Use of a Base-Labile Protected Derivative of 6-Mercaptohexanol for the Preparation of Oligonucleotides Containing a Thiol Group at the 5′-End

Abstract

The preparation of a base-labile (Dnpe) protected derivative of 6-mercaptohexanol is described. The use of the phosphoramidite derivative of this compound improves both yields and the time needed for the preparation of oligonucleotides containing a thiol group at the 5′-end.     

A Rapid Solid Phase Method for the Synthesis of 3′-Thiol Group Containing Oligonucleotides

Abstract

A rapid solid phase method for the synthesis of 3′-thiol group containing oligonucleotides is described.     

An RNA Element at the 5′-End of the Poliovirus Genome Functions as a General Promoter for RNA Synthesis

RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5′- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5′-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within “stem a” that are essential for the initiation of positive-strand RNA synthesis. These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.     

Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.Dengue virus (DENV), a member of the Flaviviridae family, is a human pathogen causing dengue fever, the most common mosquito-borne viral disease in humans. The virus has become a major international public health concern, with 3 billion people at risk for infection and an estimated 50 million dengue cases worldwide every year (28). Neither specific antiviral therapies nor licensed vaccines are available, and the biology of the virus is poorly understood.DENV is a small enveloped virus containing a positive-stranded RNA genome with a length of approximately 10.7 kb. The virus encodes one large polyprotein that is co- and posttranslationally cleaved into 10 viral proteins. The structural proteins C, prM/M, and E are located in the N terminus, followed by the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (6, 10). NS5, the largest of the viral proteins, functions as an RNA-dependent RNA polymerase (RdRP) (29). The coding region is flanked at both ends by untranslated regions (UTR). The 5′ end has a type I cap structure (m⁷GpppAmp) mediating cap-dependent translation, but the virus can switch to a noncanonical translation mechanism under conditions in which translation factors are limiting (13). Cellular mRNAs are known to circularize via a protein-protein bridge between eIF4G and eIF4E (the cap binding complex) at the 5′ end and the poly(A) binding protein (PABP) at the 3′ end, enhancing translation efficiency. Despite the fact that the DENV 3′ UTR lacks a poly(A) tail, recent findings demonstrated binding of PABP to the 3′ UTR and an effect on RNA translation, suggesting a similar mechanism (12, 26).In addition to a presumed protein-mediated genome circularization regulating viral translation, an RNA-RNA-based 5′ and 3′ (5′-3′) end interaction, which can occur in the absence of proteins, leads to circularization of the viral genome (1, 3, 4, 18, 20, 30, 33, 34). This cyclization of the genome is necessary for viral RNA replication, and thus far, two complementary sequences at the 5′ and 3′ ends have been identified (3). The first are the cyclization sequences (CS) present in the capsid-coding region at the 5′ end (5′ CS) and upstream of the 3′ stem-loop (3′ SL) in the 3′ UTR (3′ CS) (2, 4, 18, 20, 30). A second sequence, known as the 5′ upstream AUG region (5′ UAR) element in the 5′ UTR, base pairs with its complementary 3′ UAR counterpart, which is located at the bottom part of 3′ SL (1, 4, 30). Recently, the structure of the 5′ end of the DENV genome hybridized to the 3′ end was determined in solution (25), confirming previous computer-predicted structures for genome cyclization (4, 20, 30). Besides the base pairing between 5′-3′ UAR and 5′-3′ CS sequences, a third stretch of nucleotides was identified to form a double-stranded (ds) region between the 5′ and 3′ ends.In addition to RNA sequences involved in 5′-3′-end interactions that are necessary for cyclization, the 5′ end of the viral genome harbors at least two more functional RNA elements, the stem-loop A (SLA) and capsid-coding region hairpin (cHP). The SLA consists of the first 70 nucleotides (nt) of the genome, forming a stable stem-loop structure. This structure has been confirmed in several studies and identified as a promoter element for RNA synthesis that recruits the viral RdRp NS5 (16, 22). Once NS5 is bound to the SLA at the 5′ end, it is believed to be delivered to the initiation site of minus-strand RNA synthesis at the 3′ end via 5′-3′ RNA-RNA circularization. In addition, a short poly(U) tract located immediately downstream of SLA has been shown to be necessary for RNA synthesis, although it is not involved in genome circularization (22). Finally, the cHP element resides within the capsid-coding region; it directs start codon selection and is essential for RNA replication (8, 9). The cHP structure is more important than its primary sequence. For start codon selection, it is believed that the cHP stalls the scanning initiation complex over the first AUG, favoring its recognition (9). In the case of RNA replication, the cHP likely stabilizes the overall 5′-3′ panhandle structure or participates in recruitment of factors associated with the replicase machinery (8).In this study, we demonstrate that in addition to the 5′ CS and 5′ UAR sequences, a third stretch of nucleotides in the 5′ end is required for RNA replication and appears to be involved in genome circularization. This new motif is located downstream of the AUG and was therefore designated the downstream AUG region (5′ DAR) element, with the predicted counterpart in the 3′ end designated the 3′ DAR. Our results indicate that the 5′ DAR modulates RNA-RNA interaction and RNA replication, and restoring complementarity between the 5′ DAR and 3′ DAR rescues detrimental effects caused by mutations in the 5′ DAR on genome circularization and RNA replication. Although the role of the predicted 3′ DAR counterpart is less conclusive, it may serve to make other structures and sequences in the 3′ end available for 5′-3′ RNA-RNA interaction to facilitate the replication-competent conformation of the DENV genome.Furthermore, we analyzed the functional interplay of RNA elements in the viral 5′ end, showing that two separate units are formed during replication. The first consists of the SLA, and it must be located at the very 5′ end of the genome. The second unit includes UAR, DAR, cHP, and CS elements, and the positional requirements are more flexible within the DENV RNA 5′ terminus. However, all four elements in the second unit must act in concert, forming a functional tertiary RNA structure of the circularized viral genome.     

Hydrolytic Reactions of an RNA Dinucleotide Containing a 3′-S-Phosphorothiolate Linkage

Abstract

The pH-rate profiles (pH 0.2 to 9 at 90°C) for the competing hydrolytic reactions of (3′-deoxy-3′-thioinosylyl)-3′,5′-uridine (IspU) have been determined by an HPLC method.     

Solid Phase Conjugation Chemistry: Use of Alloc as a Protecting Group for 2′-Aminolinker Containing Oligonucleotides

Abstract

The drug properties of antisense and antigene oligonucleotides can he enhanced by strategic positioning of ligands capable of ameliorating these properties.^1,2 Certain ligands may improve the cellular delivery of oligomers and increase their affinity for the target gene and resistance to nucleases. The 2′-O-position is an attractive modification site.³ Oligonucleotides possessing the 2′-O-akyl modifications exhibit higher chemical stability under depurination conditions, higher stability to enzymatic cleavage by both endo- and exonucleases, and increased affinity for target mRNA. In addition, they form highly stable triple helices. Thus they promise to be versatile compounds in controlling gene expression by antisense and antigene technologies.     

Genetics and Physiology of Bacteriophage T4 3′-Phosphatase: Evidence for Involvement of the Enzyme in T4 DNA Metabolism

Mutants of bacteriophage T4D which fail to induce the deoxyribonucleotide-specific T4 3'-phosphatase have been isolated. These mutants (T4pseT) grow as well as wild-type T4 in most strains of Escherichia coli, but not in the T4-sensitive "Hospital Strain," CT196, or in a derivative strain, CTr5x. Both the formation of infectious centers and the final yield of phage are reduced by 98% when CTr5x is infected by T4pseT mutants. The growth defects are accompanied by a 50% reduction in the rate of T4 DNA synthesis, a decrease in the single-strand length of the DNA product to about one-half the mature length, and greatly reduced packaging of DNA into phage particles. Introduction of an extra-cistronic suppressor mutation (stp) into T4pseT eliminates both the requirement for the T4 3'-phosphatase in infected CTr5x and the other observed effects of the pseT mutations. The pseT gene lies between genes 63 and 31. The stp gene lies in the nonessential region between rIIB and ac. Our results suggest that 3'-phosphoryl termini can disrupt T4 DNA replication to the extent that T4 3'-phosphatase becomes required for phage production.     

Characteristics of the Deoxyribonucleic Acid of Tφ3, a Bacteriophage for Bacillus stearothermophilus

It is shown that the individual strands of bacteriophage Tphi3 DNA are intact and that heat-denatured Tphi3 DNA forms a bimodal distribution in a neutral CsCl density gradient.     

Characteristics of Tφ3, a Bacteriophage for Bacillus stearothermophilus

A bacteriophage (Tphi3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60 C, the optimal growth temperature of the host. At 60 C, the phage had a latent period of 18 min and a burst size of about 200. The phage was comparatively thermostable in broth. The halflife of Tphi3 was 400 min at 60 C, 120 min at 65 C, 40 min at 70 C, and 12 min at 75 C. The activation energy for the heat inactivation of Tphi3 was 56,000 cal. The buoyant density of Tphi3 in a cesium chloride density gradient was 1.526 g/ml. Electron micrographs of Tphi3 indicate that the phage has a head that is 57 mmu long. The dimensions and shape of the head are compatible with those of a regular icosahedron. Each edge of the head is 29 mmu long. The tail of Tphi3 is 125 mmu long and 10 mmu wide. There are about 30 cross-striations that are spaced at 3.9-mmu intervals along the tail. Under the conditions investigated, Tphi3 adsorbed slowly to the host. Only 2.8% of the phage adsorbed in 10 min at 60 C, the normal incubation temperature that was used. Tphi3 was not infective to four other thermophilic strains or to two mesophilic strains of bacteria.     

Simple methods for the 3′ biotinylation of RNA

Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3′ ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3′-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3′ ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of ϕ29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme''s proofreading activity, can cope with more extended 3′ heterogeneities.     

2′,5′-Phosphodiesterase Activity Depends Upon the Presence of a 3-Hydroxyl Moiety in the Penultimate Position of the Oligonucleotide Substrate

Abstract

3′-Deoxyadenosine-substituted analogs of 2–5A core 5′-mono-phosphate were examined for their degradation by the 2′-phosphodiesterase of mouse cells, leading to the conclusion that the 2′,5′-phosphodiesterase requires the presence of 3′-hydroxyl moiety in the penultimate nucleotide     

The Structure of Bacteriophage φCb5 Reveals a Role of the RNA Genome and Metal Ions in Particle Stability and Assembly

The structure of the Leviviridae bacteriophage φCb5 virus-like particle has been determined at 2.9 Å resolution and the structure of the native bacteriophage φCb5 at 3.6 Å. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the φCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, φCb5 particles are destabilized in solution with elevated salt concentration. The model of the φCb5 capsid provides an explanation of the salt-induced destabilization of φCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions.Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the φCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The φCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.     

The 9-Fluorenylmethyloxycarbonyl (Fmoc) Group as a 5′-O Base Labile Protecting Group in Solid Supported Oligonucleotide Synthesis

Abstract

The title strategy is discussed and applied to the synthesis of a short oligomer.     

Determination of the Group Electronegativity of CF3 Group in 3′-O-CF3-Thymidine by 1-NMR

Abstract

The interplay of enthalpy of the gauche effect (ΔH°_GE) of the [X3′-C3′-C4′-O4′] fragment in various 3′-substituted (X) 2′,3′-dideoxythymidine derivatives 1–7 and the inherent anomeric effect drives the two-state North ? South equilibrium in the constituent sugar moiety. The group electronegativity of 3′-OCF₃ substituent in Marriott's, Inamoto's and Mullay's scales has been determined from simple calibration graphs correlating the group electronegativity of various 3′-substituents (X) in 2′,3′-dideoxythymidine derivatives 1–7 with the experimental strength (ΔH°_GE) of the [X3′-C3′-C4′-O4′] gauche effect. ΔH°_GE has been experimentally determined from pseudorotational analyses of temperature-dependent ³J_HH coupling constants, and can be used as an unambiguous tool for direct experimental estimation of the group electronegativity of a specific substituent covalently attached to 3′-carbon of 2′,3′-dideoxythymidine, which can be compared, in turn, with the theoretical estimation carried out according to Marriott's or Inamoto's procedure. Inconsistency found between theoretical values in Marriott's and Inamoto's scales, on the one hand, and between our experimental estimate and the theoretical value in Marriott's scale, on the other, have been solved by refining the electronegativity scale using our experimental data for 1–7.     

Substrate Properties of Adenosine- and Uridine- 3′, 5′-bisphenylphosphonates for 3′-Nucleotidase/Nucleases

Abstract

3′, 5′-Bisphenylphosphonate and 5′-phenylphosphonate esters of adenosine and uridine were synthesized to investigate the substrate properties of the 3′, 5′-bisphenylphosphonates for 3′-nucleotidase/nucleases. The V _max/apparent K _m, values of the enzymes for them were found to be 9 to 21-fold higher than those for the corresponding nucleoside 3′-phenylphosphonates.     

The 5′-Purine-Pyrimidine-3′ Stacks are More Stabilizing in a Self-3′-Pyrinudine-Purine-5′ complementary DNA Duplex than the 5′-Purine-Purine-3′ 3′-Pyrimidine-Pyrimidine-5′ Stack

Abstract

First experimental evidence is herein reported supporting the earlier quantum chemical calculations that 5′-Punne-pyrimiidine-3′ 3′ -Pyrimidine-Punne-5 stack is more stable than 5′-Pyrimiidine-Punne-3′ 3′-Punne-Pyrimidine-5′.     

Synthesis of Guanylyl (3′→5′)-5-methylcytidine (Gpm5C), a Dinucleoside Monophosphate which is Unable to Function as a Primer in the Synthesis of RNA by the Influenza A Virus RNA Polymerase

Abstract

Guanylyl (3′→5′)-5-methylcytidine (Gpm⁵C) has been synthesized enzymatically through the use of T₁RNAse at high enzyme dilution. In contrast with GpC, the methylated dinucleoside monophosphate is shown to be inactive as a primer for RNA synthesis by the RNA-dependent RNA polymerase of Influenza A virus.