The Effect of Universal Fluorinated Nucleobases on the Catalytic Activity of Ribozymes

Abstract

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2 , containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribozyme sequence which has already been retrovirally expressed as an anti-HIV ribozyme to investigate their effect on the catalytic activity of the ribozymes. The substitution of the natural nucleosides with either 1 or 2 results only in a small decrease of the catalytic activity. The K_m value for the monosubstituted ribozyme with a 2,4-difluorobenzene is 309 nM^?1, the corresponding k_cat is 2.91 · 10^?3 min^?1. A disubstituted hammerhead ribozyme carrying one of each modification has also been synthesized. For a further stabilization of the ribozyme/substrate complex 2′-(β-aminoethoxy) modified fluorinated nucleosides 15 and 16 have been developed.     

SYNTHESIS AND PURIFICATION OF FLUORINATED BENZIMIDAZOLE AND BENZENE NUCLEOSIDE-5′-TRIPHOSPHATES

The expression “universal base” is very often used to express hybridization properties and recognition patterns of nucleosides. Their behaviour in biological applications, however, is of great interest regarding, e.g., their incorporation by polymerases. The 4,6-difluorobenzimidazole and the 2,4-difluorobenzene nucleoside analogues have proven to be universal bases that do not discriminate between the four natural nucleobases in RNA duplexes. Therefore, we synthesized the corresponding triphosphates to evaluate their behavior in polymerase catalyzed reactions and to investigate their ability to serve as substrates for the T7 RNA polymerase.     

Synthesis and purification of fluorinated benzimidazole and benzene nucleoside-5'-triphosphates

The expression "universal base" is very often used to express hybridization properties and recognition patterns of nucleosides. Their behaviour in biological applications, however, is of great interest regarding, e.g.,' their incorporation by polymerases. The 4,6-difluorobenzimidazole and the 2,4-difluorobenzene nucleoside analogues have proven to be universal bases that do not discriminate between the four natural nucleobases in RNA duplexes. Therefore, we synthesized the corresponding triphosphates to evaluate their behavior in polymerase catalyzed reactions and to investigate their ability to serve as substrates for the T7 RNA polymerase.     

Folding of the hammerhead ribozyme: pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.     

Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes

Native hammerhead ribozymes contain RNA domains that enable high catalytic activity under physiological conditions, where minimal hammerheads show little activity. However, little is known about potential differences in native versus minimal ribozyme folding. Here, we present results of photocross-linking analysis of native and minimal hammerheads containing photoreactive nucleobases 6-thioguanosine, 2,6-diaminopurine, 4-thiouridine, and pyrrolocytidine, introduced at specific sites within the catalytic core. Under conditions where catalytic activity is observed, the two substrate nucleobases spanning the cleavage site approach and stack upon G8 and G12 of the native hammerhead, two conserved nucleobases that show similar behavior in minimal constructs, have been implicated in general acid-base catalysis, and are >15 A from the cleavage site in the crystal structures. Pyrrolocytidine at cleavage site position 17 forms an efficient crosslink to G12, and the crosslinked RNA retains catalytic activity. Multiple cross-linked species point to a structural rearrangement within the U-turn, positioning residue G5 in the vicinity of cleavage site position 1.1. Intriguing crosslinks were triggered by nucleotide analogues at positions distal to the crosslinked residues; for example, 6-thioguanosine at position 5 induced a crosslink between G12 and C17, suggesting an intimate functional communication among these three nucleobases. Together, these results support a model in which the native hammerhead folds to an active structure similar to that of the minimal ribozyme, and significantly different from the crystallographic structures.     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding

Mg2+ is important for the RNase activity of the hammerhead ribozyme. To investigate the binding properties of Mg2+ to the hammerhead ribozyme, cleavage rates and CD spectra for substrates containing inosine or guanosine at the cleavage site were measured. The 2-amino group of this guanosine interfered with the rate of the cleavage reaction and did not affect the amount of Mg2+ bound to the hammerhead RNA. The kinetics and CD spectra for chemically synthesized oligoribonucleotides with a Sp or Rp phosphorothioate diester bond at the cleavage site indicated that 1 mol of Mg2+ binds to the pro-R oxygen of phosphate. The binding constant for Mg2+ was about 10(4) M-1, which represents outer-sphere complexation. The hammerhead ribozyme catalyzes the cleavage reaction via an in-line pathway. This mechanism has been proved for RNA cleavage by RNase A by using a modified oligonucleotide that has an Sp phosphorothionate bond at the cleavage site. From these results, we present the reaction pathway and a model for Mg2+ binding to the hammerhead ribozyme.     

1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes.

The incorporation of 1-deazaadenosine (c1A, 1b) into a hammerhead ribozyme and the resulting catalytic activity is described. For this purpose the phosphoramidite 2a and the 3'-phosphonate 2b as well as Fractosil-linked 1-deazaadenosine (3b) were prepared. The methoxyacetyl group was used for the 6-amino group protection and the triisopropylsilyl residue was introduced as the 2'-OH protecting group. Replacement of residues A14and A15.1 of the hammerhead ribozyme by 1-deazaadenosine resulted in a significantly reduced catalytic activity. Substitution of the A6, A9 and A13 residues has only a minor influence. The findings observed on ribozymes modified with 1-deazaadenosine were compared with those containing other adenosine analogues.     

Study of a hammerhead ribozyme containing 2'-modified adenosine residues

The improved synthesis of 2'-fluoro-2'-deoxyadenosine (2'-FA) starting from adenosine is described. This compound was converted to the phosphoramidite and incorporated into a hammerhead ribozyme RNA with the use of automated RNA synthesis techniques. Ribozymes containing 2'-deoxy-adenosine (2'-dA) were prepared in a similar manner. A kinetic rate comparison of the unmodified ribozyme with two ribozymes that had every adenosine replaced with 2'FA or 2'-dA revealed a large decrease in catalytic efficiency (kcat/Km) for the modified ribozymes resulting from a drop in kcat. The kinetic analysis of a number of partially substituted 2'-FA or 2'-dA containing hammerheads revealed that the decrease in activity was not associated with any particular residue but was the result of the accumulation of modified nucleosides within the structure.     

Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site

We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2'-hydroxyl group and protonation of the 5'-oxygen that serves as the leaving group in the cleavage reaction. We demonstrate that simultaneous substitution of two important nucleobases, G8 and G12, with 2,6-diaminopurine shifts the pH optimum of the cleavage reaction from greater than 9.5 to approximately 6.8 in two different hammerhead constructs. Controls involving substitution with other nucleobases and combinations of nucleobases at G5, G8, and/or G12 do not show this behavior. The observed changes in the pH-rate behavior are consistent with a mechanism in which N1 protonation-deprotonation events of guanine or 2,6-diaminopurine at positions 8 and 12 are essential for catalysis. Further support for the participation of G8 and G12 comes from photochemical cross-linking experiments, which show that G8 and G12 can stack upon the two substrate nucleobases at the reactive linkage, G(or U)1.1 and C17 (Heckman, J. E., Lambert, D., and Burke, J. M. (2005) Photocrosslinking detects a compact active structure of the hammerhead ribozyme, Biochemistry 44, 4148-4156). Together, these results support a model in which the hammerhead undergoes a transient conformational change into a catalytically active structure, in which stacking of G8 and G12 upon the nucleobases spanning the cleavage site provides an appropriate architecture for general acid-base catalysis. The hammerhead and hairpin ribozymes may share similarities in the organization of their active sites and their catalytic mechanism.     

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.     

Efficient ligation of the Schistosoma hammerhead ribozyme

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.     

Critical nature of a specific uridine O2-carbonyl for cleavage by the hammerhead ribozyme.

Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.     

Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study.

The ion-induced folding transitions of the hammerhead ribozyme have been analysed by fluorescence resonance energy transfer. The hammerhead ribozyme may be regarded as a special example of a three-way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end-to-end vectors as a function of magnesium ion concentration. The data support two sequential ion-dependent transitions, which can be interpreted in the light of the crystal structures of the hammerhead ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000-10 000 M-1. The second structural transition corresponds to the formation of the catalytic domain of the ribozyme, induced by a single magnesium ion with an apparent association constant of approximately 1100 M-1. The hammerhead ribozyme provides a well-defined example of ion-dependent folding in RNA.     

Synthesis and biological evaluation of phosphonated dihydroisoxazole nucleosides

Phosphonated isoxazolinyl nucleosides have been prepared via 1,3-dipolar cycloaddition reaction of nitrile oxides with corresponding vinyl or allyl nucleobases for antiviral studies. The cytotoxicity, the anti-HSV activity and the RT-inhibitory activity of the obtained compounds were evaluated and compared with those of AZT and diethyl{(1'SR,4'RS)-1'-[[(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)]-3'-methyl-2'-oxa-3'-azacyclopent-4'-yl]}methylphosphonate, a saturated phosphonated dihydroisoxazole nucleoside analogue.     

Synthesis of 2'-C-alpha-difluoromethylarauridine and its 3'-O-phosphoramidite incorporation into a hammerhead ribozyme

The 2'-C-difluoromethylated nucleoside 4 was synthesized starting from uridine. 4 was then converted to the 3'-O-phosphoramidite derivative 5 and was incorporated into a hammerhead ribozyme (7). The cleavage characteristics of the modified oligonucleotide have been analysed.     

Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain.

Several mixed DNA/RNA and 2'-O-methylribonucleotide/RNA analogues derived from the "hammerhead" domain of RNA catalysis have been prepared to study the minimum ribonucleotide requirement for catalytic activity. Oligodeoxyribonucleotides containing from seven to as few as four ribonucleotides are active in cleaving a substrate RNA. Predominantly deoxyribonucleotide-containing analogues have kcat values 20-300 and kcat/KM values approximately 100-2000 times lower than those of all-RNA ribozyme. In the case of predominantly 2'-O-methyl analogues, at least five ribonucleotides are needed to assure catalytic activity. In addition, both predominantly deoxyribonucleotide and 2'-O-methyl oligomers are at least 3 orders of magnitude more stable than an all-RNA ribozyme in incubations with RNase A and a yeast extract. These results suggest that the ribophosphate backbone is not a strict requirement for ribozyme-type catalysis. The identification of the four required ribonucleotides in the hammerhead catalytic domain provides valuable information for the rational design of chemical species having ribonuclease activities.     

Photocrosslinking detects a compact, active structure of the hammerhead ribozyme

The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S(N)2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12.     

Capture and visualization of a catalytic RNA enzyme-product complex using crystal lattice trapping and X-ray holographic reconstruction

We have determined the crystal structure of the enzyme-product complex of the hammerhead ribozyme by using a reinforced crystal lattice to trap the complex prior to dissociation and by employing X-ray holographic image reconstruction, a real-space electron density imaging and refinement procedure. Subsequent to catalysis, the cleavage site residue (C-17), together with its 2',3'-cyclic phosphate, adopts a conformation close to and approximately perpendicular to the Watson-Crick base-pairing faces of two highly conserved purines in the ribozyme's catalytic pocket (G-5 and A-6). We observe several interactions with functional groups on these residues that have been identified as critical for ribozyme activity by biochemical analyses but whose role has defied explanation in terms of previous structural analyses. These interactions may therefore be relevant to the hammerhead ribozyme reaction mechanism.