Mechanism of oligonucleotide hybridization with the 3′-terminal region of the yeast tRNAPhe

Interaction of yeast phenylalanine tRNA with oligonucleotides complementary to its 3′-terminal nucleotide sequence was thoroughly studied. Using the gel retardation technique, thermodynamic and kinetic parameters of the tRNA complexation in physiological conditions were determined. Analysis of dependences of the complex formation on the oligonucleotide concentration and incubation time showed that this process proceeds in two stages. At the first stage, a metastable complex of the oligonucleotide with the open, single-stranded sequence ACCA at the 3′ end of tRNA rapidly forms. The second stage involves a slow intramolecular rearrangement of the resulting metastable complex into a full-sized heteroduplex accompanied by the tRNA^Phe unfolding. The data gained suggest that the RNA unfolding stage is limiting in the interaction of oligonucleotides with natural RNAs. Principles of selection of optimal hybridization probes and antisense oligonucleotides are discussed.     

Study of the Mechanism of Interaction of Oligonucleotides with the 3′-Terminal Region of tRNA<Superscript>Phe</Superscript> by Computer Modeling

Three-dimensional atomic models of complexes between yeast tRNA^Phe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNA^Phe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNA^Phe destruction and formation of the final tRNA^Phe-oligonucleotide complementary complex. The primary complex formation and the following tRNA^Phe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element.     

Invasion of Strongly Binding Oligonucleotides into tRNA Structure

Abstract

Interaction of yeast tRNA^Phe with oligodeoxyribonucleotides containing 5-methylcytosine, 2-aminoadenine, and 5-propynyl-2′-deoxyuridine was investigated. The modified oligonucleotides show increased binding capacity although the association rates are similar for the modified and natural oligonucleotides. The most pronounced increase in association constant (70 times) due to the incorporation of the strongly binding units was achieved in the case of oligonucleotide complementary to the sequence 65–76 of the tRNA^phe.     

Nuclear magnetic resonance studies of codon-anticodon interaction in tRNAPhe. I. Effect of binding complementary tetra and pentanucleotides to the anticodon

The effect of codon-anticodon interaction on the structure of two tRNA^Phe species was investigated by means of nuclear magnetic resonance spectroscopy. To this end n.m.r.² spectra of yeast and Escherichia coli tRNA^Phe were recorded in the absence and the presence of the oligonucleotides U-U-C-A, U-U-C-G and U-U-C-A-G, which all contain the sequence UUC complementary to the anticodon sequence GAA. The spectra of the hydrogen-bonded protons, the methyl protons and the internucleotide phosphorous nuclei served to monitor the structure of the anticodon loop and of the tRNA in the tRNA-oligonucleotide complex. From the changes in the methyl proton spectra and in the phosphorous spectra it could be concluded that the oligonucleotides bind to the anticodon. Moreover it turned out that the binding constants obtained from these n.m.r. experiments were, within experimental error, equal to the values obtained with other techniques. Using the resonances of the protons hydrogen-bonded between the oligonucleotide and the anticodon loop the structure of the latter could be studied. In particular, binding of the pentanucleotide U-U-C-A-G, which is complementary to the five bases on the 5′ side of the anticodon loop, resulted in the resolution of four to five extra proton resonances indicating that four to five base-pairs are formed between the pentanucleotide and the anticodon loop. The formation of five base-pairs was confirmed by an independent fluorescence binding study. The resonance positions of the hydrogen-bonded protons indicate, that an RNA double helix is formed by the anticodon loop and U-U-C-A-G with the five base-pairs forming a continuous stack. This structure can be accomodated in the so-called 5′ stacked conformation of the anticodon loop, a structure that has been suggested earlier as an alternative to the familiar 3′ stacked conformation in the crystal structure models of yeast tRNA^Phe. It turned out that structural adjustments of the anticodon loop to the binding of the oligonucleotides are propagated into the anticodon stem. The relevance of these results with respect to the mechanism of protein synthesis is discussed.     

Ternary complex between elongation factor Tu•GTP and Phe-tRNA

The effect of aminoacylation and ternary complex formation with elongation factor Tu•GTP on the tertiary structure of yeast tRNA^Phe was examined by ¹H-NMR spectroscopy. Esterification of phenylalanine to tRNA^Phe does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA^Phe with elongation factor Tu•GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA^Phe upon binding to elongation factor Tu•GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA^Phe•elongation factor Tu•GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA^Phe preserves its typical L-shaped tertiary structure in the complex. At high (> 10⁻⁴ M) ligand concentrations a complex between tRNA^Phe and elongation factor Tu•GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA^Phe and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.     

Location of a platinum binding site in the structure of yeast phenylalanine transfer RNA

Trans-dichlorodiammineplatinum (II) reacts with yeast phenylalanine transfer RNA to yield a major platinum binding site. The tightly bound platinum has been located on the oligonucleotide Gm-A-A-Y-A-ψp containing the anticodon by standard fingerprinting methods using ³²P-labelled tRNA^Phe. This site corresponds to a single major platinum site identified during an X-ray crystallographic analysis of yeast tRNA^Phe. The solution studies have given confidence to the assignment of part of the 3 Å electron density map to the anticodon region of the molecular structure of yeast tRNA^Phe.     

Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe)

The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.     

Sequence-specific artificial ribonucleases. I. Bis-imidazole-containing oligonucleotide conjugates prepared using precursor-based strategy

Antisense oligonucleotide conjugates, bearing constructs with two imidazole residues, were synthesized using a precursor-based technique employing post-synthetic histamine functionalization of oligonucleotides bearing methoxyoxalamido precursors at the 5′-termini. The conjugates were assessed in terms of their cleavage activities using both biochemical assays and conformational analysis by molecular modelling. The oligonucleotide part of the conjugates was complementary to the T-arm of yeast tRNA^Phe (44–60 nt) and was expected to deliver imidazole groups near the fragile sequence C₆₁-ACA-G₆₅ of the tRNA. The conjugates showed ribonuclease activity at neutral pH and physiological temperature resulting in complete cleavage of the target RNA, mainly at the C₆₃–A₆₄ phosphodiester bond. For some constructs, cleavage was completed within 1–2 h under optimal conditions. Molecular modelling was used to determine the preferred orientation(s) of the cleaving group(s) in the complexes of the conjugates with RNA target. Cleaving constructs bearing two imidazole residues were found to be conformationally highly flexible, adopting no preferred specific conformation. No interactions other than complementary base pairing between the conjugates and the target were found to be the factors stabilizing the ‘active’ cleaving conformation(s).     

The structure of the anticodon loop of tRNAPhe from yeast as deduced from spectroscopic studies on oligonucleotides

The hexanucleotide Gm-A-A-Y-A-ψp excised from the anticodon loop of yeast tRNA^Phe and its constituent oligonucleotides have been studied by ultraviolet absorption spectroscopy, static fluorescence, and circular dichroism. Gm-Ap has a melting point of 45°C and a high melting enthalpy when compared with G-Ap; hence 2′-O-methylation seems to stabilize stacking interactions. The nucleobase Y adjacent to the 3′-side of the anticodon triplet interacts stronger with its 3′-neighboring A than with its 5′-neighboring A. It is concluded that the base Y disconnects the stack of the anticodon itself from the stack of the anticodon stem, thereby setting a reading frame for the mRNA in the course of protein biosynthesis. From the opposite signs of the short-wavelength Cotton effects in the spectra of Gm-A-A-Y-Ap and Gm-A-A-Y, it is concluded that Y after removal of its 3′ neighbor undergoes a dramatic change in its conformation. The fluorescence of the nucleobase Y upon addition of Mg²⁺ is enhanced in oligonucleotides longer than two. An identical enhancement is observed for tRNA^Phe, indicating that this Mg²⁺ effect is a property of an oligonucleotide segment and does not reflect conformational changes of the whole tRNA. The data presented here reveal that the basic structural features of the anticodon loop are already present in the hexanucleotide Gm-A-A-Y-A-ψp and are not determined by the overall structure of tRNA.     

Primary structure of Bombyx mori posterior silkgland tRNAPhe

tRNA^Phe was isolated from posterior silkgland from Bombyx mori and hydrolysed to mixtures of oligonucleotides. |³²P|5′ end labelling of the oligonucleotides and sequence study indicates that the major component of Bombyx mori tRNA^Phe is similar to mammalian tRNA^Phe, the minor component differing from the major one by one nucleotide only.     

Complex formation between transfer RNAs with complementary anticodons: use of matrix bound tRNA

It is shown that yeast tRNA^Phe, chemically coupled by its oxidized 3′CpCpA end behaves exactly as free tRNA^Phe in its ability to form a specific complex with $\text{E. coli}$ tRNA₂^Glu having a complementary anticodon. The results support models of tRNA in which the 3′CpCpA_OH end and the anticodon are not closely associated in the tertiary structure, and provide a convenient tool of general use to characterize others pairs of tRNA having complementary anticodons, as well as for highly selective purification of certain tRNA species.     

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase,and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA^Phe by tRNA nucleotidyl transferase. tRNA^Phe-C-C-2′dA and tRNA^Phe-C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA^Phe-C-C-3′dA proved to be chargeable.     

Conformational Change of the L-Shaped tRNAPhe Molecule

Abstract

Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNA^Phe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNA^Phe was measured by a singlet-singlet energy transfer. Conformational changes of tRNA^Phe with binding of tRNA^Glu ₂, which has the anticodon UUC complementary to the anticodon GAA of tRNA^Phe, were investigated. The distance obtained at the ionic strength of 100 mM K⁺ and 10 mM Mg²⁺ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNA^Glu ₂ is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ₅₅ in the TΨC loop of tRNA^Phe, Stern-Volmer quenching experiments for the probe with or without added tRNA^Glu ₂were carried out. The results showed greater access of the probe to the quencher with added tRNA^Glu ₂. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA^Glu ₂ and some structural collapse occurs at the corner of the L-shaped structure.     

Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA

Binding of Mn²⁺ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNA^Phe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNA^Phe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNA^Phe. Binding of Mn²⁺ to intact tRNA^Phe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn²⁺ to tRNA^Phe fragments (5′-$\text{1}\text{2}$, 3′-$\text{1}\text{2}$, 5′-$\text{3}\text{5}$, 3′-$\text{2}\text{5}$), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNA^Phe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

Specific oligonucleotide invasion into an end of a DNA duplex

A new phenomenon was described: a double-stranded DNA fragment interacted with a single-stranded oligonucleotide complementary to the terminal region of one strand of the duplex to yield a complex with oligonucleotide invasion. Generation of Holliday junctions by homologous linear DNA fragments was less efficient in the presence of single-stranded oligonucleotides complementary to duplex ends. The effect depended on the oligonucleotide concentration, size, and complementarity to a duplex strand. Sequence-specific complexes with single strand invasion were detected in mixtures containing radiolabeled oligonucleotides and duplexes. A single-stranded oligonucleotide invaded a duplex even when its concentration was far lower than the duplex concentration. Complexes with single strand invasion were analyzed by chemical cleavage of noncanonical base pairs. Analysis showed that an oligonucleotide interacts with the complementary region of one strand of the duplex, gradually displacing the other strand. The extent of oligonucleotide invasion into the duplex considerably varied. Oligonucleotide invasion into duplexes became more efficient with increasing oligonucleotide size.     

Specific oligonucleotide invasion into the end of DNA duplex

Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.     

Highly Efficient Site-Directed RNA Cleavage by Imidazole-Containing Conjugates of Antisense Oligonucleotides

A method has been suggested for the synthesis of conjugates of oligodeoxyribonucleotides with chemical constructs mimicking the ribonuclease A active center for directed fragmentation of RNA. The method is based on sequential addition of a linker group, 9-(methylamino)anthracene, to the 5"- or 3"-terminal phosphate of oligonucleotide, and then an imidazole-containing construct by cycloaddition. The conjugates of oligonucleotides complementary to regions 44–61 (2B–R) and 60–76 (1C–R) of yeast phenylalanine tRNA proved able to cleave tRNA^Phe under physiological conditions preferentially at the sole phosphodiester bond (C63–A64 for 2B–R and C56–G57 for 1C–R, respectively). The half-time of tRNA^Phe hydrolysis in the presence of 2B–R conjugate was 30 min at a 2B–R concentration of 10 M and several minutes at conjugate concentration of 50 M.     

Codon-anticodon interaction in tRNAPhe: II. A nuclear magnetic resonance study of the binding of the codon UUC

The effect of binding of the codon UUC to yeast tRNA^Phe was investigated by means of n.m.r.² spectroscopy and analytical ultracentrifugation. Binding of UUC to the transfer RNA anticodon tends to promote the aggregation of tRNA molecules; this is manifest from a line broadening in the n.m.r. experiments as well as from an increase in s_20,w the ultracentrifuge experiments. Such an aggregation of tRNA molecules was not observed upon addition of different oligonucleotides, as described in the accompanying paper. In addition to the general broadening observed in the n.m.r. spectra, specific resonances in the methyl proton spectrum as well as in the hydrogen-bonded proton spectrum are broadened or shifted upon binding of UUC.These results are explained on the basis of the premise that two different tRNA-UUC complexes can exist in solution. It is suggested that the binding of UUC tends to promote a disruption of the m⁷G₄₆ · m²₂G₂₂ base-pair and its neighbouring base-pairs.In studying the binding of U-U-U-U to yeast tRNA^Phe no resonances of protons hydrogen-bonded between the oligonucleotide and the tRNA could be detected at low temperatures. This indicates, that at these temperatures the lifetime of the tRNA-U-U-U-U complex is substantially shorter than the lifetime of the other tRNA-oligonucleotide complexes studied in this and the accompanying paper under these conditions.