In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3'-5' to 2'-5'

Deoxyribozymes that ligate RNA expand the scope of nucleic acid catalysis and allow preparation of site-specifically modified RNAs. Previously, deoxyribozymes that join a 5'-hydroxyl and a 2',3'-cyclic phosphate were identified by in vitro selection from random DNA pools. Here, the alternative strategy of in vitro evolution was used to transform the 8-17 deoxyribozyme that cleaves RNA into a family of DNA enzymes that ligate RNA. The parent 8-17 DNA enzyme cleaves native 3'-5' phosphodiester linkages but not 2'-5' bonds. Surprisingly, the new deoxyribozymes evolved from 8-17 create only 2'-5' linkages. Thus, reversing the direction of the DNA-mediated process from ligation to cleavage also switches the selectivity in forming the new phosphodiester bond. The same change in selectivity was observed upon evolution of the 10-23 RNA-cleaving deoxyribozyme into an RNA ligase. The DNA enzymes previously isolated from random pools also create 2'-5' linkages. Therefore, deoxyribozyme-mediated formation of a non-native 2'-5' phosphodiester linkage from a 5'-hydroxyl and a 2',3'-cyclic phosphate is strongly favored in many different contexts.     

Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages

We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region. This approach should favor formation of linear rather than branched RNA because the two RNA termini are spatially constrained by Watson-Crick base pairing during the ligation reaction. Furthermore, because native 3'-5' linkages are more stable in a duplex than isomeric non-native 2'-5' linkages, this strategy is predicted to favor the formation of 3'-5' linkages. All of the new deoxyribozymes indeed create only linear 3'-5' RNA, confirming the effectiveness of the rational design. The new deoxyribozymes ligate RNA with k(obs) values up to 0.5 h(-1) at 37 degrees C and 40 mM Mg2+, pH 9.0, with up to 41% yield at 3 h incubation. They require several specific RNA nucleotides on either side of the ligation junction, which may limit their practical generality. These RNA ligase deoxyribozymes are the first that create native 3'-5' RNA linkages, which to date have been highly elusive via other selection approaches.     

General deoxyribozyme-catalyzed synthesis of native 3'-5' RNA linkages

An elusive goal for nucleic acid enzymology has been deoxyribozymes that ligate RNA rapidly, sequence-generally, with formation of native 3'-5' linkages, and in preparatively useful yield. Using in vitro selection, we have identified Mg2+- and Zn2+-dependent deoxyribozymes that simultaneously fulfill all four of these criteria. The new deoxyribozymes operate under practical incubation conditions and have modest RNA substrate sequence requirements, specifically D downward arrowRA for 9DB1 and A downward arrowR for 7DE5 (D = A, G, or U; R = A or G). These requirements are comparable to those of deoxyribozymes such as 10-23 and 8-17, which are already widely used as biochemical tools for RNA cleavage. We anticipate that the 9DB1 and 7DE5 deoxyribozymes will find immediate practical application for RNA ligation.     

A deoxyribozyme that forms a three-helix-junction complex with its RNA substrates and has general RNA branch-forming activity

We recently used in vitro selection to identify 7S11, a deoxyribozyme that synthesizes 2',5'-branched RNA. The 7S11 DNA enzyme mediates the nucleophilic attack of an adenosine 2'-hydroxyl group at a 5'-triphosphate, forming 2',5'-branched RNA in a reaction that resembles the first step of in vivo RNA splicing. Here, we describe 7S11 characterization experiments that have two important implications for nucleic acid chemistry and biochemistry. First, on the basis of a comprehensive analysis of its substrate sequence requirements, 7S11 is shown to be generally applicable for the synthesis of a wide range of 2',5'-branched RNAs. Strict substrate sequence requirements are found at the two RNA nucleotides that directly form the branched linkage, and these requirements correspond to those nucleotides found most commonly at these two positions in natural spliced RNAs. Outside of these two nucleotides, most substrate sequences are tolerated with useful ligation activity, although rates and yields vary. Because chemical synthesis approaches to branched RNA are extremely limited in scope, the deoxyribozyme-based route using 7S11 will enable many experiments that require branched RNA. Second, comprehensive nucleotide covariation experiments demonstrate that 7S11 and its RNA substrates adopt a three-helix-junction structure in which the branch-site nucleotide is located at the intersection of the three helices. Because many natural ribozymes have multi-helix junctions, 7S11 is an interesting model system for catalytic nucleic acids.     

Evidence for a thermodynamically distinct Mg2+ ion associated with formation of an RNA tertiary structure

A folding strategy adopted by some RNAs is to chelate cations in pockets or cavities, where the ions neutralize charge from solvent-inaccessible phosphate. Although such buried Mg(2+)-RNA chelates could be responsible for a significant fraction of the Mg(2+)-dependent stabilization free energy of some RNA tertiary structures, direct measurements have not been feasible because of the difficulty of finding conditions under which the free energy of Mg(2+) chelation is uncoupled from RNA folding and from unfavorable interactions with Mg(2+) ions in other environments. In a 58mer rRNA fragment, we have used a high-affinity thermophilic ribosomal protein to trap the RNA in a structure nearly identical to native; Mg(2+)- and protein-stabilized structures differ in the solvent exposure of a single nucleotide located at the chelation site. Under these conditions, titration of a high affinity chelation site takes place in a micromolar range of Mg(2+) concentration, and is partially resolved from the accumulation of Mg(2+) in the ion atmosphere. From these experiments, we estimate the total and site-specific Mg(2+)-RNA interaction free energies over the range of accessed Mg(2+) concentrations. At 0.1 mM Mg(2+) and 60 mM K(+), specific site binding contributes ～-3 kcal/mol of the total Mg(2+) interaction free energy of ～-13 kcal/mol from all sources; at higher Mg(2+) concentrations the site-binding contribution becomes a smaller proportion of the total (-4.5 vs -33 kcal/mol). Under approximately physiological ionic conditions, the specific binding site will be saturated but will provide only a fraction of the total free energy of Mg(2+)-RNA interactions.     

Deoxyribozymes that synthesize branched and lariat RNA

Branched RNA molecules with a 2',5'-phosphodiester linkage are important biochemical intermediates. Lariat RNA is a particular type of branched RNA that is formed during intron splicing in vivo. Synthesis of branched and lariat RNA is challenging, and there are few general approaches that are applicable in vitro. Here we report the identification of divalent metal-dependent deoxyribozymes (DNA enzymes) that synthesize branched and lariat RNA. In vitro selection was used to obtain deoxyribozymes that selectively join an internal RNA 2'-hydroxyl with a 5'-terminal triphosphate in a convenient "binding arms" format. At least 85% yield of 2',5'-branched RNA is obtained at 37 degrees C and 20 mM Mn2+, pH 7.5 in 相似文献   

Nonenzymatic, template-directed ligation of oligoribonucleotides is highly regioselective for the formation of 3'-5' phosphodiester bonds

We have found that nonenzymatic, template-directed ligation reactions of oligoribonucleotides display high selectivity for the formation of 3'-5' rather than 2'-5' phosphodiester bonds. Formation of the 3'-5'-linked product is favored regardless of the metal ion catalyst or the leaving group, and for several different ligation junction sequences. The degree of selectivity depends on the leaving group: the ratio of 3'-5'- to 2'-5'-linked products was 10-15:1 when the 5'-phosphate was activated as the imidazolide, and 60-80:1 when the 5'-phosphate was activated by the formation of a 5'-triphosphate. Comparison of oligonucleotide ligation reactions with previously characterized single nucleotide primer extension reactions suggests that the strong preference for 3'-5'-linkages in oligonucleotide ligation is primarily due to occurence of ligation within the context of an extended Watston-Crick duplex. The ability of RNA to correctly self-assemble by template-directed ligation is an intrinsic consequence of its chemical structure and need not be imposed by an external catalyst (i.e., an enzyme polymerase); RNA therefore provides a reasonable structural basis for a self-replicating system in a prebiological world.     

Selective stabilization of natively folded RNA structure by DNA constraints

Learning how native RNA conformations can be stabilized relative to unfolded states is an important objective, for both understanding natural RNAs and improving the design of artificial functional RNAs. Here we show that covalently attached double-stranded DNA constraints (ca. 14 base pairs in length) can significantly stabilize the native conformation of an RNA molecule. Using the P4-P6 domain of the Tetrahymena group I intron as the test system, we identified pairs of RNA sites where attaching a DNA duplex is predicted to be structurally compatible with only the folded state of the RNA. The DNA-constrained RNAs were synthesized and shown by nondenaturing polyacrylamide gel electrophoresis (native PAGE) to have substantial decreases in their Mg2+ midpoints ([Mg2+]1/2 values). These changes are equivalent to free energy stabilizations as large as DeltaDeltaGdegrees = -2.5 kcal/mol, which is approximately 14% of the total tertiary folding energy. For comparison, the sole modification of P4-P6 previously reported to stabilize this RNA is a single-nucleotide deletion (DeltaC209) that provides only 1.1 kcal/mol of stabilization. Our findings indicate that nature has not completely optimized P4-P6 RNA folding. Furthermore, the DNA constraints are designed not to interact directly and extensively with the RNA, but rather more indirectly to modulate the relative stabilities of folded and unfolded RNA states. The successful implementation of this strategy to further stabilize a natively folded RNA conformation suggests an important element of modularity in stabilization of RNA structure, with implications for how nature might use other molecules such as proteins to stabilize specific RNA conformations.     

Pseudoknot preorganization of the preQ1 class I riboswitch

To explore folding and ligand recognition of metabolite-responsive RNAs is of major importance to comprehend gene regulation by riboswitches. Here, we demonstrate, using NMR spectroscopy, that the free aptamer of a preQ(1) class I riboswitch preorganizes into a pseudoknot fold in a temperature- and Mg(2+)-dependent manner. The preformed pseudoknot represents a structure that is close to the ligand-bound state and that likely represents the conformation selected by the ligand. Importantly, a defined base pair mutation within the pseudoknot interaction stipulates whether, in the absence of ligand, dimer formation of the aptamer competes with intramolecular pseudoknot formation. This study pinpoints how RNA preorganization is a crucial determinant for the adaptive recognition process of RNA and ligand.     

Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes

Although DNA has not been found responsible for biological catalysis, many artificial DNA enzymes have been created by "in vitro selection." Here we describe a new selection approach to assess the influence of four common divalent metal ions (Ca(2+), Cu(2+), Mg(2+), and Mn(2+)) on sequence diversity, metal specificity, and catalytic proficiency of self-phosphorylating deoxyribozymes. Numerous autocatalytic DNA sequences were isolated, a majority of which were selected using Cu(2+) or Mn(2+) as the divalent metal cofactor. We found that Cu(2+)- and Mn(2+)-derived deoxyribozymes were strictly metal specific, while those selected by Ca(2+) and Mg(2+) were less specific. Further optimization by in vitro evolution resulted in a Mn(2+)-dependent deoxyribozyme with a k(cat) of 2.8 min(-1). Our findings suggest that DNA has sufficient structural diversity to facilitate efficient catalysis using a broad scope of metal cofactor utilizing mechanisms.     

Deoxyribozymes: DNA catalysts for bioorganic chemistry

Deoxyribozymes are DNA molecules with catalytic activity. For historical and practical reasons, essentially all reported deoxyribozymes catalyze reactions of nucleic acid substrates, although this is probably not a fundamental limitation. In vitro selection strategies have been used to identify many deoxyribozymes that catalyze RNA cleavage, RNA and DNA ligation, and a variety of covalent modification reactions of nucleic acid substrates. Many deoxyribozymes are capable of catalysis with substantial rate enhancements reaching up to 10(10)-fold over background, and their very high selectivities would often be difficult or impossible to achieve using traditional organic synthesis approaches. This report summarizes the current utility and potential future applications of deoxyribozymes from the bioorganic chemistry perspective.     

The structure of a TNA-TNA complex in solution: NMR study of the octamer duplex derived from alpha-(L)-threofuranosyl-(3'-2')-CGAATTCG

TNA (alpha-( l)-threofuranosyl-(3'-2') nucleic acid) is a nucleic acid in which the ribofuranose building block of the natural nucleic acid RNA is replaced by the tetrofuranose alpha-( l)-threose. This shortens the repetitive unit of the backbone by one bond as compared to the natural systems. Among the alternative nucleic acid structures studied so far in our laboratories in the etiological context, TNA is the only one that exhibits Watson-Crick pairing not only with itself but also with DNA and, even more strongly, with RNA. Using NMR spectroscopy, we have determined the structure of a duplex consisting entirely of TNA nucleotides. The TNA octamer (3'-2')-CGAATTCG forms a right-handed double helix with antiparallel strands paired according to the Watson-Crick mode. The dominant conformation of the sugar units has the 2'- and 3'-phosphodiester substituents in quasi-diaxial position and corresponds to a 4'-exo puckering. With 5.85 A, the average sequential P i -P i+1 distances of TNA are shorter than for A-type DNA (6.2 A). The helix parameters, in particular the slide and x-displacement, as well as the shallow and wide minor groove, place the TNA duplex in the structural vicinity of A-type DNA and RNA.     

Probing essential nucleobase functional groups in aptamers and deoxyribozymes by nucleotide analogue interference mapping of DNA

Nucleotide analogue interference mapping of DNA (dNAIM) is here introduced as a new nonenzymatic interference-based approach that enables high-throughput identification of essential nucleobase functional groups in DNA aptamers and in the catalytic core of deoxyribozymes. Nucleobase-modified ribonucleotides are statistically incorporated into DNA by solid-phase synthesis, employing the 2'-OH group as a chemical tag for analysis of interference effects. This method is exemplified on an AMP-binding DNA aptamer and was further used to identify indispensable nucleobase functional groups for DNA-catalyzed RNA-ligation by the Mg(2+)-dependent deoxyribozymes 7S11 and 9DB1. dNAIM should prove broadly useful for facile structural probing of functional DNA for which active and inactive variants can be separated based on catalytic or ligand-binding activities.     

Preferred RNA binding sites for a threading intercalator revealed by in vitro evolution

In pursuit of small molecules capable of controlling the function of RNA targets, we have explored the RNA binding properties of peptide-acridine conjugates (PACs). In vitro evolution (SELEX) was used to isolate RNAs capable of binding the PAC Ser-Val-Acr-Arg, where Acr is an acridine amino acid. The PAC binds RNA aptamers selectively and with a high degree of discrimination over DNA. PAC binding sites contain the base-paired 5'-CpG-3' sequence, a known acridine intercalation site. However, RNA structure flanking this sequence causes binding affinities to vary over 30-fold. The preferred site (K(D) = 20 nM) contains a base-paired 5'-CpG-3' step flanked on the 5' side by a 4 nt internal loop and the 3' side by a bulged U. Several viral 5'- and 3'-UTR RNA sequences that likely form binding sites for this PAC are identified.     

Synthesis of amine- and thiol-modified nucleoside phosphoramidites for site-specific introduction of biophysical probes into RNA

For studies of RNA structure, folding, and catalysis, site-specific modifications are typically introduced by solid-phase synthesis of RNA oligonucleotides using nucleoside phosphoramidites. Here, we report the preparation of two complete series of RNA nucleoside phosphoramidites; each has an appropriately protected amine or thiol functional group. The first series includes each of the four common RNA nucleotides, U, C, A, and G, with a 2'-(2-aminoethoxy)-2'-deoxy substitution (i.e., a primary amino group tethered to the 2'-oxygen by a two-carbon linker). The second series encompasses the four common RNA nucleotides, each with the analogous 2'-(2-mercaptoethoxy)-2'-deoxy substitution (i.e., a tethered 2'-thiol). The amines are useful for acylation and reductive amination reactions, and the thiols participate in displacement and oxidative cross-linking reactions, among other likely applications. The new phosphoramidites will be particularly valuable for enabling site-specific introduction of biophysical probes and constraints into RNA.     

Fast cleavage kinetics of a natural hammerhead ribozyme

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.     

Magnesium fluctuations modulate RNA dynamics in the SAM-I riboswitch

Experiments demonstrate that Mg(2+) is crucial for structure and function of RNA systems, yet the detailed molecular mechanism of Mg(2+) action on RNA is not well understood. We investigate the interplay between RNA and Mg(2+) at atomic resolution through ten 2-μs explicit solvent molecular dynamics simulations of the SAM-I riboswitch with varying ion concentrations. The structure, including three stemloops, is very stable on this time scale. Simulations reveal that outer-sphere coordinated Mg(2+) ions fluctuate on the same time scale as the RNA, and that their dynamics couple. Locally, Mg(2+) association affects RNA conformation through tertiary bridging interactions; globally, increasing Mg(2+) concentration slows RNA fluctuations. Outer-sphere Mg(2+) ions responsible for these effects account for 80% of Mg(2+) in our simulations. These ions are transiently bound to the RNA, maintaining interactions, but shuttled from site to site. Outer-sphere Mg(2+) are separated from the RNA by a single hydration shell, occupying a thin layer 3-5 ? from the RNA. Distribution functions reveal that outer-sphere Mg(2+) are positioned by electronegative atoms, hydration layers, and a preference for the major groove. Diffusion analysis suggests transient outer-sphere Mg(2+) dynamics are glassy. Since outer-sphere Mg(2+) ions account for most of the Mg(2+) in our simulations, these ions may change the paradigm of Mg(2+)-RNA interactions. Rather than a few inner-sphere ions anchoring the RNA structure surrounded by a continuum of diffuse ions, we observe a layer of outer-sphere coordinated Mg(2+) that is transiently bound but strongly coupled to the RNA.     

Montmorillonite catalysis of RNA oligomer formation in aqueous solution. A model for the prebiotic formation of RNA

Oligomers of adenylic acid of up to the 11-mer in length are formed by the reaction of the phosphorimidazolide of adenosine (ImpA) in pH 8 aqueous solution at room temperature in the presence of Na(+)-montmorillonite. These oligomers are joined by phosphodiester bonds in which the 3',5'-linkage predominates over the 2',5'-linkage by a 2:1 ratio. Reaction of a 9:1 mixture of ImpA, A5'ppA results in the formation of oligomers with a 3:1 ratio of 3',5'- to 2',5'-linked phosphodiester bonds. A high proportion of these oligomers contain the A5'ppA grouping. A5'ppA reacts much more rapidly with ImpA than does 5'-ADP (ppA) or 5'-ATP (pppA). The exchangeable cation associated with the montmorillonite effects the observed catalysis with Li+, Na+, NH4+, and Ca2+ being the more effective while Mg2+ and Al3+ are almost ineffective catalysts. 2',5'-Linked oligomers, up to the tetramer in length, are formed using UO2(2+)-montmorillonite. The structure analysis of individual oligomer fractions was performed by selective enzymatic and KOH hydrolytic studies followed by HPLC analysis of the reaction products. It is concluded from the composition of the oligomers that the rate of addition ImpA to a 3'-terminus containing a 2',5'-linkage is slower than the addition to a nucleoside joined by a 3',5'-linked phosphodiester bond. The potential importance of mineral catalysis of the formation of RNA and other oligomers on primitive Earth is discussed.     

Driving in vitro selection of anti-HIV-1 TAR aptamers by magnesium concentration and temperature

In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.     

Chiral hybrid metal-organic dendrimers

[structure: see text] New chiral terpyridines containing Frechét-type dendrons have been readily synthesized by coupling dendritic benzyl bromide and 4'-[6-(2,2'-dihydroxy-1,1'-binaphthyl)]-2,2':6'2' '-terpyridine. These chiral dendritic terpyridines were used to efficiently construct high molecular weight hybrid metal-organic dendrimers based on the Ru(II)-bis(terpy) linkage. Preliminary fluorescence measurements show generation-dependent fluorescence quenching behavior of 3,5-dimethoxybenzyl peripherals by the [Ru(terpy)(2)](2+) unit.