A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids

In site-directed spin labeling (SDSL), a nitroxide moiety containing a stable, unpaired electron is covalently attached to a specific site within a macromolecule, and structural and dynamic information at the labeling site is obtained via electron paramagnetic resonance (EPR) spectroscopy. Successful SDSL requires efficient site-specific incorporation of nitroxides. Work reported here presents a new method for facile nitroxide labeling at the 5' terminus of nucleic acids of arbitrary sizes. T4-polynucleotide kinase was used to enzymatically substitute a phosphorothioate group at the 5' terminus of a nucleic acid, and the resulting phosphorothioate was then reacted with an iodomethyl derivative of a nitroxide. The method was successfully demonstrated on both chemically synthesized and naturally occurring nucleic acids. The attached nitroxides reported duplex formation as well as tertiary folding of nucleic acids, indicating that they serve as a valid probe in nucleic acid studies.     

Acridine spin labels as probes for nucleic acids.

Adridine spin labels, 4-[9-(6-chloro-2-methoxy)-acridylamino]- 2,2,6,6-tetramethyl-1-piperidinyloxy (I) and 4-(9-acridylamino)- 2,2,6,6-tetramethyl-1-piperidinyloxy (II), have been synthesized and their interaction with nucleic acids studied by means of electron spin resonance (ESR). The ESR spectra of labels I and II in the presence of calf thymus DNA were characteristic of highly immobilized nitroxide radicals with maximum hyperfine splittings (2T_ˌˌ) of 58.7 and 55.5 G, respectively. The melting temperature (T_m) of DNA, determined in the presence of labels I and II by the ESR technique, were closely similar to those obtained by spectrophotometric methods. The ESR spectrum of label I bound to calf liver RNA and yeast RNA indicated that the nitroxide group of this label was highly mobile. These results suggest that spin labels I and II are suitable noncovalent probes for nucleic acids.     

The C nucleotide at the mature 5′ end of the Escherichia coli proline tRNAs is required for the RNase E cleavage specificity at the 3′ terminus as well as functionality

Proline tRNA 3′-maturation in Escherichia coli occurs through a one-step RNase E endonucleolytic cleavage immediately after the CCA determinant. This processing pathway is distinct from the 3′-end maturation of the other tRNAs by avoiding the widespread use of 3′ → 5′ exonucleolytic processing, 3′-polyadenylation and subsequent degradation. Here, we show that the cytosine (C) at the mature 5′-terminus of the proK and proL tRNAs is required for both the RNase E cleavage immediately after the CCA determinant and their functionality. Thus, changing the C nucleotide at the mature 5′-terminus of the proL and proK tRNAs to the more common G nucleotide led to RNase E cleavages 1–4 nucleotides downstream of the CCA determinant. Furthermore, the 5′-modified mutant tRNAs required RNase T and RNase PH for their 3′-maturation and became substrates for polyadenylation and degradation. Strikingly, the aminoacylation of the 5′-modified proline tRNAs was blocked due to the change in the recognition element for prolyl-tRNA-synthetase. An analogous modification of the pheV 5′-mature terminus from G to C nucleotide did not support cell viability. This result provides additional support for the importance of first nucleotide of the mature tRNAs in their processing and functionality.     

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R² = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.     

Effects of oxygen on the metabolism of nitroxide spin labels in cells

The products of the reduction of nitroxides in cells are the corresponding hydroxylamines, which cells can oxidize back to the nitroxides in the presence of oxygen. Both the reduction of nitroxides and the oxidation of hydroxylamines are enzyme-mediated processes. For lipid-soluble nitroxides, the rates of reduction are strongly dependent on the intracellular concentration of oxygen; severely hypoxic cells reduce nitroxides more rapidly than cells supplied with oxygen. In contrast, the rates of oxidation of hydroxylamines increase smoothly with increasing intracellular oxygen concentration up to 150 microM. In order to separate the effects on the rates of metabolism of nitroxides due directly to oxygen from effects due to the redox state of enzymes, we studied the cells under conditions in which each of these variables could be changed independently. Oxygen affects the metabolism of these nitroxides primarily by interacting with cytochrome c oxidase to change the redox state of the enzymes in the respiratory chain. Our results are consistent with the conclusions that in these cells reduction of lipophilic nitroxides occurs at the level of ubiquinone in the respiratory chain in mitochondria, and oxidation of the corresponding hydroxylamines occurs at the level of cytochrome c oxidase.     

Binding of antibodies to nitroxide spin labels and to the corresponding hydroxylamines.

The affinities of rabbit antibodies directed against the spin-label nitroxide group have been found to be of the order of 10⁶ 1/mole for a number of low molecular weight water soluble haptens. It is shown that the same antibodies have almost equal binding affinities to corresponding hydroxylamines.     

Light-directed 5′→3′ synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3′→5′ direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5′→3′ direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5′→3′ direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150 000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R² = 0.998). We have also shown that the 3′ ends of array probes are available for sequence-specific primer extension and ligation reactions.     

A bridged nucleic acid, 2′,4′-BNACOC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2′,4′-BNACOC monomers and RNA-selective nucleic-acid recognition

Recently, we synthesized pyrimidine derivatives of the 2′-O,4′-C-methylenoxymethylene-bridged nucleic-acid (2′,4′-BNA^COC) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNA^COC) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNA^COC consisting of 2′,4′-BNA^COC monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2′,4′-BNA^COC monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNA^COC/BNA^COC duplex possessed excellent thermal stability and that the BNA^COC increased thermal stability with a complementary RNA strand. On the other hand, BNA^COC/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNA^COC generally improved the sequence selectivity with Watson–Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNA^COC formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.     

Aminonucleosides and their derivatives. IVI Synthesis of the 3′-amino-3′-deoxynucleoside 5′-phosphates

A new procedure has been developed for the synthesis of 3′-amino-3′-deoxyribonucleosides of adenine, cytosine and uracil by condensing the trimethylsilylated bases with peracylated 3-azido-3-deoxyribose derivative. The azido group could subsequently be reduced to amino. The 5′-phosphates of these nucleosides have been prepared and the analogues have been tested for their ability to stimulate the ribosome-catalyzed reaction of 3′(2′)-O-(N-formylmethionyl)adenosine 5′-phosphate with phenylalanyl-tRNA.     

Uncoupling of Protein and Ribonucleic Acid Synthesis by 5′,5′,5′-Trifluoroleucine in Salmonella typhimurium

The addition of 5',5',5'-trifluoroleucine (fluoroleucine) to leucine auxotrophs of Salmonella typhimurium permitted protein but not ribonucleic acid (RNA) synthesis to continue after leucine depletion. The uncoupling of the formation of these macromolecules by fluoroleucine was apparent if RNA and protein synthesis was measured either by the uptake of radioactive precursors or by direct chemical determinations. The analogue did not appear to be an inhibitor of RNA formation, since it was as effective as leucine in permitting RNA synthesis in a leucine auxotroph upon the addition of small amounts of chloramphenicol. In contrast to these data, fluoroleucine allowed continued protein and RNA formation in a leucine auxotroph of Escherichia coli strain W. In addition, contrary to the results obtained with S. typhimurium, the analogue replaced leucine for repression of the leucine bio-synthetic enzymes as well as the isoleucine-valine enzymes. We propose that these ambivalent effects of fluoroleucine on repression and RNA and protein synthesis in the two strains are due to differences in the ability of the analogue to attach to the various species of leucine transfer RNA.     

Effect of nucleoside 5′-di- and 5′-tri-phosphates on pancreatic ribonuclease activity

1. ADP, ATP and GDP inhibited the phosphotransferase activity, the release of cyclic nucleotides from RNA, of ribonuclease. No significant inhibition was elicited by pyrimidine 5'-nucleoside diphosphates, CDP and UDP. 2. Inhibition by ADP, AMP, adenosine, adenine, NAD and NADP was insignificant at the concentrations tested. Small inhibition was observed with high concentrations of AMP and only when soluble RNA was the substrate. 3. Inhibition by ADP was found to be ;uncompetitive'. 4. Results seem to indicate that at least for optimum inhibition the polyphosphate of the purine nucleoside is essential. They further suggest that the inhibitor acts by combining with the enzyme only when the enzyme is bound to the substrate.     

5-Bromouridylyl-(3′ → 5′)-Adenosine Isolated from 5-Bromouracil-Induced Filaments of Escherichia coli

A dinucleoside monophosphate was isolated from 5-bromouracil-induced filaments of a thymine auxotroph of Escherichia coli K-12. The dinucleoside monophosphate was fractioned from a [(14)C]5-bromouracil-labeled perchloric acid extract using Dowex-1-formate ion-exchange chromatography. Sephadex chromatography revealed its molecular weight to be 710. Snake venom phosphodiesterase digest of the dinucleoside monophosphate yielded [(14)C]5-bromouridine and adenosine 5'-monophosphate. The presence of [(14)C]5-bromouracil in bacterial ribonucleic acid indicates that ribonucleic acid, which had incorporated 5-bromouracil, was the probable source of this dinucleoside monophosphate, 5-bromouridylyl-(3' --> 5')-adenosine.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Interfering with nucleotide excision by the coronavirus 3′-to-5′ exoribonuclease

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3′-to-5′ proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3′-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3′-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.     

A simple and reliable 5′-RACE approach

A novel approach for the amplification of cDNA ends is described. It requires only minimal amounts of material, a simple cDNA synthesis reaction and a single PCR reaction to amplify the desired 5′- or 3′-ends of a certain cDNA of interest. It combines the so called CapFinder approach with solid phase cDNA synthesis, thus almost eliminating background problems usually associated with 5′-RACE protocols. This approach could be used to generate complete 5′-ends of numerous cDNAs using only one cDNA synthesis reaction. In combination with LA PCR, several kilobases of unknown 5′-ends could be amplified. It is easy to perform, quick, inexpensive and reliable, which should enable it to replace most currently used 5′-RACE protocols.     

Effects of Adenosine 3′,5′-Monophosphate and Adenosine 5′-Monophosphate on Glycogen Degradation and Synthesis in Dictyostelium discoideum

Data are presented demonstrating that the presence in vivo of adenosine 3',5'-monophosphate (3',5'-AMP) causes a rapid depletion of glycogen storage material in the cellular slime mold. The effect of adenosine 5'-monophosphate (5'-AMP) is twofold, stimulating both glycogen degradation and synthesis. In pseudoplasmodia, cell-free extracts appear to contain at least two species of glycogen phosphorylase, one of which is severely inhibited by glucose-1-phosphate and another which is only partially inhibited by this hexose-phosphate. In some cases, 5'-AMP partially overcomes the inhibition by glucose-1-phosphate. Data presented here also indicate the existence of two forms of glycogen synthetase, the total activity of which does not change during 10 hr of differentiation from aggregation to culmination. During this period there is a quantitative conversion of glucose-6-phosphate-independent enzyme activity to glucose-6-phosphate-dependent activity. It is suggested that one effect of 3',5'-AMP is closely related to enzymatic processes involved in the rapid conversion of glycogen to cell wall material and other end products accumulating during sorocarp construction.     

Efficient methods for attaching non-radioactive labels to the 5'' ends of synthetic oligodeoxyribonucleotides.

The syntheses are described of two types of linker molecule useful for the specific attachment of non-radioactive labels such as biotin and fluorophores to the 5' terminus of synthetic oligodeoxyribonucleotides. The linkers are designed such that they can be coupled to the oligonucleotide as a final step in solid-phase synthesis using commercial DNA synthesis machines. Increased sensitivity of biotin detection was possible using an anti-biotin hybridoma/peroxidase detection system.     

3′,5′-Cyclic Adenosine Monophosphate-Requiring Mutants of Escherichia coli

Mutants that require exogenous 3',5'-cyclic adenosine monophosphate (cAMP) for exponential growth were isolated from strains deficient in adenyl cyclase. Studies of one strain showed that cAMP is not incorporated into macromolecules; instead, it seems to have a regulatory function, i.e., in media lacking cAMP, cells form ribonucleic acid (RNA) and protein at linear rather than exponential rates. The exact lesion is not known; ribosomes, messenger RNA, and the beta and beta' subunits of RNA polymerase continue to be made in absence of added cAMP.     

Cyclic 3′,5′-Adenosine Monophosphate Phosphodiesterase of Escherichia coli

The cyclic 3',5'-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia coli has been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier.