Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem–loop structured probes

Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem–loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.     

Loop-mediated isothermal amplification of DNA

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10⁹ copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.     

DNA microarrays with stem–loop DNA probes: preparation and applications

We have developed DNA microarrays containing stem–loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem–loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10–30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3′-deletion sets from a target and evaluated the use of stem–loop DNA arrays for detecting p53 mutations in the deletion set. The stem–loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.     

RNA recognition by the DNA end-binding Ku heterodimer

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem–loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem–loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.     

Binding of human SLBP on the 3'-UTR of histone precursor H4-12 mRNA induces structural rearrangements that enable U7 snRNA anchoring

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3′ end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem–loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem–loop binding protein (SLBP), which binds to the stem–loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3′-untranslated region (3′-UTR) of mouse histone pre-mRNAs H4–12, H1t and H2a–614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3′-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4–12 pre-mRNAs 3′ end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.     

Discriminating duplex and hairpin oligonucleotides using chemical shifts: application to the anticodon stem–loop of Escherichia coli tRNAPhe

A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G·U wobble base pair formed between a ¹⁵N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D ¹⁵N–¹H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin–duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of ~20 µM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem–loop of Escherichia coli tRNA^Phe and suggests that this hairpin has a 3 nt loop.     

RNA stem–loop enhanced expression of previously non-expressible genes

The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem–loop (stem length, 7 bp; ΔG₀ = –9.9 kcal/mol) in front of various gene sequences. In each case, the stem–loop was inserted 15 nt downstream from the start codon. Insertion of the stem–loop allowed in vitro expression of five previously non-expressible genes and enhanced the expression of all other genes investigated. Analysis of the RNA structure proved that the stem–loop was formed in vitro, and demonstrated that stabilization of the ribosome binding site is due to stem–loop introduction. By theoretical RNA structure analysis we showed that the inserted RNA stem–loop suppresses long-range interactions between the translation initiation domain and gene-specific mRNA sequences. Thus the inserted RNA stem–loop supports the formation of a separate translational initiation domain, which is more accessible to ribosome binding.     

Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem-loop structured probes

Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem-loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.     

The role of phosphate groups in the VS ribozyme-substrate interaction

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme–substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem–loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.     

Properties and Anti-HIV Activity of Nicked Dumbbell Oligonucleotides

Abstract

We have designed a new type of oligodeoxyribonucleotide. These oligodeoxyribonucleotides form two hairpin loop structures with base pairs (sense and antisense) in the double helical stem at the 3′ and 5′-ends (nicked dumbbell oligonucleotides). The nicked dumbbell oligonucleotides are molecules with free ends that are more resistant to exonuclease attack. Furthermore, the nicked dumbbell oligonucleotide containing phosphorothioate (P=S) bonds in the hairpin loops has increased nuclease resistance, as compared to the unmodified nicked oligonucleotide. The binding of the nicked dumbbell oligonucleotide to RNA is lower than that of a single-stranded DNA. We also describe the anti-HIV activity of nicked dumbbell oligonucleotides.

     

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.     

Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA

The synthesis of a 5′-O-BzH–2′-O-ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G₁₉₀₆–C₁₉₂₄) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudouridine at the stem–loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.     

Molecular dynamics reveals the stabilizing role of loop closing residues in kissing interactions: comparison between TAR-TAR* and TAR-aptamer

A RNA aptamer (R06) raised against the trans- activation responsive (TAR) element of HIV-1 was previously shown to generate a loop–loop complex whose stability is strongly dependent on the selected G and A residues closing the aptamer loop. The rationally designed TAR* RNA hairpin with a loop sequence fully complementary to the TAR element, closed by U,A residues, also engages in a loop–loop association with TAR, but with a lower stability compared with the TAR–R06 complex. UV absorption monitored thermal denaturation showed that TAR–TAR*(GA), in which the U,A kissing residues were exchanged for G,A, is as stable as the selected TAR–R06 complex. Consequently, we used the TAR–TAR* structure deduced from NMR studies to model the TAR–R06 complex with either GA, CA or UA loop closing residues. The results of the molecular dynamics trajectories correlate well with the thermal denaturation experiments and show that the increased stability of the GA variant results from an optimized stacking of the bases at the stem–loop junction and from stable interbackbone hydrogen bonds.     

Modifications of the 3'-UTR stem-loop of infectious bursal disease virus are allowed without influencing replication or virulence

Many questions regarding the initiation of replication and translation of the segmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) remain to be solved. Computer analysis shows that the non-polyadenylated extreme 3′-untranslated regions (UTRs) of the coding strand of both genomic segments are able to fold into a single stem–loop structure. To assess the determinants for a functional 3′-UTR, we mutagenized the 3′-UTR stem–loop structure of the B-segment. Rescue of infectious virus from mutagenized cDNA plasmids was impaired in all cases. However, after one passage, the replication kinetics of these viruses were restored. Sequence analysis revealed that additional mutations had been acquired in most of the stem–loop structures, which compensated the introduced ones. A rescued virus with a modified stem–loop structure containing four nucleotide substitutions, but preserving its overall secondary structure, was phenotypically indistinguishable from wild-type virus, both in vitro (cell culture) and in vivo (chickens, natural host). Sequence analysis showed that the modified stem–loop structure of this virus was fully preserved after four serial passages. Apparently, it is the stem–loop structure and not the primary sequence that is the functional determinant in the 3′-UTRs of IBDV.     

Red light-activated phosphorothioate oligodeoxyribonucleotides

Hairpin-structured phosphorothioate oligodeoxyribonucleotides containing a singlet oxygen-sensitive linker in the loop were prepared. These compounds do not bind complementary nucleic acids in the dark. Upon irradiation with red light in the presence of chlorine e6 the linker within these compounds is cleaved and a single-stranded oligodeoxyribonucleotide is produced. The latter compound is an efficient binder of complementary nucleic acids. This is the first example of ‘caged’ phosphorothioate oligodeoxyribonucleotides, whose nucleic acid binding ability is triggered by red light.     

The 5' stem-loop regulates expression of collagen alpha1(I) mRNA in mouse fibroblasts cultured in a three-dimensional matrix

The stability of collagen α1(I) mRNA is regulated by its 5′ stem–loop, which binds a cytoplasmic protein in a cap-dependent manner, and its 3′-untranslated region (UTR), which binds αCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen α1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse–human collagen α1(I) mRNA with a wild-type 5′ stem–loop decayed faster than the same mRNA with a mutated stem–loop. When the 5′ stem–loop was placed in a heterologous mRNA, the mRNA accumulated to a lower level in cells grown in gel than in cells grown on plastic. This suggests that the 5′ stem–loop down-regulates collagen α1(I) mRNA. Protein binding to the 5′ stem–loop was reduced in cells grown in gel, which was associated with destabilization of the collagen α1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen α1(I) 5′ stem–loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen α1(I) mRNA. Binding of αCP, a protein involved in stabilization of collagen α1(I) mRNA, was unchanged by the culture conditions.     

寡聚脱氧核苷酸的结构与抗降解特性的研究

合成了４段具有不同高级结构或不同修饰的寡聚脱氧核苷酸,检查它们在２０％血清中的稳定性．发现：（１）寡核苷酸主要被血清中的３′外切核酸酶降解,未经修饰的线性寡核苷酸降解严重;（２）末端部分硫代修饰的寡核苷酸稳定性明显提高;（３）自身互补形成的配对结构可有效保护３′末端．具有４个以上（含４个）ＧＣ对的３′端发夹结构寡核苷酸,其抗核酸酶的能力几乎与硫代修饰的寡核苷酸相当．     

The C-terminal extension of Lsm4 interacts directly with the 3′ end of the histone mRNP and is required for efficient histone mRNA degradation

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem–loop sequence, which is bound to the stem–loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3′ end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3′hExo, forms a ternary complex with SLBP and the stem–loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3′hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3′hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.