Small non-coding RNAs as magic bullets.

RNA interference (RNAi) - inhibition of gene expression by small, non-coding RNAs [small interfering RNAs (siRNAs) or microRNAs (miRNAs)] - has changed our view of regulation of expression dramatically. The application of siRNAs for both functional analysis of genes and medication raises several questions. These include the design of the double-stranded oligonucleotides, their preparation and introduction into cells or animals either as chemically synthesized entities or as transcripts from a suitable vector. Delivery of the oligonucleotides, choice of vector, chemical modification to stabilize against nucleases and avoidance of side effects (e.g. stimulation of interferons) are major challenges. Work to identify the multiple targets of miRNAs is still in its infancy, and a clear distinction between siRNAs and miRNAs is difficult in some instances. Moreover, transcriptional silencing by RNAi is poorly understood; it is evident that the siRNA machinery is involved but the details await clarification. Given the multitude of interactions of the small non-coding RNAs revealed so far, we should be prepared to encounter, as yet, undiscovered interactions and mechanisms.

Synthesis and Properties of Diuridine Phosphate Analogues Containing Thio and Amino Modifications.

Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).

Phosphorothioates, essential components of therapeutic oligonucleotides.

Phosphorothioates have found their usefulness in the general area of oligonucleotide therapeutic applications. Initially this modification was introduced into the antisense methodology because of the nuclease resistance of the phosphorothioate linkage in comparison with that of the phosphate linkage. However, as experimental data accumulated, it was detected that this chemical modification also facilitates cellular uptake and bioavailibity in vivo. Thus, today the majority of therapeutic oligonucleotides contain this modification. This review will discuss the historical development of this modification and present some of its chemical properties where they differ from those of the phosphate group. The antisense application will be discussed in the original context with cleavage of the target mRNA, but other target RNAs such as microRNAs and long noncoding RNAs will also be covered. It continues with applications where the target RNA should not be cleaved. A brief presentation of decoy oligonucleotides will be included, as well as some miscellaneous applications. Cellular uptake is a crucial step for oligonucleotides to reach their target and will be briefly reviewed. Lastly, a most surprising recent observation is the presence of phosphorothioate groups in bacterial DNA where functions still remain to be fully determined.

Modification of cognitive performance in schizophrenia by complexin 2 gene polymorphisms.

CONTEXT: Schizophrenia is the collective term for a heterogeneous group of mental disorders with a still obscure biological basis. In particular, the specific contribution of risk or candidate gene variants to the complex schizophrenic phenotype is largely unknown. OBJECTIVE: To prepare the ground for a novel "phenomics" approach, a unique schizophrenia patient database was established by GRAS (Göttingen Research Association for Schizophrenia), designed to allow association of genetic information with quantifiable phenotypes.Because synaptic dysfunction plays a key role in schizophrenia, the complexin 2 gene (CPLX2) was examined in the first phenotype-based genetic association study (PGAS) of GRAS [corrected] DESIGN: Subsequent to a classic case-control approach, we analyzed the contribution of CPLX2 polymorphisms to discrete cognitive domains within the schizophrenic population. To gain mechanistic insight into how certain CPLX2 variants influence gene expression and function, peripheral blood mononuclear cells of patients, Cplx -null mutant mice, and transfected cells were investigated. SETTING: Coordinating research center (Max Planck Institute of Experimental Medicine) and 23 collaborating psychiatric centers all over Germany. PARTICIPANTS: One thousand seventy-one patients with schizophrenia (DSM-IV) examined by an invariant investigator team, resulting in the GRAS database with more than 3000 phenotypic data points per patient, and 1079 healthy control subjects of comparable ethnicity. Main Outcome Measure Cognitive performance including executive functioning, reasoning, and verbal learning/memory. RESULTS: Six single-nucleotide polymorphisms, distributed over the whole CPLX2 gene, were found to be highly associated with current cognition of schizophrenic subjects but only marginally with premorbid intelligence. Correspondingly, in Cplx2 -null mutant mice, prominent cognitive loss of function was obtained only in combination with a minor brain lesion applied during puberty, modeling a clinically relevant environmental risk ("second hit") for schizophrenia. In the human CPLX2 gene, 1 of the identified 6 cognition-relevant single-nucleotide polymorphisms, rs3822674 in the 3' untranslated region, was detected to influence microRNA-498 binding and gene expression. The same marker was associated with differential expression of CPLX2 in peripheral blood mononuclear cells. CONCLUSIONS: The PGAS allows identification of marker-associated clinical/biological traits. Current cognitive performance in schizophrenic patients is modified by CPLX2 variants modulating posttranscriptional gene expression.

The versatility of oligonucleotides as potential therapeutics.

Oligonucleotides can in a variety of ways inhibit gene expression by interfering with translation. Oligonucleotides that are complementary to a target mRNA, antisense oligonucleotides, can prevent translation either by cleaving the target or by physically blocking the process. Additionally, oligonucleotides can correct the undesired splicing of pre-mRNA. RNA interference using double-stranded oligoribonucleotides also results in cleavage of the target mRNA. Catalytically competent ribozymes and DNAzymes can have the same effect. Even with no RNA as target, oligonucleotides can be selected as aptamers to bind to any protein to inhibit its activity. Moreover, oligonucleotides can act as decoys particularly for transcription factors to prevent binding to the promoter. A different mode of action is the activation of Toll-like receptors to induce an immune response. Several pathways for drug development are still in their infancy, for example microRNAs and antagomirs.

Selection of hammerhead ribozyme variants with low Mg2+ requirement: importance of stem-loop II.

Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg(2+) concentrations were in vitro selected from a library with 18 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had the same sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mM Mg(2+) concentration in the context of several substrates with differences in the lengths of stem I and III, including the well-characterised HH16 substrate and a derivative thereof with a GUA triplet at the cleavage site, as well as a substrate used previously in a related study. For the HH16 substrate, a change of base pair 10.2-11.2 to C-G in stem II further improved activity by about 2.5-fold to 0.8 min(-1) (at 1 mM Mg(2+) concentration, 25 degrees C, pH 7.5). Interestingly, this very active variant was not identified by the selection procedure. Changing loop II from UUUU to GCAA or extension of stem II to three or four base pairs reduced the cleavage rate by 2.0-2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so far at low Mg(2+) concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degrees C and pH 7.5 in monophasic reactions, with endpoints between 75 and 90 %. Such constructs promise to be advantageous for the inhibition of gene expression in vivo.