RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to ...employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.
Oligonucleotides modified by a 2'-deoxy-2'-(
-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding
-(methoxy)oxazolidine-linked ...oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ''switched off-state''. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the
-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-β-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary
-acetyl-d-galactosamine-cluster with a β-Ala-H unit has been synthesized. These aldehydes were conjugated via
(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19-47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to β-Ala-H residue resulted in a faster cleavage of the
-(methoxy)oxazolidine-linker at pH 5, being comparable (T
ca 7 h) to hydrazone-based structures.
2′‐O‐(4‐Trifluoromethyl‐triazol‐1‐yl)methyl reporter groups have been incorporated into guanosine‐rich RNA models (including a known bistable Qd/Hp RNA and two G‐rich regions of mRNA of human prion ...protein, PrP) and applied for the 19F NMR spectroscopic characterization of plausible G‐quadruplex/hairpin (Qd/Hp) transitions in these RNA structures. For the synthesis of the CF3‐labeled RNAs, phosphoramidite building blocks of 2′‐O‐(4‐CF3‐triazol‐1‐yl)methyl nucleosides (cytidine, adenosine, and guanosine) were prepared and used as an integral part of the standard solid‐phase RNA synthesis. The obtained 19F NMR spectra supported the usual characterization data (obtained by UV‐ and CD‐melting profiles and by 1H NMR spectra of the imino regions) and additionally gave more detailed information on the Qd/Hp transitions. The molar fractions of the secondary structural species (Qd, Hp) upon thermal denaturation and under varying ionic conditions could be determined from the intensities and shifts of the 19F NMR signals. For a well‐behaved Qd/Hp transition, thermodynamic parameters could be extracted.
Trifluoromethylated NMR probe: 19F NMR spectroscopy, together with 2′‐O‐(4‐CF3‐triazol‐1‐yl)methyl reporter groups, has been demonstrated to be a valuable tool for the determination of molar fractions of the conformers in Hp/Qd RNA transitions (see scheme; Qd, Ss, and Hp represent G‐quadruplex, single‐strand, and hairpin structures, respectively).
Glycosidic (β-1''→6, 3' and 4') site isomers of neomycin B (i.e., neobiosamine (β-1''→6, 3' and 4') neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used ...as a common starting material to obtain neobiosamine glycosyl donor and 6, 3',4'-tri-
-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4'-, 5,3',4'- and 5,6,3'-tri-
-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.
In this report, we investigate the efficiency and selectivity of a Zn
-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are ...varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that form 4-nt bulges, we identified systems with an improved efficacy (an estimated half-life of ca 7-8 h as compared to 11-12 h for sequences studied earlier) as well as systems with an improved site selectivity (up to over 70% cleavage at a single site as compared to 50-60% with previous targets sequences). For targets forming 3-nt bulges, the enhancement compared to previous systems was even more pronounced. Compared to a starting point of targets forming 3-nt AAA bulges (half-lives of ca 21-24 h), we could identify target sequences that were cleaved with half-lives three times lower (ca 7-8 h), i.e., at rates similar to those found for the fastest 4-nt bulge system. In addition, with the 3-nt bulge RNA target site selectivity was improved even further to reach well over 80% cleavage at a specific site.
Triplex‐forming peptide nucleic acids (TFPNAs) were targeted to double‐helical regions of 19F‐labeled RNA hairpin models (a UA‐rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, ...miR‐215). In addition to conventional UV‐ and circular dichroism (CD)‐based detection, binding was monitored by 19F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple‐helical peptide nucleic acid (PNA)/RNA and (PNA)2/RNA binding modes could be obtained. γ‐(R)‐Hydroxymethyl‐modified thymine‐1‐yl‐ and 2‐aminopyridin‐3‐yl‐acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ‐(R)‐hydroxymethyl group on binding was studied. An appropriate pattern of γ‐(R)‐hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR‐215 model.
Seeing into the complex: The stoichiometry of the binding, thermal denaturation of complexes, and transition between triple‐helical peptide nucleic acid (PNA)/microRNA and (PNA)2/microRNA complexes were characterized by 19F NMR spectroscopy (see figure).
4'-C-Azidomethylthymidine 3'-(H-phosphonate) monomer (10) was synthesized in high yield and three such monomers were incorporated by the H-phosphonate coupling into a 15-mer oligodeoxyribonucleotide. ...The unmodified 2'-deoxynucleosides could be coupled by either the H-phosphonate or phosphoramidite chemistry, indicating that the Staudinger reaction between the azido group and the phosphoramidite reagent severely hampered the coupling only when it took place intramolecularly. After chain assembly, three alkynyl group bearing ligands, viz., propargyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside (2), N-{4-N-(trifluoroacetyl)aminomethylbenzyl}-4-pentynamide (3) and N (1), N (3), N (2')-tris(trifluoroacetyl)-N (6')-(4-pentynoyl)neamine (4), were conjugated to the azido groups of the oligonucleotide by click chemistry both on a solid support and in solution. The products were deprotected by conventional ammonolysis and purified by HPLC chromatography. Melting temperature studies revealed that the mannose conjugated oligonucleotides formed more stable duplexes with 2'-O-methyl RNA than with DNA strand. With 2'-O-methyl RNA, a slight destabilization compared to an unmodified sequence was observed at low ionic strength, while at high salt content, the manno-conjugation was stabilizing.
Pseudouridimycin (PUM), a selective inhibitor of bacterial RNA polymerase has been previously detected in microbial-extracts of two strains of Streptomyces species (strain ID38640 and ID38673). Here, ...we isolated PUM and its deoxygenated analogue desoxy-pseudouridimycin (dPUM) from Streptomyces albus DSM 40763, previously reported to produce the metabolite strepturidin (STU). The isolated compounds were characterized by HRMS and spectroscopic techniques and they selectively inhibited transcription by bacterial RNA polymerase as previously reported for PUM. In contrast, STU could not be detected in the cultures of S. albus DSM 40763. As the reported characteristics reported for STU are almost identical with that of PUM, the existence of STU was questioned. We further sequenced the genome of S. albus DSM 40763 and identified a gene cluster that contains orthologs of all PUM biosynthesis enzymes but lacks the enzymes that would conceivably allow biosynthesis of STU as an additional product.
5′‐O‐(4,4′‐Dimethoxytrityl)thymidine was attached to a pentaerythrityl‐derived core, and the resulting tetravalent nucleoside cluster and the next dendritic generations served as a soluble support ...for the synthesis of short oligo‐2′‐deoxyribonucleotides in solution. Couplings using a small excess (1.5 equiv. per 5′‐OH group) of the standard phosphoramidite building blocks proved efficient, and the products could be purified by quantitative precipitation from methanol. Ammonolysis released nearly homogeneous oligonucleotides (CCT, GCT, ACT, and AGCCT) in high yields.
A liquid‐phase method for the synthesis of short oligonucleotides is described that is based on the efficient precipitation of tetrahedrally branched oligonucleotides in methanol.
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