Tethered naphthalene diimide-based intercalators for DNA triplex stabilization

The synthesis and triplex stabilizing properties of oligodeoxyribonucleotides functionalized at the 5′- and/or 3′-termini with a naphthalene diimide-based (NDI) intercalator is described. The NDI intercalator was prepared in a single step from the corresponding dianhydride and was attached to the 5′-terminus of an oligodeoxyribonucleotide following a reverse coupling procedure. The DMT protecting group was removed and the sequence phosphitylated to generate the phosphoramidite derivative on the 5′-terminus of the support-bound oligodeoxyribonucleotide. The NDI intercalator with a free hydroxyl was then added in the presence of tetrazole. Attachment of the NDI to the 3′-terminus relied upon a tethered amino group that could be functionalized first with the naphthalene dianhydride, which was subsequently converted to the diimide. Using both procedures, an oligonucleotide conjugate was prepared having the NDI intercalator at both the 5′- and 3′-termini. Thermal denaturation studies were used to determine the remarkable gain in stability for triplexes formed when the NDI-conjugated oligonucleotide was present as the third strand in the complex.     

Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA

At the ends of bacteriophage λ DNA, the 5′-terminated strands are 12 nucleotides longer than the 3′-terminated strands. The complete sequence of deoxynucleotides in both the protruding 5′-terminated single strands of λ DNA has been determined by partial repair and by complete repair followed by sequencing of isolated oligonucleotides. Starting from the 5′-end of the left-hand cohesive end, the 12 nucleotides are in the sequence dpGpGpGpCpGpGpCpGpApCpCpT. The sequence from the right-hand cohesive end is exactly complementary to that from the left-hand end.     

Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E.coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3′- or 5′-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5′-terminus of the 3′ cleaved fragment, but is unable to remove DHU remaining on the 3′-terminus of the cleaved 5′ fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3′-terminus of a cleaved 5′ fragment, but are unable to remove DHU remaining on the 5′-terminus of a cleaved 3′ fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.     

RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour

We have probed the effects of altering buffer conditions on the behaviour of two aptamer RNAs for the bacteriophage MS2 coat protein using site-specific substitution of 2′-deoxy-2-aminopurine nucleotides at key adenosine positions. These have been compared to the wild-type operator stem–loop oligonucleotide, which is the natural target for the coat protein. The fluorescence emission spectra show a position and oligonucleotide sequence dependence which appears to reflect local conformational changes. These are largely similar between the differing oligonucleotides and deviations can be explained by the individual features of each sequence. Recognition by coat protein is enhanced, unaffected or decreased depending on the site of substitution, consistent with the known protein–RNA contacts seen in crystal structures of the complexes. These data suggest that the detailed conformational dynamics of aptamers and wild-type RNA ligands for the same protein target are remarkably similar.     

The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

The influence of locked nucleic acid (LNA) residues on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes is reported. Optical melting studies indicate that LNA incorporated into an otherwise 2′-O-methyl RNA oligonucleotide usually, but not always, enhances the stabilities of complementary duplexes formed with RNA. Several trends are apparent, including: (i) a 3′ terminal U LNA and 5′ terminal LNAs are less stabilizing than interior and other 3′ terminal LNAs; (ii) most of the stability enhancement is achieved when LNA nucleotides are separated by at least one 2′-O-methyl nucleotide; and (iii) the effects of LNA substitutions are approximately additive when the LNA nucleotides are separated by at least one 2′-O-methyl nucleotide. An equation is proposed to approximate the stabilities of complementary duplexes formed with RNA when at least one 2′-O-methyl nucleotide separates LNA nucleotides. The sequence dependence of 2′-O-methyl RNA/RNA duplexes appears to be similar to that of RNA/RNA duplexes, and preliminary nearest-neighbor free energy increments at 37°C are presented for 2′-O-methyl RNA/RNA duplexes. Internal mismatches with LNA nucleotides significantly destabilize duplexes with RNA.     

PCR hot start using primers with the structure of molecular beacons (hairpin-like structure)

A new technique of PCR hot start using oligonucleotide primers with a stem–loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3′-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3′-end were added to the 5′-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem–loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.     

Sequence analysis of a PM2-DNA anti-Z-IgG-binding region

An anti-Z-antibody-binding region between PM2-DNA map units 0.05 and 0.18, containing approx. 25% of the bound PM2 antibody molecules (1,2) has been sequenced. Analysis of this PM2 DNA sequence from map units 0.00 to 0.175 demonstrates that alternating purine/pyrimidine tracts capable of adopting the left-handed conformation are present within this antibody-binding region. Longer (GC)n-rich tracts are clustered together and comprise seven alternating purine/pyrimidine-rich areas (48%–84%) ranging from 19 to 142 nucleotides in length. The DNA located between these alternating purine/pyrimidine-rich areas exhibit a low level (0%–19%) of this sequence arrangement. There is a very strong correlation between the alternating purine/pyrimidine-rich areas and the anti-Z-DNA-IgG-binding sites. Nucleotides 1461–1583 of the PM2-DNA genome encode the bacteriophage capsid protein IV. One of the PM2 left-handed sites is located within this protein-coding sequence; a B-to-Z transition within this site may be involved in protein-IV gene regulation in vivo.     

Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements

The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95°C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5′ end adjacent to the single-strand–duplex junction. Deletion of the free 3′ end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3′ end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5′ end failed to block cleavage, although this substrate was refractory to cleavage by the 5′-3′ exonuclease activity of Taq DNA polymerase. For verification, the free 5′ end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5′ single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion X174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.     

RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

Here, we show that Escherichia coli Ribonuclease III cleaves specifically the RNA genome of hepatitis C virus (HCV) within the first 570 nt with similar efficiency within two sequences which are ~400 bases apart in the linear HCV map. Demonstrations include determination of the specificity of the cleavage sites at positions C²⁷ and U³³ in the first (5′) motif and G⁴³⁹ in the second (3′) motif, complete competition inhibition of 5′ and 3′ HCV RNA cleavages by added double-stranded RNA in a 1:6 to 1:8 weight ratio, respectively, 50% reverse competition inhibition of the RNase III T7 R1.1 mRNA substrate cleavage by HCV RNA at 1:1 molar ratio, and determination of the 5′ phosphate and 3′ hydroxyl end groups of the newly generated termini after cleavage. By comparing the activity and specificity of the commercial RNase III enzyme, used in this study, with the natural E.coli RNase III enzyme, on the natural bacteriophage T7 R1.1 mRNA substrate, we demonstrated that the HCV cuts fall into the category of specific, secondary RNase III cleavages. This reaction identifies regions of unusual RNA structure, and we further showed that blocking or deletion of one of the two RNase III-sensitive sequence motifs impeded cleavage at the other, providing direct evidence that both sequence motifs, besides being far apart in the linear RNA sequence, occur in a single RNA structural motif, which encloses the HCV internal ribosome entry site in a large RNA loop.     

2′-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF

Synthetic oligonucleotides are used to regulate gene expression through different mechanisms. Chemical modifications of the backbone of the nucleic acid and/or of the 2′ moiety of the ribose can increase nuclease stability and/or binding affinity of oligonucleotides to target molecules. Here we report that transfection of 2′-F-modified phosphorothioate oligonucleotides into cells can reduce the levels of P54nrb and PSF proteins through proteasome-mediated degradation. Such deleterious effects of 2′-F-modified oligonucleotides were observed in different cell types from different species, and were independent of oligonucleotide sequence, positions of the 2′-F-modified nucleotides in the oligonucleotides, method of delivery or mechanism of action of the oligonucleotides. Four 2′-F-modified nucleotides were sufficient to cause the protein reduction. P54nrb and PSF belong to Drosophila behavior/human splicing (DBHS) family. The third member of the family, PSPC1, was also reduced by the 2′-F-modified oligonucleotides. Preferential association of 2′-F-modified oligonucleotides with P54nrb was observed, which is partially responsible for the protein reduction. Consistent with the role of DBHS proteins in double-strand DNA break (DSB) repair, elevated DSBs were observed in cells treated with 2′-F-modified oligonucleotides, which contributed to severe impairment in cell proliferation. These results suggest that oligonucleotides with 2′-F modifications can cause non-specific loss of cellular protein(s).     

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.     

High-Mobility Group 1/2 Proteins Are Essential for Initiating Rolling-Circle-Type DNA Replication at a Parvovirus Hairpin Origin

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5′ end of the DNA at the nick and providing a 3′ hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5′) MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3′) viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.     

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5′–3′-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5′-overhang. A 3′-single-stranded tail arising from the same end of the duplex as the 5′-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5′-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5′-terminal overhangs as well as to remove nucleotides from the 5′-termini enables it to effectively process the 5′-termini of Okazaki fragments before they are ligated.     

Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP?Mg2+ and 5′-OH oligonucleotide and a product complex with GDP?Mg2+ and 5′-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5′ end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5′-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg²⁺ and a 5′-OH oligonucleotide and a product complex with GDP•Mg²⁺ and a 5′-PO₄ oligonucleotide. The O5′ nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5′-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38—hereby implicated as the essential general base catalyst that abstracts a proton from the 5′-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the ‘reverse kinase’ reaction by donating a proton to the O5′ leaving group of the 5′-PO₄ strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.     

Identification of Specific Nucleotide Sequences within the Conserved 3′-SL in the Dengue Type 2 Virus Genome Required for Replication

The flavivirus genome is a positive-stranded ~11-kb RNA including 5′ and 3′ noncoding regions (NCR) of approximately 100 and 400 to 600 nucleotides (nt), respectively. The 3′ NCR contains adjacent, thermodynamically stable, conserved short and long stem-and-loop structures (the 3′-SL), formed by the 3′-terminal ~100 nt. The nucleotide sequences within the 3′-SL are not well conserved among species. We examined the requirement for the 3′-SL in the context of dengue virus type 2 (DEN2) replication by mutagenesis of an infectious cDNA copy of a DEN2 genome. Genomic full-length RNA was transcribed in vitro and used to transfect monkey kidney cells. A substitution mutation, in which the 3′-terminal 93 nt constituting the wild-type (wt) DEN2 3′-SL sequence were replaced by the 96-nt sequence of the West Nile virus (WN) 3′-SL, was sublethal for virus replication. An analysis of the growth phenotypes of additional mutant viruses derived from RNAs containing DEN2-WN chimeric 3′-SL structures suggested that the wt DEN2 nucleotide sequence forming the bottom half of the long stem and loop in the 3′-SL was required for viability. One 7-bp substitution mutation in this domain resulted in a mutant virus that grew well in monkey kidney cells but was severely restricted in cultured mosquito cells. In contrast, transpositions of and/or substitutions in the wt DEN2 nucleotide sequence in the top half of the long stem and in the short stem and loop were relatively well tolerated, provided the stem-loop secondary structure was conserved.     

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5′-ends and an amino group at their 3′-ends in good yield (up to 72 OD units/µmol for a 19mer phosphorothioate). Syntheses of 3′-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2′-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3′-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5′-terminal S-acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3′-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligonucleotide; the 5′-dithiopyridyl group was then quantitatively reduced to give a free thiol group which was then substituted by reaction with an Nα-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide–oligonucleotide conjugate.     

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.