Natural products have failed to meet the urgent need for drug discovery in recent decades due to limited resources, necessitating new strategies for re-establishing the key role of natural products in hit screening. This work introduced DNA-encoding techniques into the synthesis of phenolic acid-focused libraries containing 32 000 diverse compounds. Online selection of the library using immobilized angiotensin II type I receptor (AT1R) resulted in seven phenolic acid derivatives. The half-maximal concentration (IC50) of hit 1 for the right shift of the [125I]-Sar1-AngII competition curve was 19.6 nM. Pharmacological examination of renovascular hypertensive rats demonstrated that hit 1 significantly lowered the blood pressure of the animals without changing their heart rates. These results were used to create a general strategy for rapid and unbiased discovery of hits derived from natural products with high throughput and efficiency.

Oligonucleotides can be designed or evolved to bind to specific DNA, RNA, protein, or small molecule targets and thereby alter the biological function of the target. The therapeutic potential of oligonucleotides targeted to intracellular molecules will depend largely on their ability to be taken up by the cells of interest, as well as their subsequent subcellular distribution. Here we describe methods to characterize the extent and mechanism of cellular uptake of AS1411, an aptamer oligonucleotide that has progressed to human clinical trials and which is also widely used by researchers as a cancer-targeting ligand.

The development of aluminum salts (alum) as vaccine adjuvants was an empirical process with little understanding of the mechanism of action and, with decades of use, it has become clear that there is a need for alternatives where alum-based adjuvants are suboptimal. Oligonucleotides containing unmethylated CpG sequences represent one alternative as they are potent stimulators of the vertebrate innate immune system through activation of Toll-like receptor-9. This chapter outlines the methods used by Dynavax Technologies to progress a CpG-containing oligonucleotide sequence termed 1018 through preclinical and clinical testing as an adjuvant for immunization against hepatitis B virus (HBV). 1018 is a short (22-mer) oligonucleotide sequence containing CpG motifs active in both rodents and primates. Preclinical testing of hepatitis B surface antigen (HBsAg) + 1018 in comparison to HBsAg + alum demonstrated induction of substantially higher antibody titers and a favorable safety profile for 1018. Most importantly, clinical studies with HBsAg vaccination consistently demonstrate more rapid induction of protective antibody titers with 1018 compared to alum in all populations studied, including groups that are harder to immunize such as the elderly and immunocompromised individuals. These studies represent the basis for use of the CpG-motif-containing oligonucleotide 1018 as an improved adjuvant for HBsAg immunogenicity. HBsAg + 1018 (HEPLISAV-B™) is currently in late-stage clinical testing for prophylactic immunization against HBV.

Using information from wild-type and mutant Vibrio vulnificus nuclease (Vvn) and I-PpoI homing endonuclease co-crystallized with different oligodeoxynucleotides, we have built the complex of Vvn with a DNA octamer and carried out a series of simulations to dissect the catalytic mechanism of this metallonuclease in a stepwise fashion. The distinct roles played in the reaction by individual active site residues, the metal cation and water molecules have been clarified by using a combination of classical molecular dynamics simulations and quantum mechanical calculations. Our results strongly support the most parsimonious catalytic mechanism, namely one in which a single water molecule from bulk solvent is used to cleave the phosphodiester bond and protonate the 3\'-hydroxylate leaving group.

BACKGROUND: Nuclear factor-kappa B (NF-kappaB) is well known for playing a pivotal role in restenosis after percutaneous coronary intervention (PCI).
RESULTS: This is the first report to demonstrate an effect of NF-kappaB decoy oligodeoxynucleotides (ODN) to prevent restenosis after PCI after a 4-year observation using a coronary computed tomography (CT) scan. We showed that the decoy treatment suppressed neointimal formation after stent implantation compared to that in the same artery.
CONCLUSIONS: Thus, for the first time, we demonstrate the clinical usefulness of the CT scan to reveal the effects of NF-kappaB decoy ODN transfer after PCI.

Specific protein-nucleic acid interactions are of paramount importance for the propagation, maintenance and expression of genetic information. Restriction endonucleases serve as model systems to study the mechanisms of DNA recognition by proteins. SsoII is a Type II restriction endonuclease that recognizes the double stranded sequence downward arrow CCNGG and cleaves it in the presence of Mg(2+)-ions, as indicated. SsoII shows sequence similarity over a stretch of approximately 70 amino acid residues with several other restriction endonucleases that recognize a similar sequence as SsoII (Cfr10I, EcoRII, NgoMIV, PspGI). In NgoMIV this stretch is involved in DNA recognition and cleavage, as shown by the crystal structure analysis of an enzyme-product complex. To find out whether the presumptive DNA recognition region in SsoII is indeed in contact with DNA we have photocrosslinked SsoII with an oligodeoxyribonucleotide in which the first guanine of the recognition sequence was replaced by 5-iodouracil. Following digestion by trypsin, the peptide-oligodeoxyribonucleotide conjugate was purified by Fe(3+)-IMAC and then incubated with hydrogen fluoride, which hydrolyzes the oligodeoxyribonucleotide to yield the peptide-deoxyuridine conjugate. The site of photocrosslinking was identified by MALDI-TOF-MS and MALDI-TOF-MS/MS to be Trp189, adjacent to Arg188, which aligns with Arg194 in NgoMIV, involved in recognition of the second guanine in the NgoMIV recognition sequence G downward arrow CCGGC. This result confirms previously published conclusions drawn on the basis of a mutational analysis of SsoII. The methodology that was employed here can be used in principle to identify the DNA binding site of any protein.

The restriction endonuclease EcoRV has been characterized in structural and functional terms in great detail. Based on this detailed information we employed a structure-guided approach to engineer variants of EcoRV that should be able to discriminate between differently flanked EcoRV recognition sites. In crystal structures of EcoRV complexed with d(CGGGATATCCC)(2) and d(AAAGATATCTT)(2), Lys104 and Ala181 closely approach the two base pairs flanking the GATATC recognition site and thus were proposed to be a reasonable starting point for the rational extension of site specificity in EcoRV [Horton,N.C. and Perona,J.J. (1998) J. Biol. Chem., 273, 21721-21729]. To test this proposal, several single (K104R, A181E, A181K) and double mutants of EcoRV (K104R/A181E, K104R/A181K) were generated. A detailed characterization of all variants examined shows that only the substitution of Ala181 by Glu leads to a considerably altered selectivity with both oligodeoxynucleotide and macromolecular DNA substrates, but not the predicted one, as these variants prefer cleavage of a TA flanked site over all other sites, under all conditions tested. The substitution of Lys104 by Arg, in contrast, which appeared to be very promising on the basis of the crystallographic analysis, does not lead to variants which differ very much from the EcoRV wild-type enzyme with respect to the flanking sequence preferences. The K104R/A181E and K104R/A181K double mutants show nearly the same preferences as the A181E and A181K single mutants. We conclude that even for the very well characterized restriction enzyme EcoRV, properties that determine specificity and selectivity are difficult to model on the basis of the available structural information.

Recently, the gene for the determination of maleness has been identified in the sex-determining region on the short arm of the Y chromosome (SRY) between the Y-chromosomal pseudoautosomal boundary (PABY) and the ZFY gene locus. Experiments with transgenic mice confirmed that SRY is a part of the testis-determining factor (TDF). We describe a sporadic case of a patient with intersexual genitalia and the histological finding of ovotestes in the gonad, which resembles the mixed type of gonadal tissue without primordial follicle structures. The karyotype of the patient was 46,XY. By PCR amplification, we tested for the presence of PABY, SRY, and ZFY by using DNA isolated from peripheral blood leukocytes and for the presence of SRY by using DNA obtained from histological gonadal slices. The SRY products of both DNA preparations were further analyzed by direct sequencing. All three parts of the sex-determining region of the Y chromosome could be amplified from leukocytic DNA. The patient\'s and the father\'s SRY sequences were identical with the published sequence. In the SRY PCR product of gonadal DNA, the wild-type and two point mutations were present in the patient\'s sequence, simulating a heterozygous state of a Y-chromosomal gene: one of the mutations was silent, while the other encoded for a nonconservative amino acid substitution from leucine to histidine. Subcloning procedures showed that the two point mutations always occurred together. The origin of the patient\'s intersexuality is a postzygotic mutation of the SRY occurring in part of the gonadal tissue. This event caused the loss of the testis-determining function in affected cells.

We report here the second evidence of retrotransposition of L1, which was found inserted into the dystrophin gene of a patient, causing Duchenne muscular dystrophy (DMD). When the PCR was used to amplify a region of the dystrophin gene encompassing exon 44 from genomic DNA of two Japanese brothers with DMD, it was found to be approximately 600 bp larger than expected. Both the normal and the abnormally large products were amplified from the DNA of their mother. However, the maternal grandparents did not have the abnormal allele, and the mutation must therefore have occurred in the mother. Analysis of nucleotide sequence of the amplified product from a patient disclosed that the insertion was present zero to two bases upstream from the 3\' end of exon 44 and that two to four bases of the exon sequence were deleted from the insertion site. The insertion sequence was found to be composed of 606-608 bp and to be almost identical to the inverse complement of 3\' portion of the L1 retrotransposon consensus sequence. The dystrophin gene transcript from peripheral lymphocytes of one of the patients was analyzed by using reverse transcription/semi-nested PCR. The size of the amplified product encompassing exon 42 to 46 was smaller than expected. Sequencing of the amplified product disclosed that the sequence of exon 43 was directly joined to that of exon 45. Exon 44 of the transcript was thus shown to be skipped during splicing. This novel mutation of the dystrophin gene has important implications regarding retrotransposition of an active L1 element and provides a new insight into the origins of mutations in the dystrophin gene.

The Wolfram syndrome (MIM 222300) is a disease of unknown origin consisting of diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Here we report on a generalized deficiency of the mitochondrial respiratory enzyme activities in skeletal muscle and lymphocyte homogenate of a girl suffering from the Wolfram syndrome. In addition, we provide evidence for a 7.6-kilobase pair heteroplasmic deletion (spanning nucleotides 6465-14135) of the mitochondrial DNA in the two tissues and show that directly repeated sequences (11 bp) were present in the wild-type mitochondrial genome at the boundaries of the deletion. Neither of the patient\'s parents was found to bear rearranged molecules. This study supports the view that a respiratory chain defect can present with insulin-dependent diabetes mellitus as the onset symptom. It also suggests that a defect of oxidative phosphorylation should be considered when investigating other cases of Wolfram syndrome, especially because this syndrome fulfills the criteria for a genetic defect of the mitochondrial energy supply: (a) an unexplained association of symptoms (b) with early onset and rapidly progressive course, (c) involving seemingly unrelated organs and tissues.