress conditions as part of the plant defence mechanism. Other homologues are developmentally regulated and their expression varies in different plant organs. The PR-10 proteins are encoded by multigene families, have a weight of about 17 kDa and are found in the cytosol. In yellow lupin, nine different homologues have been identified and divided into two subclasses, LlPR-10.1 and LlPR-10.2. Within each subclass the sequence identity is about 75-91%, while across the subclasses it is only 59-60%. Here, the crystal structure of a yellow lupin PR-10 protein from the second subclass, LlPR-10.2A, is presented. The structure was solved by molecular replacement and refined to R = 0.205 using 1.9 A resolution data. The general fold of LlPR-10.2A resembles that of the other PR-10 proteins and consists of a long C-terminal alpha-helix surrounded by a seven-stranded antiparallel beta-sheet, with two shorter alpha-helices located between strands beta1 and beta2. The most variable part of the structure, the C-terminal helix, is strongly kinked towards the beta-sheet core in both LlPR-10.2A molecules present in the asymmetric unit. This unexpected feature reduces the size of the hydrophobic cavity observed in other PR-10 proteins that is reported to be the ligand-binding site. As in other PR-10 structures, a surface loop located near the entrance to the cavity shows very high structural conservation and stability despite the high glycine content in its sequence.

Nucleoside 5-O-phosphorothioates are formed in vivo as primary products of hydrolysis of oligo(nucleoside phosphorothioate)s (PS-oligos) that are applied as antisense therapeutic molecules. The biodistribution of PS-oligos and their pharmacokinetics have been widely reported, but little is known about their subsequent decay inside the organism. We suggest that the enzyme responsible for nucleoside 5-O-monophosphorothioate ((d)NMPS) metabolism could be histidine triad nucleotide-binding protein 1 (Hint-1), a phosphoramidase belonging to the histidine triad (HIT) superfamily that is present in all forms of life. An additional, but usually ignored, activity of Hint-1 is its ability to catalyze the conversion of adenosine 5-O-monophosphorothioate (AMPS) to 5-O-monophosphate (AMP). By mutagenetic and biochemical studies, we defined the active site of Hint-1 and the kinetic parameters of the desulfuration reaction (P-S bond cleavage). Additionally, crystallographic analysis (resolution from 1.08 to 1.37 Å ) of three engineered cysteine mutants showed the high similarity of their structures, which were not very different from the structure of WT Hint-1. Moreover, we found that not only AMPS but also other ribonucleoside and 2-deoxyribonucleoside phosphorothioates are desulfurated by Hint-1 at the following relative rates: GMPS > AMPS > dGMPS > CMPS > UMPS > dAMPS Ͼ Ͼ dCMPS > TMPS, and during the reaction, hydrogen sulfide, which is thought to be the third gaseous mediator, was released. (Fig. 1). 5Ј-O-Phosphorothioates of ribonucleosides (NMPSNMPS and dNMPS (together denoted (d)NMPS)) are formed during the enzymatic hydrolysis of oligo(nucleoside phosphorothioate) (PS-oligos) that contain a sulfur atom attached in non-bridging positions to the phosphorus atom at each or selected internucleotide bond(s). Synthetic PS-oligos have been developed as antisense probes for genomic research and medicinal applications (1, 2). These oligonucleotides are promising therapeutic molecules because they are much more stable against nucleolytic degradation in blood and various cellular systems than their natural, unmodified counterparts (3-5). Their hydrolysis in plasma, kidney, and liver proceeds mainly from the 3Ј end, resulting in the appearance of the mononucleoside 5Ј-phosphorothioates identified in urine from PS-oligo-injected animals (6, 7). (d)NMPS may exert cytotoxic effects affecting cell proliferation, DNA or RNA synthesis, and other unknown processes (8, 9). Recently, the phosphorothioate DNA segments have been identified in bacterial DNA (10), which makes investigations into PS-oligo metabolism even more important.Although several reports have been published on the biodistribution of PS-oligos, little is known about the metabolism of the products of their degradation in vivo. It has been suggested that extracellular dNMPS and dNMP can be converted to the corresponding nucleoside by 5Ј-nucleotidase (ecto-5Ј-NT) (9). This membrane-bound enzyme preferably releases adenosine from extracellular AMP, but other purine and pyrimid...