RNA Linkage Research Articles

Human vault-associated non-coding RNAs bind to mitoxantrone, a chemotherapeutic compound

Human vaults are the largest cytoplasmic ribonucleoprotein and are overexpressed in cancer cells. Vaults reportedly function in the extrusion of xenobiotics from the nuclei of resistant cells, but the interactions of xenobiotics with the vault-associated proteins or non-coding RNAs have never been directly observed. In the present study, we show that vault RNAs (vRNAs), specifically the hvg-1 and hvg-2 RNAs, bind to a chemotherapeutic compound, mitoxantrone. Using an in-line probing assay (spontaneous transesterification of RNA linkages), we have identified the mitoxantrone binding region within the vRNAs. In addition, we analyzed the interactions between vRNAs and mitoxantrone in the cellular milieu, using an in vitro translation inhibition assay. Taken together, our results clearly suggest that vRNAs have the ability to bind certain chemotherapeutic compounds and these interactions may play an important role in vault function, by participating in the export of toxic compounds.

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.

Characterization of a fluorescence-signaling and RNA-cleaving deoxyribozyme

DNA enzymes (deoxyribozymes or DNAzymes) are single-stranded DNA molecules with catalytic function. Previously, we isolated several RNA-cleaving deoxyribozymes with fluorescence-signaling properties. These special DNA molecules are capable of cleaving an RNA linkage embedded within a DNA sequence and flanked by a pair of deoxyribothymidines modified with a fluorophore (fluorescein) and a quencher (dabcyl). Here we report on the sequence truncation and secondary structure characterization of one such deoxyribozyme known as pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicates that pH6DZ1 has a four-way junction-like secondary structure comprising four short duplexes, three hairpin loops, and three inter-helical unpaired elements.

Synthesis of 2‘-C-Difluoromethylribonucleosides and Their Enzymatic Incorporation into Oligonucleotides

[Chemical reaction: See text] Nucleosides bearing a branched ribose have significant promise as therapeutic agents and biotechnological and biochemical tools. Here we describe synthetic entry into a new subclass of these analogues, 2'-C-beta-difluoromethylribonucleosides. We constructed the glycosylating agent 4 in three steps from 1,3,5-tri-O-benzoyl-alpha-D-ribofuranose 1. The key steps included nucleophilic addition of difluoromethyl phenyl sulfone to 2-ketoribose 2 followed by mild and efficient reductive desulfonation. Ribofuranose 4 glycosylated bis(trimethylsilyl)uracil directly, giving difluoromethyluridine 7 efficiently after deprotection. Conversion of 4 to the corresponding ribofuranosyl bromide allowed efficient access to C, A, and G analogues. A related approach starting from methyl D-ribofuranose offered synthetic entry into the diastereomeric manifold, 2'-C-alpha-difluoromethyl-arabino-alpha-pyrimidine. To incorporate 2'-C-beta-difluoromethyluridine into an oligodeoxynucleotide we converted 7 to the bisphosphate and carried out successive ligation reactions using T4 RNA ligase and T4 DNA ligase. Analogous to natural RNA linkages, the resulting oligonucleotide undergoes hydroxide-catalyzed backbone scission at the difluoromethyluridine residue via internal transphosphorylation.

Parallel Selections In Vitro Reveal a Preference for 2′–5′ RNA Ligation upon Deoxyribozyme-Mediated Opening of a 2′,3′-Cyclic Phosphate

We previously used in vitro selection to identify Mg(2+)-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5'-hydroxyl group with a 2',3'-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2'-5' phosphodiester bonds rather than native 3'-5' linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2'-5' linkages. This includes deoxyribozymes with an arrangement that favors 3'-5' linkages for a different chemical reaction, that of a 2',3'-diol plus 5'-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2'-5' RNA linkages upon opening of a 2',3'-cyclic phosphate with a 5'-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2',3'-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.

Zn2+-Dependent Deoxyribozymes That Form Natural and Unnatural RNA Linkages

We report Zn(2+)-dependent deoxyribozymes that ligate RNA. The DNA enzymes were identified by in vitro selection and ligate RNA with k(obs) up to 0.5 min(-)(1) at 1 mM Zn(2+) and 23 degrees C, pH 7.9, which is substantially faster than our previously reported Mg(2+)-dependent deoxyribozymes. Each new Zn(2+)-dependent deoxyribozyme mediates the reaction of a specific nucleophile on one RNA substrate with a 2',3'-cyclic phosphate on a second RNA substrate. Some of the Zn(2+)-dependent deoxyribozymes create native 3'-5' RNA linkages (with k(obs) up to 0.02 min(-)(1)), whereas all of our previous Mg(2+)-dependent deoxyribozymes that use a 2',3'-cyclic phosphate create non-native 2'-5' RNA linkages. On this basis, Zn(2+)-dependent deoxyribozymes have promise for synthesis of native 3'-5'-linked RNA using 2',3'-cyclic phosphate RNA substrates, although these particular Zn(2+)-dependent deoxyribozymes are likely not useful for this practical application. Some of the new Zn(2+)-dependent deoxyribozymes instead create non-native 2'-5' linkages, just like their Mg(2+) counterparts. Unexpectedly, other Zn(2+)-dependent deoxyribozymes synthesize one of three unnatural linkages that are formed upon the reaction of an RNA nucleophile other than a 5'-hydroxyl group. Two of these unnatural linkages are the 3'-2' and 2'-2' linear junctions created when the 2'-hydroxyl of the 5'-terminal guanosine of one RNA substrate attacks the 2',3'-cyclic phosphate of the second RNA substrate. The third unnatural linkage is a branched RNA that results from attack of a specific internal 2'-hydroxyl of one RNA substrate at the 2',3'-cyclic phosphate. When compared with the consistent creation of 2'-5' linkages by Mg(2+)-dependent ligation, formation of this variety of RNA ligation products by Zn(2+)-dependent deoxyribozymes highlights the versatility of transition metals such as Zn(2+) for mediating nucleic acid catalysis.

Cooperation of metal-ion fixation and target-site activation for efficient site-selective RNA scission

Iminodiacetate-DNA conjugates and acridine-DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)-iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine-DNA conjugate.

Directing the Outcome of Deoxyribozyme Selections To Favor Native 3‘−5‘ RNA Ligation

Previous experiments have identified numerous RNA ligase deoxyribozymes, each of which can synthesize either 2',5'-branched RNA, linear 2'-5'-linked RNA, or linear 3'-5'-linked RNA. These products may be formed by reaction of a 2'-hydroxyl or 3'-hydroxyl of one RNA substrate with the 5'-triphosphate of a second RNA substrate. Here the inherent propensities for nucleophilic reactivity of specific hydroxyl groups were assessed using RNA substrates related to the natural sequences of spliceosome substrates and group II introns. With the spliceosome substrates, nearly half of the selected deoxyribozymes mediate a ligation reaction involving the natural branch-point adenosine as the nucleophile. In contrast, mostly linear RNA is obtained with the group II intron substrates. Because the two sets of substrates differ at only three nucleotides, we conclude that the location of the newly created ligation junction in DNA-catalyzed branch formation depends sensitively on the RNA substrate sequences. During the experiment that led primarily to branched RNA, we abruptly altered the selection strategy to demand that the deoxyribozymes create linear 3'-5' linkages by introducing an additional selection step involving the 3'-5'-selective 8-17 deoxyribozyme. Although no 3'-5' linkages (<or=1%) were detectable in the pool products at the point that the 3'-5' selection pressure was applied, deoxyribozymes that specifically create 3'-5' linkages quickly emerged within a few selection rounds. Our success in obtaining 3'-5' linkages via this approach shows that the outcome of deoxyribozyme selection experiments can be dramatically redirected by strategic changes in the selection procedure, even at a late stage. These results relate to natural selection, in which abrupt environmental variation can provide a rapid change in selection pressure. Linear 3'-5' RNA linkages are an important practical objective because the native backbone is desirable in site-specifically modified ribozymes assembled by ligation. Therefore, this new approach to obtain 3'-5'-selective RNA ligase deoxyribozymes is particularly important for ongoing selection efforts.

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.

MRNA molecules containing murine leukemia virus packaging signals are encapsidated as dimers.

Prior work by others has shown that insertion of psi (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3' untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV psi or with psi sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the psi region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Psi(+) mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops.

Pseudorotation of natural and chemically modified biological phosphoranes: implications for RNA catalysis.

Biological phosphates form the anionic backbone linkage in DNA and RNA and play a central role in the regulation of cellular processes including signaling, respiration, replication, and translation. Consequently, the study of the chemistry of biological phosphates is an area of great importance. Of particular interest are the mechanisms by which RNA can catalyze fairly complicated reactions such as the transphosphorylation and hydrolysis of phosphodiester bonds. A useful experimental strategy to probe the catalytic mechanisms of RNA enzymes is the study of thio effects: changes in the reaction rate that occur upon substitution of key phosphate oxygen atoms with sulfur atoms. Kinetic analysis of thio effects provides insight into the specific role that these oxygen positions play in catalysis. Theoretical methods are powerful tools to aid the interpretation of kinetic data through characterization of the structure and energetics of transition states and intermediates along competing reaction paths. The dominant reaction path for transphosphorylation in RNA (Scheme 1) proceeds via an in-line attack of an activated 2’-hydroxy group of the RNA sugar ring on the reactive phosphate group to produce a pentavalent phosphorane transition state or intermediate, followed by the cleavage of the P-O bond to produce a 2’,3’-cyclic phosphate. Experimental and theoretical data suggest a dianionic oxyphosphorane transphosphorylation intermediate is kinetically indistinguishable from a transition state and is too short-lived to undergo other processes such as protonation or pseudorotation. As the pH is lowered, acid-catalyzed migration products begin to emerge, which result from pseudorotation of a singly or doubly protonated phosphorane intermediate (Scheme 2). The ratio of products resulting from phosphate hydrolysis/ transphosphorylation and isomerization (migration) and their pH dependence involve a balance between the endoand exo-

Structure-function correlations derived from faster variants of a RNA ligase deoxyribozyme.

We previously reported the in vitro selection of several Mg2+-dependent deoxyribozymes (DNA enzymes) that synthesize a 2'-5' RNA linkage from a 2',3'-cyclic phosphate and a 5'-hydroxyl. Here we subjected the 9A2 deoxyribozyme to re-selection for improved ligation rate. We found two new DNA enzymes (7Z81 and 7Z48) that contain the catalytic core of 7Q10, a previously reported small deoxyribozyme that is unrelated in sequence to 9A2. A third new DNA enzyme (7Z101) is unrelated to either 7Q10 or 9A2. The new 7Z81 and 7Z48 DNA enzymes have ligation rates over an order of magnitude higher than that of 7Q10 itself and they have additional sequence elements that correlate with these faster rates. Our findings provide insight into structure-function relationships of catalytic nucleic acids.

Engineering DNA aptamers and DNA enzymes with fluorescence-signaling properties

Abstract Single-stranded DNA molecules with ligand-binding ability and catalytic function, referred to as DNA aptamers and DNA enzymes, respectively, are special DNA sequences isolated from random-sequence DNA libraries by “in vitro selection”. These two new classes of artificial DNA molecules have the potential of being used as molecular tools in a variety of innovative applications ranging from biosensing to gene regulation. Our laboratory is interested in engineering fluorescence-signaling DNA aptamers and DNA enzymes that can be widely exploited for detection-directed applications. In this article, we will first discuss our recent efforts on the rational design of a new class of signaling aptamers denoted “structure- switching signaling aptamers”, which report target binding by switching structures from DNA/DNA duplex to DNA/target complex. We will then describe the in vitro selection of fluorescence-signaling DNA enzymes that exhibit a synchronized catalysis-signaling capability by cleaving a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by closely spaced fluorophore-quencher pair. Potential utilities of these signaling DNA molecules will also be discussed.

An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling.

DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.

Non-covalent ternary systems (DNA-acridine hybrid/DNA/lanthanide(iii)) for efficient and site-selective RNA scission

The target phosphodiester linkage in RNA is activated by the combination of acridine-attached DNA and unmodified DNA, so that the RNA is site-selectively hydrolysed at this linkage by free LuIII ion.

Relationship between internucleotide linkage geometry and the stability of RNA.

The inherent chemical instability of RNA under physiological conditions is primarily due to the spontaneous cleavage of phosphodiester linkages via intramolecular transesterification reactions. Although the protonation state of the nucleophilic 2'-hydroxyl group is a critical determinant of the rate of RNA cleavage, the precise geometry of the chemical groups that comprise each internucleotide linkage also has a significant impact on cleavage activity. Specifically, transesterification is expected to be proportional to the relative in-line character of the linkage. We have examined the rates of spontaneous cleavage of various RNAs for which the secondary and tertiary structures have previously been modeled using either NMR or X-ray crystallographic data. Rate constants determined for the spontaneous cleavage of different RNA linkages vary by almost 10,000-fold, most likely reflecting the contribution that secondary and tertiary structures make towards the overall chemical stability of RNA. Moreover, a correlation is observed between RNA cleavage rate and the relative in-line fitness of each internucleotide linkage. One linkage located within an ATP-binding RNA aptamer is predicted to adopt most closely the ideal conformation for in-line attack. This linkage has a rate constant for transesterification that is approximately 12-fold greater than is observed for an unconstrained linkage and was found to be the most labile among a total of 136 different sites examined. The implications of this relationship for the chemical stability of RNA and for the mechanisms of nucleases and ribozymes are discussed.

Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2‘-Hydroxyl Group

A detailed understanding of the susceptibility of RNA phosphodiesters to specific base-catalyzed cleavage is necessary to approximate the stability of RNA under various conditions. In addition, quantifying the rate enhancements that can be produced exclusively by this common cleavage mechanism is needed to fully interpret the mechanisms employed by ribonucleases and by RNA-cleaving ribozymes. Chimeric DNA/RNA oligonucleotides were used to examine the rates of hydroxide-dependent degradation of RNA phosphodiesters under reaction conditions that simulate those of biological systems. Under neutral or alkaline pH conditions, the dominant pathway for RNA degradation is an internal phosphoester transfer reaction that is promoted by specific base catalysis. As expected, increasing the concentration of hydroxide ion, increasing the concentration of divalent magnesium, or raising the temperature accelerates strand scission. In most instances, the identities of the nucleotide bases that flank the target RNA linkage have a negligible effect on the pKa of the nucleophilic 2‘-hydroxyl group, and only have a minor effect on the maximum rate constant for the transesterification reaction. Under representative physiological conditions, specific base catalysis of RNA cleavage generates a maximum rate enhancement of ∼100 000-fold over the background rate of RNA transesterification. The kinetic parameters reported herein provide theoretical limits for the stability of RNA polymers and for the proficiency of RNA-cleaving enzymes and enzyme mimics that exclusively employ a mechanism of general base catalysis.

The synthesis and hybridization properties of an oligonucleotide containing hexafluoroacetone ketal internucleotide linkages

The hexafluoroketal functionality is isosteric with the phosphate linkage in DNA and RNA. A thymidine:thymidine dimer bearing a hexafluoroacetone ketal internucleotide linkage has been synthesized from hexafluoroacetone using a Mitsunobu reaction. The resulting dimer has been incorporated into an oligonucleotide and has been shown to result in poorer binding to a complementary DNA and RNA relative to an unmodified control.

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

Chemical Cleavage of 5′-Linked Protein from Tobacco Ringspot Virus Genomic RNAs and Characterization of the Protein–RNA Linkage

Each of the two genomic RNAs of tobacco ringspot nepovirus is known to have a 5′-linked protein, the VPg. We report a simplified analysis of the covalent VPg-RNA connection that allowed us to identify the 5′ nucleotide residue of each genomic RNA and its phosphodiester link to a specific serine residue of the VPg, without resorting toin vivolabeling with32P,in vitroradioiodination, or separation of the two genomic RNAs. Unfractionated genomic RNA was incubated with an oligodeoxyribonucleotide specific for the 5′ region of either RNA 1 or RNA 2 and ribonuclease H. Reaction products were 3′-end-labeled and were fractionated by gel electrophoresis. The most highly labeled product derived from each genomic RNA was identified as a VPg-oligoribonucleotide (VPg-5′-oligo) by its sensitivity to proteinase. In a presumed β-elimination reaction that apparently was more rapid than phosphodiester cleavage, incubation in alkaline sodium bicarbonate released a rapidly migrating product, 5′-oligo. Phosphatase-treated 5′-oligo accepted 5′-label in a polynucleotide kinase-catalyzed reaction, and uridylate was identified as the 5′ terminal residue for both RNA 1 and RNA 2. Results from Edman degradation of the VPg suggest that the VPg is linked at serine 5 to the 5′ uridylate of each genomic RNA.