Complementary Oligoribonucleotides Research Articles

Oligoarginine peptides slow strand annealing and assist non-enzymatic RNA replication.

The nonenzymatic replication of RNA is thought to have been a critical process required for the origin of life. One unsolved difficulty with nonenzymatic RNA replication is that template-directed copying of RNA results in a double-stranded product; following strand separation, rapid strand reannealing outcompetes slow nonenzymatic template copying, rendering multiple rounds of RNA replication impossible. Here we show that oligoarginine peptides slow the annealing of complementary oligoribonucleotides by up to several thousand-fold; however, short primers and activated monomers can still bind to template strands, and template-directed primer extension can still occur within a phase-separated condensed state, or coacervate. Furthermore, we show that within this phase, partial template copying occurs even in the presence of full-length complementary strands. This method for enabling further rounds of replication suggests one mechanism by which short, non-coded peptides could have enhanced early cellular fitness, potentially explaining how longer, coded peptides, i.e. proteins, came to prominence in modern biology.

Stereocontrolled Solid-Phase Synthesis of Phosphate/Phosphorothioate (PO/PS) Chimeric Oligodeoxyribonucleotides on an Automated Synthesizer Using an Oxazaphospholidine-Phosphoramidite Method.

Stereocontrolled solid-phase synthesis of phosphate/phosphorothioate chimeric oligodeoxyribonucleotides (PO/PS-ODNs) was achieved by integrating the conventional phosphoramidite method into a previously developed oxazaphospholidine method for the stereocontrolled synthesis of P-chiral oligonucleotides. P-Stereodefined PO/PS-ODNs with mixed sequences (up to 12-mers) were obtained in good yields and high stereoselectivities by reacting different combinations of monomers (conventional phosphoramidites/diastereopure nucleoside 3'-O-oxazaphospholidines), activators (ETT/CMPT), capping reagents (Pac2O/CF3COIm), and oxidizing/sulfurizing reagents (TBHP/POS) on an automated synthesizer. A thermal denaturation study examined the resultant diastereopure PO/PS-ODN 12-mers with three consecutive (Rp)- or (Sp)-PS-linkages at the internal or terminal regions of the molecules. We found that (Rp)-PO/PS-ODNs can only moderately destabilize duplexes with complementary oligoribonucleotides (ORNs) compared with their unmodified ODN counterparts (ΔTm = -0.4 °C per modification). In contrast, (Sp)-PO/PS-ODNs have larger destabilizing effects (ΔTm = -1.2 to -0.8 °C per modification). Although smaller destabilizing effects were observed when the (Sp)-PS-linkages were incorporated into the terminal regions of the molecule, there was a weaker correlation between the location of an incorporated (Rp)-PS-linkage and its destabilizing effect.

Cross-Linking Antisense Oligodeoxyribonucleotides with a Photoresponsive α-Chloroaldehyde Moiety for RNA Point Mutations.

Because point mutations in GTPase-coding genes have been reported to be responsible for the transformation of cells, anticancer reagents that react effectively and sequence selectively with target RNAs having a point mutation are highly desired. In this study, we developed novel photo-cross-linking oligodeoxyribonucleotides ((pro)PCA-ODNs) that had a caged α-chloroaldehyde group conjugated to a 2-methylpropanediyl backbone ((pro)PCA) in the middle of the strand. A kinetic study of the deprotection reaction of (pro)PCA-ODN revealed that the bis(2-nitrobenzyl)acetal group was completely deprotected within 1 min. Photo-cross-linking studies of (pro)PCA-ODNs with complementary oligoribonucleotides (ORNs) revealed that (pro)PCA-ODNs reacts efficiently and selectively with the target ORNs that have an adenosine or cytidine residue at a frontal position of the (pro)PCA residue without adverse effects of bases adjacent to the mutation site.

An expanded seed sequence definition accounts for full regulation of the hid 3′ UTR by bantam miRNA

MicroRNAs (miRNAs) are an abundant class of approximately 22 nucleotide (nt) long noncoding RNAs that negatively regulate gene expression post-transcriptionally through imperfect base-pairing interactions with sequences in the target messenger RNA (mRNA). We examined the interactions of the bantam miRNA with the 3' untranslated region (UTR) of the hid mRNA, and a synthetic derivative, in Drosophila S2 cells in order to define the relative contributions of proposed bantam binding sites. The contribution of the bantam miRNA to repression of reporter constructs carrying different 3' UTRs was evaluated by measuring derepression of reporter expression following the transfection of bantam complementary oligoribonucleotides (anti-bantam). Systematic excision of bantam miRNA target sequences in the hid 3' UTR identified by commonly used miRNA target prediction programs failed to relieve repression to the extent predicted by the anti-bantam experiment. However, removal of additional bantam complementary sequences (with a "seed" match to nucleotide 3-9) derepressed the reporter constructs to the full extent, arguing for a less narrow definition of the seed sequence. Further support for the potential contribution of the 3-9 seed register to microRNA-mediated gene regulation is provided by the experimental validation of several novel bantam targets identified with a more relaxed search algorithm.

Microarray-based label-free detection of RNA using bispyrene-modified 2′-O-methyl oligoribonucleotide as capture and detection probe

A novel oligonucleotide microarray that can detect RNAs without fluorescent labeling of sample RNAs was developed. As a capture and detection probe, bispyrene-modified 2'-O-methyl oligoribonucleotide (OMUpy2), whose fluorescence was dramatically increased when hybridized with its complementary RNA, was adopted. Fluorescence of the OMUpy2 tethered on the glass surface was enhanced as much as 22-fold by the addition of complementary oligoribonucleotide.

FASTDXL: A Generalized Screen to Trap Disulfide-Stabilized Complexes for Use in Structural Studies

Structural studies of macromolecular complexes have produced extraordinary insights into a wide variety of biological processes. Unfortunately, as structural biologists pursue larger and more challenging assemblies, weakly stable and/or nonspecific interactions can become significant roadblocks to structure determination. We have developed a rapid and effective pool-based screen, termed FASTDXL (focused array screening technique for disulfide X-linking), to produce and identify disulfide-stabilized protein-nucleic acid assemblies. A significant strength of FASTDXL is that it can take advantage of prior structural knowledge about molecular interactions, but does not necessarily rely upon it. A detailed application of the approach to the difficult problem of trapping a bacterial primase-ssDNA complex is described, validating the method as a route toward obtaining diffracting crystals suitable for structure determination.

Solid-phase detection of RNA using bispyrene-modified RNA probe

Bispyrene modified 2'-O-methyl oligoRNA (OMUpy2), which is a useful fluorescent probe for specific RNA detection, was immobilized on a glass substrate via various linker molecules to develop a more convenient RNA detection chip for gene expression study. As the fluorescence intensity of OMUpy2 enhanced greatly when it hybridized with its complementary RNA, it was expected that RNA detection by OMUpy2-immobilized chip (RNA-chip) required neither fluorescent labels to target nucleic acid nor washing protocol after the hybridization. As the linker molecules, oligo(dT), agarose, and poly(ethylene glycol) (PEG) were chosen, and the fluorescence from the chip was measured by a fluorescence microscope. Results indicated that the RNA-chip was able to detect the complementary oligoribonucleotide (cORN) without target labeling and washing protocols. Furthermore, the background emission was reduced as the length of the linker increased. In the case of PEG linker, 25-fold enhancement of the fluorescence intensity of the OMUpy2 was observed upon the addition of cORN.

Binary Hammerhead Ribozymes with Improved Catalytic Activity

A new design of binary hammerhead ribozymes displaying high catalytic activity and nucleolytic stability is described. These catalytic structures consist of two partially complementary oligoribonucleotides, capable of assembling into the hammerhead-like structure without tetraloop II on binding to the RNA target. A series of these binary ribozymes targeting the translation initiation region of multiple drug resistance gene mdr1 mRNA was synthesized and assessed in terms of catalytic activity under single and multiple reaction turnover conditions. Enhanced nuclease resistance of the binary ribozymes was achieved by incorporation of 2'-modified nucleotides at selected positions, along with addition of a 3'-3'-linked thymidine cap. The new binary ribozymes exhibit higher RNA cleavage activity than their full-length analogs because of faster dissociation of cleavage products. Furthermore, an excess of one of the ribozyme strands provides the possibility to unfold structured regions of the target RNA and facilitate productive complex formation.

Synthesis and Properties of Oligodeoxyribonucleotides Containing 2′‐O‐(2,3‐Dihydroxypropyl)‐ and 2′‐O‐(2‐Oxoethyl)arabinouridine Residues.

2’-O-(2,3-Dihydroxypropyl)arabinouridine-containing oligodeoxyribonucleotides were synthesized starting from a new modified nucleoside, viz., 2’-O-(2,3-dihydroxypropyl)arabinouridine, and the corresponding 3’-phosphoramidite. Oxidation of these oligodeoxyribonucleotides with sodium periodate afforded oligonucleotides containing 2’-O-(2-oxoethyl)arabinouridine residues. Subsequent modification of the aldehyde-containing oligonucleotides involved the reactions with 9-hydrazinoacridine and N-aminooxyacetyl peptide and reductive amination by 4-(1-pyrenyl)butyrohydrazide and biotin hydrazide. Thermal stabilities of duplexes of modified oligodeoxyribonucleotides with complementary oligodeoxyribonucleotides are slightly lower than those of natural duplexes. Duplexes with complementary oligoribonucleotides are substantially destabilized.

Synthesis and properties of oligodeoxyribonucleotides containing 2’-O-(2,3-dihydroxypropyl)- and 2’-O-(2-oxoethyl)arabinouridine residues

2’-O-(2,3-Dihydroxypropyl)arabinouridine-containing oligodeoxyribonucleotides were synthesized starting from a new modified nucleoside, viz., 2’-O-(2,3-dihydroxypropyl)arabinouridine, and the corresponding 3’-phosphoramidite. Oxidation of these oligodeoxyribonucleotides with sodium periodate afforded oligonucleotides containing 2’-O-(2-oxoethyl)arabinouridine residues. Subsequent modification of the aldehyde-containing oligonucleotides involved the reactions with 9-hydrazinoacridine and N-aminooxyacetyl peptide and reductive amination by 4-(1-pyrenyl)butyrohydrazide and biotin hydrazide. Thermal stabilities of duplexes of modified oligodeoxyribonucleotides with complementary oligodeoxyribonucleotides are slightly lower than those of natural duplexes. Duplexes with complementary oligoribonucleotides are substantially destabilized.

Synthesis and properties of fluorescent-labeled oligonucleotides containing amide-linked dinucleoside with pyrene

Oligonucleotides containing 2'-pyrene modified uridine (Upy) exhibit the increase of the pyrene monomer emission when hybridize with the complementary RNA. In order to examine the effect of restricted C3'-endoconformation of the Upy residue for the enhancement of the pyrene monomer emission, an oligodeoxyribonucleotide containing amide-linked dinucleoside with a pyrene group at the sugar residue (UapyODN) was synthesized by use of 3'-carboxymethyl-5'-O-dimethoxytrityl-2'-O-(1-pyrenylmethyl)-3'-deoxyuridine. NMR analysis indicated that the furanose ring of the uridine derivative was fixed to C3'-endo conformation. The UapyODN showed increase of fluorescence intensity to 7-fold at 375 nm when hybridized with complementary oligoribonucleotide. This result suggests that the UapyODN can detect the target RNA.

Sequence-Selective Cleavage of Oligoribonucleotides by 3d Transition Metal Complexes of 1,5,9-Triazacyclododecane-Functionalized 2‘-O-Methyl Oligoribonucleotides

2'-O-Methyl oligoribonucleotides bearing a 3'-[2,6-dioxo-3,7-diaza-10-(1,5,9-triazacyclododec-3-yl)decyl phospate conjugate group have been shown to cleave in slight excess of Zn(2+) ions complementary oligoribonucleotides at the 5'-side of the last base-paired nucleotide. The cleavage obeys first-order kinetics and exhibits turnover. The acceleration compared to the monomeric Zn(2+) 1,5,9-triazacyclododecane chelate is more than 100-fold. In addition, 2'-O-methyl oligoribonucleotides having the 1,5,9-triazacyclododec-3-yl group tethered to the anomeric carbon of an intrachain 2-deoxy-beta-d-erythro-pentofuranosyl group via a 2-oxo-3-azahexyl, 2,6-dioxo-3,7-diazadecyl, or 2,9-dioxo-3,10-diazatridecyl linker have been studied as cleaving agents. These cleave as zinc chelates a tri- and pentaadenyl bulge opposite to the conjugate group approximately 50 times as fast as the monomeric chelate and show turnover. The cleavage rate is rather insensitive to the length of linker. Interestingly, a triuridyl bulge remains virtually intact in striking contrast to a triadenyl bulge. Evidently binding of the zinc chelate to a uracil base prevents its catalytic action. Replacement of Zn(2+) with Cu(2+) or Ni(2+) retards the cleaving activity of all the cleaving agents tested.

Preparation of azacrown-functionalized 2'-O-methyl oligoribonucleotides, potential artificial RNases.

An improved synthesis for 3-(3-aminopropyl)- and 3-(3-mercaptopropyl)-1,5,9-triazacyclododecane has been developed and alternative methods for their conjugation to oligonucleotides have been described. Accordingly, the 3-aminopropyl azacrown and its N-(3-aminopropanoyl)-3-aminopropyl analogue have been tethered to the 3'-terminus of a 2'-O-methyloligoribonucleotide by aminolytic cleavage of the thioester linker utilized for the chain assembly. Studies on a monomeric model compound verify that the reaction proceeds solely by the attack of the primary amino group. 5'-Conjugation has been achieved by introducing a 2-benzylthio-2-oxoethyl group to the 5'-terminus as a phosphoramidite reagent and cleaving the thioester bond with the 3-aminopropyl azacrown. For intrachain conjugation, a phosphoramidite reagent derived from 1-deoxy-1-(2-benzylthio-2-oxoethyl)-beta-d-erythro-pentofuranose has been inserted in a desired position within the chain and subjected to on-support aminolysis with the 3-aminopropyl azacrown or its N-(3-aminopropanoyl)-3-aminopropyl and N-(6-aminohexanoyl)-3-aminopropyl analogues. The 3-mercaptopropyl-derivatized azacrown has been tetherd by a disulfide bond to a 3'-(3-mercaptoalkyl)phosphate-tailed oligonucleotide. The 3'- and intrachain-tethered conjugates have been shown to cleave as their Zn(II) chelate complementary oligoribonucleotide sequences.

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis.

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.

Inhibition of human immunodeficiency virus type 1 replication by P-stereodefined oligo(nucleoside phosphorothioate)s in a long-term infection model

Oligo(nucleoside phosphorothioate)s (S-ODNs), if prepared by conventional methods, consist of a mixture of diastereomers by virtue of the asymmetry of the phosphorus atom involved in the internucleotide linkages. This may affect the stability of the complexes formed between S-ODNs and complementary oligoribonucleotides, which is commonly accepted as the most important factor in determining the efficacy of an antisense approach. Using HIV-1-infected MOLT-4 cells via a long-term culture approach, we studied the influence of the P-chirality sense of stereodefined 28mer oligo(nucleoside phosphorothioate)s, [All-Rp]-S-ODN- gag-28-AUG and [All-Sp]-S-ODN- gag-28-AUG, complementary to the sequence starting at the AUG initiation codon of the gag mRNA of HIV-1, upon the anti-HIV-1 activity. The [All-Sp]-S-ODN- gag-28-AUG at a low concentration of 0.5 μM can completely suppress HIV-1 gag p24 antigen expression in HIV-1-infected MOLT-4 clone 8 cells for 32 days. Cells treated with [All-Rp]-S-ODN- gag-28-AUG (0.5 μM) showed a high level of the antigen expression at day 16. Furthermore, satisfactory suppression could not be achieved from a random [Mix]-S-ODN- gag-28-AUG, consisting of a diastereomeric mixture of the oligonucleotides. Our results suggest that chemotherapy based upon the use of stereodefined antisense [All-Sp] S-ODN may be a more effective method for reducing the viral burden in HIV-1-infected individuals.

Characterization of RNA structure by bis-pyrene-labeled 2'-O-methyloligonucleotides.

Properties of 2'-O-methyloligoribonucleotides containing two consecutive 2'-O-(1-pyrenylmethyl)uridine were investigated as a fluorescent probe to search the single strand regions of RNA. The bis-pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy2) induced the formation of pyrene dimer upon hybridization with the complementary oligoribonucleotides and showed remarkable appearance of broad structureless fluorescence at 480 nm. Contrarily, when OMUpy2 was hybridized with the complementary oligodeoxyribonucleotides, such enhancement of fluorescence was scarcely observed. When various OMUpy2 were applied to E. coli 5S-rRNA, the fluorescence intensity at 480 nm was varied in a sequence specific manner.

Study on RNA structure by pyrene-labeled 2'-O-methyloligoribonucleotides.

Properties of 2'-O-methyloligoribonucleotides containing 2'-O-(1-pyrenylmethyl)uridine were investigated as the fluorescent probe to search the single strand regions on RNA secondary and tertiary structure. The pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) showed remarkable increase of fluorescence intensity to 333-fold at 375 nm when hybridized with the complementary oligoribonucleotide. When OMUpy, complementary to loop or stem regions, was applied to E. coli 5S-rRNA, the fluorescence intensities were increased in a sequence specific manner. The difference of the fluorescence intensities corresponds to the higher-order structure of 5S-rRNA, suggesting that pyrene-labled 2'-O-methyloligoribonucleotide can be applicable to search single strand regions of RNA.

Synthesis, biochemical and biological studies on oligonucleotides bearing a lipophilic dimethoxytrityl group.

Dimethoxytritylphosphono-oligonucleotide conjugates have been prepared. They are totally resistant to nucleases present in human serum and do not affect cleavage of a complementary oligoribonucleotide by RNase H. Conjugates possessing a phosphate backbone gave better antisense inhibition of expression of plasminogen activator inhibitor type-1 within endothelial cells as compared with unconjugated oligonucleotides.

Binding affinity and specificity of Escherichia coli RNase H1: impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids.

In this study we report for the first time the binding affinity of RNase H1 for oligonucleotide duplexes. We used a previously described 17-mer antisense sequence [Monia, B. P., Johnston, J. F., Ecker, D. J., Zounes, M. A., Lima, W. F., & Freier, S. M. (1992) J. Biol. Chem. 267, 19954-19962] hybridized to a complementary oligoribonucleotide to evaluate both the binding affinity and the catalytic rate of RNase H1. The dissociation constants (Kd) of RNase H1 for the various substrates tested were determined by inhibition analysis using chemically modified noncleavable oligonucleotide heteroduplexes. Catalytic rates were determined using heteroduplex substrates containing chimeric antisense oligonucleotides composed of a five-base deoxynucleotide sequence flanked on either side by chemically modified nucleotides. We find that the enzyme preferentially binds A-form duplexes: RNase H bound A-form duplexes (RNA:RNA and DNA:RNA) approximately 60-fold tighter than B-form duplexes (DNA:DNA) and approximately 300-fold tighter than single-strand oligonucleotides. The enzyme exhibited equal affinity for both the wild type (RNA:DNA) oligonucleotide substrate and heteroduplexes containing various 2'-sugar modifications, while the cleavage rates for these chemically modified substrates were without exception slower than for the wild type substrate. The introduction of a single positively charged 2'-propoxyamine modification into the chimeric antisense oligonucleotide portion of the heteroduplex substrate resulted in both decreased binding affinity and a slower rate of catalysis by RNase H. The cleavage rates for heteroduplexes containing single-base mismatch sequences within the chimeric oligonucleotide portion varied depending on the position of the mismatch but had no effect on the binding affinity of the enzyme. These results offer further insights into the physical binding properties of the RNase H-substrate interaction as well as the design of effective antisense oligonucleotides.

Helical structure formation between complementary oligonucleotides. Minimum chain length required for the template-directed synthesis of oligonucleotides.

Helix formation between various combinations of 3'-5' linked oligoribouridylates and oligoriboadenylates from dimer to dodecamer has been studied to gain information on the chain-length requirement for the template-directed condensation of oligoribonucleotides. We have measured the helix formation under high oligoribonucleotide concentration in the presence of magnesium ion at 0-50 degrees C by UV or CD, as many model processes of oligoribonucleotides replication have been carried out under such conditions. Adenylic acid, (pA), diadenylic acid, (pA)2, or triadenylic acid, (pA)3, forms a helix with poly(U) or oligo(U) with a chain length of more than eight. On the other hand, neither uridylic acid, (pU), nor diuridylic acid. (pU)2, can form a helix with oligo(A) or poly(A). Triuridylic acid, (pU)3, or the longer oligo(U) forms a helix with oligo(A) with a chain length of over six. The results suggest that a trimer is the minimum unit as an incorporating nucleotide for conducting any set of nonenzymatic template-directed synthesis, A --> U and U --> A, as the nonenzymatic template-directed condensation of oligoribonucleotides correlates well with the results of helix formation of complementary oligoribonucleotides. We have further found the partial helix formation between 2'-5' linked decauridylate, (pU)10, and pA or 2'-5' linked (pA)2 at 0 degrees C, which indicates the possibility of the template activity of long 2'-5' linked oligonucleotides for the nonenzymatic oligonucleotide synthesis.