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PubMed1989

Studies of interactions of porphyrins with transfer RNA by high-resolution NMR.

The interactions of tetra-4N-methylpyridyl porphyrin and its zinc(II), copper(II) and manganese(III) complexes with brewer's yeast type V phenylalanine specific tRNA have been evaluated by high-resolution NMR. Differences in chemical shifts have been noted for three proton resonances in response to the presence of small quantities of the free base and the zinc and copper complexes. The protons giving rise to these signals are located on bases T54 and psi 55, both of which are involved in the primary intraloop and interloop hydrogen bonds that hold the D and T psi C loops together in the tertiary structure. In addition, broadening of specific resonances due to hydrogen bonding protons in the D stem at low ratios of porphyrin to tRNA indicates that the association of porphyrins increases the rate of imino proton exchange. The titration of the tRNA with the manganese(III) complex did not reveal shifts or specific broadening comparable to the other porphyrins at low ratios. The changes induced in the NMR spectrum of tRNA by porphyrins define their site of interaction with the polynucleotide. This site, at the outside of the elbow-bend in the tRNA 'L', is different from the locus of binding in tRNA for other classical DNA intercalators. Furthermore, a new mode of binding may be involved that is neither intercalative nor simply electrostatic.

Binding Sites↗
PubMed2000

Prothymosin alpha is a nonspecific facilitator of nuclear processes: studies of run-on transcription.

The effect of prothymosin alpha on transcriptional elongation has been examined. The addition of prothymosin alpha to COS-1 and NIH3T3 cell nuclei engaged in run-on transcription stimulated RNA synthesis approximately two- to threefold in a dose-dependent manner. Polyglutamic acid or a random polypeptide composed of glutamic acid, alanine, and tyrosine, did not substitute for prothymosin alpha. Enhanced transcription occurred in the presence of high and low doses of actinomycin D and in the presence of alpha-amanitin, but not in nuclear extracts. The stimulatory effect was dependent on a limiting concentration of one nucleoside triphosphate and was nearly abrogated by saturating levels of precursors. In the presence of Sarkosyl, which itself increases transcription, prothymosin alpha was almost ineffectual. The data are consistent with a model in which prothymosin alpha does not interact directly with polymerases but, instead, nonspecifically decreases the barriers to diffusion of charged molecules in electrostatically charged environments.

3T3 Cells↗
PubMed2003

Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations.

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.

Archaeal Proteins↗
PubMed1996

Magnesium ions are required by Bacillus subtilis ribonuclease P RNA for both binding and cleaving precursor tRNAAsp.

The multiple roles Mg2+ plays in ribozyme-catalyzed reactions in stabilizing RNA structure, enhancing the affinity of bound substrates, and increasing catalysis are delineated for the RNA component of ribonuclease P (RNase P RNA) by a combination of steady-state kinetics, transient kinetics, and equilibrium binding measurements. Divalent metal ions cooperatively increase the affinity of Bacillus subtilis RNase P RNA for B. subtilis tRNA(Asp) more than 10(3)-fold, consistent with at least two additional magnesium ions binding to the RNase P RNA.tRNA complex. Monovalent cations also decrease KD(tRNA) and reduce, but do not eliminate, the dependence on magnesium ions, demonstrating that nonspecific electrostatic shielding is not sufficient to explain the requirement for high salt. Both di- and monovalent cations promote the high affinity of tRNA by forming contacts in the binary complex that reduce the dissociation rate constant for tRNA. Additionally, the hyperbolic dependence of the hydrolytic rate constant on the concentration of magnesium with a K1/2 approximately equal to 36 mM suggests that a third low-affinity divalent metal ion stabilizes the transition state for pre-tRNA cleavage. Furthermore, many (about 100) magnesium ions bind independently to RNase P RNA with higher affinity than the K1/2 of any of the functionally characterized magnesium binding sites. Therefore, the magnesium binding sites that have differential affinity in either the "folded" species or binary complex are a small subset of the total number of associated magnesium ions. In summary, the importance of magnesium bound to RNase P RNA can be separated functionally into three crucial roles: at least three sites stabilize the folded RNA tertiary structure [Pan. T. (1995) Biochemistry 34, 902-909], at least two sites enhance the formation of complexes of RNase P RNA with pre-tRNA or tRNA, and at least one site stabilizes the transition state for pre-tRNA cleavage.

Bacillus subtilis↗
PubMed1997

Affinities and selectivities of divalent cation binding sites within an RNA tertiary structure.

A 58 nucleotide fragment of Escherichia coli large subunit ribosomal RNA, nucleotides 1051 to 1108, adopts a specific tertiary structure normally requiring both monovalent (NH4+ or K+) and divalent (Mg2+) ions to fold; this ion-dependent structure is a prerequisite for recognition by ribosomal protein L11. Melting experiments have been used to show that a sequence variant of this fragment, GACG RNA, is able to adopt a stable tertiary structure in the presence of 1.6 M NH4Cl and absence of divalent ions. The similarity of this high-salt structure to the tertiary structure formed under more typical salt conditions (0.1 M NH4Cl and several mM MgCl2) was shown by its following properties: (i) an unusual ratio of hyperchromicity at 260 nm and 280 nm upon unfolding, (ii) selectivity for NH4+ over K+ or Na+, (iii) stabilization by L11 protein, and (iv) further stabilization by added Mg2+. Delocalized electrostatic interactions of divalent ions with nucleic acids should be very weak in the presence of >1 M monovalent salt; thus stabilization of the tertiary structure by low (<1 mM) Mg2+ concentrations in these high-salt conditions suggests that Mg2+ binds at specific site(s). GACG RNA tertiary structure unfolding in 1.6 M NH4Cl (Tm approximately 39 degrees C) is distinct from melting of the secondary structure (centered at approximately 72 degrees C), and it has been possible to calculate the free energy of tertiary structure stabilization upon addition of various divalent cations. From these binding free energies, ion-RNA binding isotherms for Mn2+, Mg2+, Ca2+, Sr2+ and Ba2+ have been obtained. All of these ions bind at two sites: one site favors Mg2+ and Ba2+ and discriminates against Ca2+, while the other site favors binding of smaller ions over larger ones (Mg2+ >Ca2+ >Sr2+ >Ba2+). Weak cooperative or anticooperative interactions between the sites, also dependent on ion radius, may also be taking place.

Barium↗
PubMed2003

Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens.

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP-RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic supernatants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic supernatants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

Archaeal Proteins↗
PubMed2003

Motifs in nucleic acids: molecular mechanics restraints for base pairing and base stacking.

In building and refining nucleic acid structures, it is often desirable to enforce particular base pairing and/or base stacking interactions. Energy-based modeling programs with classical molecular mechanics force fields do not lend themselves to the easy imposition of penalty terms corresponding to such restraints, because the requirement that two bases lie in or near the same plane (pairing) or that they lie in parallel planes (stacking) cannot be easily expressed in terms of traditional interactions involving two atoms (bonds), three atoms (angles), or four atoms (torsions). Here we derive expressions that define a collection of pseudobonds and pseudoangles through which molecular mechanics restraints for base pairing and stacking can be imposed. We have implemented these restraints into the JUMNA package for modeling DNA and RNA structures. JUMNA scripts can specify base pairing with a variety of standard geometries (Watson-Crick, Hoogsteen, wobble, etc.), or with user-defined geometries; they can also specify stacking arrangements. We have also implemented "soft-core" functions to modify van der Waals and electrostatic interactions to avoid steric conflicts in particularly difficult refinements where two backbones need to pass through one another. Test cases are presented to show the utility of the method. The restraints could be adapted for implementation in other molecular mechanics packages.

Base Pairing↗
PubMed2001

Reviewing efficacy of alternative water treatment techniques.

This section is designed to provide a brief summary of some of the findings. A good deal of work has been conducted by Mr N. L. Pavey and the team at BSRIA, Bracknell. The BSRIA publications are an excellent source of further information. Ultraviolet radiation: UV radiation of wavelength 254 nm destroys bacteria by a mechanism of damaging nucleic acids by producing thymine dimers which disrupt DNA replication [Gavdy and Gavdy, 1980]. L. pneumophila has been reported as sensitive to UV dosages of 2,500-7,000 uW.s/cm2 [Antopol & Ellner, 1979; Knudson, 1985]. Antopol and Ellner [1979] examined the susceptibility of L. pneumophila to UV dosage. Their results indicated that 50% of the organisms were killed by 380 uWs/cm2 and 90% were killed by 920 uWs/cm2. Kills of 99 and 99.9% were obtained using 1,840 and 2,760 uWs/cm2 respectively. Muraca et al [1987] showed that continuous UV irradiation resulted in a 5 logarithm decrease in waterborne L. pneumophila in a circulating system. Gilpin [1984] reported that in laboratory buffer solutions, exposure to 1 uW of UV radiation per cm2 achieved a 50% kill of L longbeachae in 5 minutes, L. gormanii in 2-30 minutes and L pneumophila in 17 minutes. Exposure times for 99% kills for L. longbeachae, L pneumophila and L. Gormanii were 33, 48 and 63 minutes respectively. The same research worker conducted experiments using a 3 litre circulating water system, connected to a stainless steel housing containing a UV source. The UV lamp output was 7 ergs/mm2 per second per 100 cm at 254 nm. L. pneumophila was killed within 15 seconds, that is within their first pass through the system. Continuous disinfection with UV has the advantages of imparting no taste, odour or harmful chemical by-products and requires minimal operation and maintenance [Muraca et al 1988]. Keevil et al [1989] state that UV irradiation fails to clear systems of biofilm because of poor penetration into microflocs of the micro-organisms. Copper/silver ionisation: A recent study of full scale hot water test rigs incorporating copper-silver ionisation systems has been reported by Pavey, 1996. Copper and silver ions were introduced into the water by electrolysis. One of the principal mechanisms of biocidal action of these ions is thought to be cell penetration. The positively charged copper ions form electrostatic bonds with negatively charged sites on the cell wall. The cell membrane is thus distorted, allowing ingress of silver ions which attack the cell by binding at specific sites to DNA, RNA, respiratory enzymes and cellular protein, causing catastrophic failure of the life support systems of the cell. Silver and copper ion concentrations of 40 and 400 ug/L respectively were effective against planktonic Legionellae in cold water systems and hot water systems containing soft water. In hard water, the ionisation was ineffective due to the inability to control silver ion concentrations. This was caused by scaling of the electrodes and silver ion complexation by the high concentration of dissolved solids. Bosch et al [1993] had earlier extended the application of copper-silver disinfection to human enteric viruses in water, such as adenovirus, rotavirus, hepatitis A virus, and poliovirus. Their work showed that copper and silver ions in the presence of reduced levels of free chlorine did not ensure the total elimination of viral pathogens from water. In the case of an amoeba, Naegleria fowleria [responsible for primary amoebic meningoencephalitis], Cassells et al [1995] have demonstrated that a combination of silver and copper ions were ineffective at inactivating the amoebae at 80 and 800 ug/L respectively. However addition of 1.0 mg/L free chlorine produced a synergistic effect, with superior inactivation relative to either chlorine or silver-copper in isolation. A similar synergy was reported by Yahya et al [1989] in their study of Staphylococcus sp. and Pseudomonas aeruginosa. Yahya et al [1992] also suggested an additive or synergistic effect in the inactivation of coliphage MS-2 and poliovirus. Other techniques: There are a number of other techniques. We have conducted trials of most of these in the control of Legionella sp., but these fall out of the scope of this article, and as such less emphasis has been placed on them here. Ozonation: Ozone [O3] is an oxidising gas, generated electrically from oxygen [O2]. L. pneumophila can be killed at < 1 mg/L of ozone [Edelstien et al 1982]. Muraca et al [1987] found that 1-2 mg/L of continuous ozone over a six hour contact time, produced a 5 logarithm decrease of L. pneumophila. The effectiveness of ozone treatment against a range of bacteria and coliphages has been studied Botzenhart et al [1993]. E. coli was least resistant to ozone, followed by MS 2-coliphage and PhiX 174-coliphage, with L. pneumophila and Bacillus subtilis spores being the most resistant. (ABSTRACT TRUNCATED)

Chlorine Compounds↗
PubMed1993

On the reversal by deoxyribonucleic acid of the binding of adriamycin to cardiolipin-containing liposomes.

The binding of the cytotoxin adriamycin (doxorubicin) to phospholipids, DNA, and RNA was investigated using (i) fluorescence quenching by this drug of liposomes containing pyrene-labeled phospholipids and (ii) monomolecular cardiolipin films on an air/water interface. In accordance with previous studies, our fluorescence experiments revealed that the acidic phospholipids, phosphatidylglycerol, phosphatidylmethanol, and phosphatidic acid all have high and comparable affinities to adriamycin mainly due to electrostatic interactions whereas binding to phosphatidylcholine was much weaker. Highest affinity, however, was possessed by cardiolipin. Addition of 4 mM CaCl2 reduced the binding of adriamycin to the above lipids. Moreover, in the presence of calcium the affinity of the drug to the different lipids was similar. Ca2+ concentrations > 100 microM began to reverse the binding of adriamycin to cardiolipin-containing liposomes whereas lower concentrations had only an insignificant effect. The association of adriamycin with DNA, RNA, and cardiolipin was then compared by observing the reversal of the cytotoxin-cardiolipin association by nucleic acids. The affinity of adriamycin was found to decrease in the sequence DNA > cardiolipin > RNA with relative affinities of 7.8, 2.3, and 1, respectively. Penetration of the drug into cardiolipin monolayers spread on an air/water interface resulted in an increase in surface pressure pi whereas only a very weak increase was observed using dimyristoylphosphatidylcholine films. Removal of adriamycin from lipid monolayers was achieved by adding DNA into the aqueous subphase. Similar to the fluorescence quenching studies increase in pi after the inclusion of adriamycin into the aqueous subphase was significantly reduced in the presence of Ca2+ concentrations > 100 microM, whereas lower concentrations had only an insignificant effect. Residual drug-phospholipid interactions in the lipid monolayer observed in the presence of Ca2+ were also reversed by DNA.

Binding Sites↗
PubMed2006

RNA recognition and recognition control of alpha-peptide ribonucleic acids containing arginine residue by external factors.

A novel nucleic acid model using alpha-peptide ribonucleic acid (alpha-PRNA) possessing alternative alpha-PRNA/lysine and alpha-PRNA/arginine sequences, where the lysine or arginine residue are expected to stabilize the complex not only by the conventional hydrogen-bonding interactions between the complementary nucleobase pairs but also through the electrostatic interactions between basic amino acid side-chain groups and DNA's phosphate anions on the backbone. These alpha-PRNAs form stable sequence-specific complex with the complementary DNA.

Arginine↗
PubMed2005

Solution structure of the symmetric coiled coil tetramer formed by the oligomerization domain of hnRNP C: implications for biological function.

During active cell division, heterogeneous nuclear ribonucleoprotein (hnRNP) C is one of the most abundant proteins in the nucleus. hnRNP C exists as a stable tetramer that binds about 230 nucleotides of pre-mRNA and functions in vivo to package nascent transcripts and nucleate assembly of the 40 S hnRNP complex. Previous studies have shown that monomers lacking or possessing mutant oligomerization domains bind RNA with low affinity, strongly suggesting a cooperative protomer-RNA binding mode. In order to understand the role of the oligomerization domain in defining the biological functions and structure of hnRNP C tetramers, we have determined the high-resolution NMR structure of the oligomerization interface that is formed at the core of the complex, examining specific molecular interactions that drive assembly and contribute to the structural integrity of the tetramer. The determined structure reveals an antiparallel four-helix coiled coil, where classically described knobs-into-holes packing interactions at interhelical contact surfaces are optimized so that side-chains interdigitate to create an even distribution of hydrophobic surfaces along the core. While the stoichiometry of the complex appears to be primarily specified by occlusion of hydrophobic surfaces, particularly the interfacial residue L198, from solvent, helix orientation is primarily determined by electrostatic attractions across helix interfaces. The creation of potential interaction surfaces for other hnRNP C domains along the coiled coil exterior and the assembly of oligomerization interfaces in an antiparallel orientation shape the tertiary fold of full-length monomers and juxtapose RNA-binding elements at distal surfaces of the tetrameric complex in the quaternary assembly. In addition, we discuss the specific challenges encountered in structure determination of this symmetric oligomer by NMR methods, specifically in sorting ambiguous interatomic distance constraints into classes that define different elements of the coiled coil structure.

Amino Acid Sequence↗
PubMed2000

The structure of the yrdC gene product from Escherichia coli reveals a new fold and suggests a role in RNA binding.

The yrdC family of genes codes for proteins that occur both independently and as a domain in proteins that have been implicated in regulation. An example for the latter case is the sua5 gene from yeast. SuaS was identified as a suppressor of a translation initiation defect in cytochrome c and is required for normal growth in yeast (Na JG, Pinto I, Hampsey M, 1992, Genetics 11:791-801). However, the function of the Sua5 protein remains unknown; Sua5 could act either at the transcriptional or the posttranscriptional levels to compensate for an aberrant translation start codon in the cyc gene. To potentially learn more about the function of YrdC and proteins featuring this domain, the crystal structure of the YrdC protein from Escherichia coli was determined at a resolution of 2.0 A. YrdC adopts a new fold with no obvious similarity to those of other proteins with known three-dimensional (3D) structure. The protein features a large concave surface on one side that exhibits a positive electrostatic potential. The dimensions of this depression, its curvature, and the fact that conserved basic amino acids are located at its floor suggest that YrdC may be a nucleic acid binding protein. An investigation of YrdC's binding affinities for single- and double-stranded RNA and DNA fragments as well as tRNAs demonstrates that YrdC binds preferentially to double-stranded RNA. Our work provides evidence that 3D structures of functionally uncharacterized gene products with unique sequences can yield novel folds and functional insights.

Amino Acid Sequence↗
PubMed2003

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

Binding Sites↗
PubMedDate not supplied

Structural basis for topoisomerase I inhibition by nucleoside analogs.

Nucleoside analogs such as 1-beta-D-arabinofuranosyl cytidine (AraC) and 2',2'-difluoro deoxycytidine (dFdC) are important components of the anticancer chemotherapeutic arsenal and are among the most effective anticancer drugs currently available. Although both AraCTP and dFdCTP impede DNA replication through pausing of DNA polymerases, both nucleoside analogs are ultimately incorporated into replicated DNA and interfere in DNA-mediated processes. Our laboratories are investigating the structural basis for the poisoning of topoisomerase I (top1) due to antipyrimidine incorporation into duplex DNA. We recently reported that both AraC and dFdC induce formation of top1 cleavage complexes, and poisoning of top1 contributes to the anticancer activities of both these drugs. Recent NMR and thermodynamic studies from our laboratories provide insight into the mechanism by which AraC and dFdC poison top1. NMR studies from our laboratories have revealed that the arabinosyl sugar of AraC adopted a C2'-endo conformation. Although this is a B-type sugar pucker characteristic of duplex DNA, the conformation is rigid, and this lack of flexibility probably contributes to inhibition of the religation step of the top1 reaction. In contrast to AraC, NMR studies revealed dFdC adopted a C3' endo sugar pucker characteristic of RNA, rather than DNA duplexes. dFdC substitution enhanced formation of top1 cleavage complexes, but did not inhibit religation. The enhancement of top1 cleavage complexes most likely results from a combination of conformational and electrostatic effects. The structural effects of dFdC and AraC are being further investigated in duplex DNA with well-defined top1 cleavage sites to analyze more specifically how these structural perturbations lead to enzyme poisoning.

Antimetabolites, Antineoplastic↗
PubMedDate not supplied

[How do ribosome binding sites recognize the functional state of tRNA?].

The order of relative affinity of three different functional form of tRNA (aminoacyl-tRNA, peptidyl-tRNA and deacylated tRNAOH) was established for three sites of the ribosome. The affinity increases for A-site in consecutive order: tRNAOH less than AcPhe-tRNA less than aa-tRNA; for P-site with messenger RNA: AcPhe-tRNA less than aa-tRNA less than tRNAOH; without messenger RNA: aa-tRNA less than AcPhe-tRNA less than tRNAOH; for E-site: (AcPhe-tRNA, aa-tRNA) much less than much less than tRNAOH. The dependence of association constants versus magnesium concentration for all forms of tRNA conforms the equation: delta lg Ka/delta lg[Mg2+] = n. Number "n" varies in the range 1 to 8 depending on the site of adsorption the form of tRNA and the presence of mRNA. Such magnesium dependence of affinity of tRNAs shows that the electrostatic interactions play an important role in the recognition of functional forms of tRNA by ribosomal sites.

Binding Sites↗
PubMed1996

2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

Exonucleases↗
PubMed2003

Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme.

Several small ribozymes carry out self-cleavage at a specific phosphodiester bond to yield 2',3'-cyclic phosphate and 5'-hydroxyl termini. Prior mechanistic and structural studies on the HDV ribozymes led to the proposal that the pK(a) of C75 is shifted toward neutrality, making it an effective general acid. Recent mechanistic studies on the hairpin ribozyme have led to models in which protonation of G8 is required for phosphodiester cleavage, either for general acid catalysis or for electrostatic stabilization. Inspection of recent crystal structures of the hairpin ribozyme, including a complex with a vanadate transition state mimic, suggests an alternative model involving general acid-base catalysis with G8 serving as the general base and A38 as the general acid. This model is consistent with the literature on the hairpin ribozyme, including pH-rate profiles of wild-type and mutant ribozymes and solvent isotope effects. General mechanistic considerations for RNA catalysis suggest that the penalty for having general acids and bases with pK(a)s removed from neutrality is not as severe as expected. These considerations suggest that general acid-base catalysis may be a common mechanistic strategy of RNA enzymes.

Animals↗