Poly(U)-agarose affinity chromatography: specific, sensitivity selectivity, and affinity of binding

These studies were done to determine four basic intrinsic properties of poly(U)-agarose affinity columns. Specificity of binding studies demonstrated that binding to these columns is highly specific with greater than 90% complementary binding and less than or equal to 3% noncomplementary binding. Sensitivity of binding studies indicated that a minimum sequence of 10 adenylates is required for detectable complementary binding. Selectivity of binding studies revealed that nonsequential adenylates in native RNAs and randomly distributed adenylates in synthetic poly(A)-poly(C) co-polymers did not bind to poly(U)-agarose affinity columns. Whereas, affinity of binding studies demonstrated that A=U complementary base pairing is independent of chain-lengths of greater than or equal to 25 adenylates and dependent of chain-lengths of less than 25 adenylates. Thus the data demonstrates that poly(U)-agarose affinity chromatography is scientifically sound and expedient for the detection and isolation of poly(A)-containing cellular and viral RNAs.     

Purification of Avian Myeloblastosis Virus DNA Polymerase by Affinity Chromatography on Polycytidylate-Agarose

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.     

Hydrophobic affinity chromatography of nucleic acids and proteins.

5' tritylated oligonucleotides binding hydrophobically to low trityl cellulose/sepharose (< 15 microMTr/ml) retain their hydrogen-bonding specificities for complementary sequences. This, constitutes a novel mode of attaching affinity ligands to solid supports, is more convenient than existing methods, and proceeds with 100% yield. The salt, dielectric constant and temperature dependence of these non-covalently anchored ligands permits the isolation of a variety of RNAs including fibroin mRNA. Medium trityl sepharose (15-40 microM Tr/ml) has a high binding specificity for poly A and poly A containing mRNA, equivalent to dT cellulose. Most proteins, including nucleic acid enzymes, bind to these columns and retain enzymatic activity, thus mimicking enzymes attached covalently to solid phases. A number of in vivo counterparts to this hydrophobically determined specificity are noted, as are homologies to nitro-cellulose filters.     

Affinity separation of polyribonucleotide-binding human blood proteins

Using affinity columns with immobilized poly(A), poly(G), poly(U), poly(C), and poly(A).poly(U) and poly(G) x poly(C) duplexes several polyribonucleotide-binding blood plasma proteins have been captured. Albumin and keratins K1 and K2e have been detected to bind polypurine tracts. The in vitro glycated albumin binds poly(A) and poly(G) more efficiently than the unmodified protein. The major polypyrimidine-binding blood plasma protein (28 kDa) can catalyze the hydrolysis of poly(U).     

Probing the binding of two sugar bearing anticancer agents aristololactam-β-(D)-glucoside and daunomycin to double stranded RNA polynucleotides: a combined spectroscopic and calorimetric study

The plant alkaloid aristololactam-β-d-glucoside and the anticancer chemotherapy drug daunomycin are two sugar bearing DNA binding antibiotics. The binding of these molecules to three double stranded ribonucleic acids, poly(A)·poly(U), poly(I)·poly(C) and poly(C)·poly(G), was studied using various biophysical techniques. Absorbance and fluorescence studies revealed that these molecules bound non-cooperatively to these ds RNAs with the binding affinities of the order 10(6) for daunomycin and 10(5) M(-1) for aristololactam-β-d-glucoside. Fluorescence quenching and viscosity studies gave evidence for intercalative binding. The binding enhanced the melting temperature of poly(A)·poly(U) and poly(I)·poly(C) and the binding affinity values evaluated from the melting data were in agreement with that obtained from other techniques. Circular dichroism results suggested minor conformational perturbations of the RNA structures. The binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived from calorimetry were in agreement with that obtained from spectroscopic data. Daunomycin bound all the three RNAs stronger than aristololactam-β-d-glucoside and the binding affinity varied as poly(A)·poly(U) > poly(I)·poly(C) > poly(C)·poly(G). The temperature dependence of the enthalpy changes yielded negative values of heat capacity changes for the complexation suggesting substantial hydrophobic contribution to the binding process. Furthermore, an enthalpy-entropy compensation behavior was also seen in all systems. These results provide new insights into binding of these small molecule drugs to double stranded RNA sequences.     

Binding of the plant alkaloid aristololactam-β-d-glucoside and antitumor antibiotic daunomycin to single stranded polyribonucleotides

Background

Interaction of the plant alkaloid aristololactam-β-d-glucoside and the antitumor drug daunomycin with single stranded RNAs poly(G), poly(I), poly(C) and poly(U) has been investigated.

Methods

Biophysical techniques of absorption, fluorescence, competition dialysis, circular dichroism, and microcalorimetry have been used.

Results

Absorption and fluorescence studies have revealed noncooperative binding of ADG and DAN to the single stranded RNAs. The binding affinity of ADG varied as poly(G) > poly(I) > > poly(C) > poly(U). The affinity of DAN was one order higher than that of ADG and varied as poly(G) > poly(I) > poly(U) > poly(C). This binding preference was further confirmed by competition dialysis assay. The thermodynamics of the binding was characterised to be favourable entropy and enthalpic terms but their contributions were different for different systems. The major non-polyelectrolytic contribution to the binding revealed from salt dependent data appears to be arising mostly from stacking of DAN and ADG molecules with the bases leading to partial intercalation to single stranded RNA structures. Small negative heat capacity values have been observed in all the four cases.

Conclusions

This study presents the comparative structural and thermodynamic profiles of the binding of aristololactam-β-d-glucoside and daunomycin to single stranded polyribonucleotides.

General significance

These results suggest strong, specific but differential binding of these drug molecules to the single stranded RNAs and highlight the role of their structural differences in the interaction profile.     

A basic isozyme of yeast glyceraldehyde-3-phosphate dehydrogenase with nucleic acid helix-destabilizing activity

A nucleic acid helix-destabilizing protein has been purified from Saccharomyces cerevisiae using affinity chromatographic techniques. Crude protein extracts at low ionic strength (approx. 0.05 M) were applied sequentially to tandem columns of native DNA-cellulose, aminophenyl-phosphoryl-UMP-agarose, poly(I . C)-agarose, poly(U)-cellulose and denatured DNA-cellulose. The 2 M NaCl eluant of the poly(U)-cellulose column was dialyzed to low ionic strength and recycled through native DNA-cellulose, poly(I . C)-agarose and poly(U)-cellulose. Purified helix-destabilizing protein eluted from the poly(U)-cellulose between 0.1 and 0.5 M NaCl. On the basis of enzymatic activity, immunological cross-reactivity, mobility on SDS gels, amino acid analysis and preliminary peptide mapping experiments, this material was identified as an isozymic fraction of glyceraldehyde-3-phosphate dehydrogenase. The major crystallizable isozyme of this enzyme from yeast is, however, considerably more acidic than the helix-destabilizing protein, and displays significantly lower helix-destabilizing activity. Stoichiometric levels of the isolated protein at low (approx. 0.01) ionic strength depress the Tm of poly(A-U) and poly [d(A-T)] by as much as 28 and 22 degrees C, respectively. Longer double helices, poly(A . U) and Clostridium perfringens DNA are also denatured by the helix-destabilizing protein, but at relatively slow rates. The binding of this protein to [3H]-poly(U) on nitrocellulose filters in [Na+]-dependent, with a 50% reduction at 0.09 M NaCl. Based on its effect on the circular dichroism spectrum of poly(A), the protein was shown to distort the conformation of the polynucleotide chain. An analogous protein from mammalian cells, P8, was also shown to depress poly(A-U) Tm.     

The binding of spermine to polynucleotides and complementary oligonucleotides at near physiological ionic strength

The binding of [14C] spermine to polynucleotides has been studied by equilibrium dialysis and the data analysed by Scatchard plots. The binding of spermine to poly(A) shows a binding site for 1 spermine/140 nucleotides when measured in 0.2M NaCl at 5 degrees C. Poly(C) also has a similar sites; on the other hand poly(U) and poly(G) each have a binding site for 1 spermine/12 nucleotides. The addition of complementary di- or trinucleotides to either poly(A) or poly(U) affects their ability to bind spermine, in particular the high affinity site on poly(A) is no longer detectable. The effect of spermine, spermidine and putrescine on the binding of polynucleotides to complementary di- and trinucleotides was also studied. Spermine markedly increased the binding of both ApA and of ApApA to poly(U) whereas spermidine and putrescine had very little effect. In contrast spermine had little effect on the binding of either UpU or UpUpU to poly(A). These results suggest that spermine binding to oligo- and polynucleotides is dependent on the particular nucleotide combination involved and that spermine may therefore be able to act selectively within cells.     

Direct demonstration of a complementarity between mRNA and double-stranded sequences of pre-mRNA

The total poly(A)-containing mRNA from mouse liver or Ehrlich ascites carcinoma cells was annealed with denatured ds RNA prepared from heavy nuclear ³H-labeled pre-mRNA of the same tissue. The hybrids formed were detected by binding of complexes to poly(U)-Sepharose columns through the poly(A) of mRNA. With this technique, about 30% of labeled ds RNA was bound to poly(U)-Sepharose after annealing it with an mRNA excess. The proportion of hybrid material detected by RNase treatment was two to three times lower than that obtained by poly(U)-Sepharose binding. The length of the RNase-stable acid precipitable hybrid material consisted of heterogeneous sequences of 10–100 nucleotides long when cytoplasmic, and 10–60 nucleotides long when polysomal mRNA was used in the hybridization reaction. The results obtained show that at least some of the mRNA molecules contain sequences complementary to one of the branches of the pre-mRNA hairpins. These results are compatible with the idea that the hairpin-like sequences in pre-mRNA are localized between mRNA and the non-informative part of the precursor molecule.     

Translation of mRNA for glutamate dehydrogenase and spectrophotometric procedure to follow the enzyme biosynthesis.

Heterogeneous poly (A)-mRNA fraction was isolated from rat liver microsomes using phenol-chloroform extraction, millipore filtration and poly (U)-agarose affinity chromatography. Obtained fractions were characterized with respect to their secondary structure and poly (A) content. Isolated poly (A)-mRNA fraction contained high template activity for glutamate dehydrogenase in cell-free systems with microsomes or polysomes. A spectrophotometric procedure to follow enzyme biosynthesis was also developed.     

Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).     

Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase.

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.     

Affinity purification of monoclonal antibodies,using a bifunctional oligosaccharide hapten

A bifunctional hapten was synthesized consisting of a blood group A active tetrasaccharide (A-tetra) and a blood group Le^a active pentasaccharide. lacto-N-fucopentaose II (LNF II), linked to each other with a phenylaminothiourea spacer connecting the reducing ends (A-tetra-LNF II). The hapten was demonstrated to retain both blood group A and Le^a activity and could be easily bound to both monoclonal anti-A and anti-Le^a affinity columns. Due to the strong temperature dependence of the two antibodies in their binding to oligosaccharides, the bifunctional hapten could be utilized to achieve easy desorption in the final step of affinity purification of either monoclonal anti-Le^a or anti-A. The system is postulated to have general applicability in affinity purification of any ligate that binds with an avidity too high to achieve non-denaturing desorption.To whom correspondence should be addressed.     

The effect of the antibiotic edeine on tRNA interaction with A, P and E sites of Escherichia coli 70S ribosomes

Edeine inhibits poly(U)-dependent binding of tRNAPhe to the P and A sites simultaneously, both on 30S subunits and 70S ribosomes. Hence, edeine cannot be considered as antibiotic, "complementary" to tetracycline for selective adsorption of tRNA only to the P or to the A site. Further, edeine decreases the affinity constant of tRNAPhe for the P-site by more than two orders of magnitude, no matter poly(U) is present or not. Neither edeine nor tetracycline affect interaction of deacylated tRNAPhe with the E-site of E. coli 70S ribosomes.     

Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity.

The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3'-end sequences. Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity. By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified. While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding. Finally, we found a terminal transferase activity associated with the purified enzyme. However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.     

Characterization of the essential activities of Saccharomyces cerevisiae Mtr4p, a 3'->5' helicase partner of the nuclear exosome

Mtr4p belongs to the Ski2p family of DEVH-box containing proteins and is required for processing and degradation of a variety of RNA substrates in the nucleus. In particular, Mtr4p is required for creating the 5.8 S ribosomal RNA from its 7 S precursor, proper 3'-end processing of the U4 small nuclear RNA and some small nucleolar RNAs, and degradation of aberrant mRNAs and tRNAs. In these studies we have shown that Mtr4p has RNA-dependent ATPase (or dATPase) activity that is stimulated effectively by likely substrates (e.g. tRNA) but surprisingly weakly by poly(A). Using an RNA strand-displacement assay, we have demonstrated that Mtr4p can, in the presence of ATP or dATP, unwind the duplex region of a partial duplex RNA substrate in the 3'-->5' direction. We have examined the ability of Mtr4p to bind model RNA substrates in the presence of nucleotides that mimic the stages (i.e. ATP-bound, ADP-bound, and nucleotide-free) of the unwinding reaction. Our results demonstrate that the presence of a non-hydrolyzable ATP analog allows Mtr4p to discriminate between partial duplex RNA substrates, binding a 3'-tailed substrate with 5-fold higher affinity than a 5'-tailed substrate. In addition, Mtr4p displays a marked preference for binding to poly(A) RNA relative to an oligoribonucleotide of the same length and a random sequence. This binding exhibits apparent cooperativity and different dynamic behavior from binding to the random single-stranded RNA. This unique binding mode might be employed primarily for degradation.