Diels--Alder bioconjugation of diene-modified oligonucleotides.

In an effort to offer complementary technology for covalent biomolecule modification (bioconjugation), we have developed a method that exploits the aqueous acceleration of Diels--Alder reactions for this purpose. Three different diene phosphoramidite reagents have been synthesized that enable diene modification of synthetic oligonucleotides prepared by the phosphoramidite method. Clean and efficient Diels--Alder cycloaddition of these diene oligonucleotides with maleimide dieneophiles was carried out, and the labeled oligonucleotide bioconjugates were characterized by HPLC and electrospray mass spectrometry. Dieneophile stoichiometry, temperature, and pH are all parameters that were shown to influence the efficiency of the process.

Tc-99m-labeling of modified RNA.

The synthesis of Tc-99m-labeled, modified RNA is reported. This new class of radiopharmaceuticals is of potential interest as target specific imaging agents. The preparation of N3S-RNA was achieved by coupling protected MAG2-units to amino modified ON's. The N3S-RNA was Tc-99m-labeled with 90-95% radiochemical yield and specific activities of 37MBq/nmol leading to 1:1-Tc-99m-N3S-aptamers.

Purification of a highly modified RNA-aptamer. Effect of complete denaturation during chromatography on product recovery and specific activity.

To evaluate RNA-aptamers as potential drug candidates, efficient and scaleable purification protocols are needed. Because aptamers are highly structured and rigid molecules, denaturation during the purification process is a critical aspect to obtain a pure and active product. A two-step chromatographic procedure was developed to purify a synthetic anti-VEGF aptamer at the preparative scale. A reversed-phase chromatographic step was optimized with a highly hydrophobic ion pairing reagent, followed by ion-exchange chromatography in which heat and a chaotropic salt were used. Because of the presence of 2'-modified ribose, denaturation conditions had to be optimized in both chromatographic steps to achieve a fully active molecule.

Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis.

A new activator for the coupling of phosphoramidites to the 5'-hydroxyl group during oligonucleotide synthesis is introduced. The observed time to complete coupling is twice as fast with 4, 5-dicyanoimidazole (DCI) as the activator, compared with 1 H -tetrazole. The effectiveness of DCI is thought to be based on its nucleophilicity. DCI is soluble in acetonitrile up to 1.1 M at room temperature and can be used as the sole coupling activator during routine automated solid phase synthesis of oligonucleotides. The addition of 0.1 M N -methylimidazole to 0.45 M 1 H -tetrazole also results in higher product yields during oligonucleotide synthesis than observed with 1 H -tetrazole alone.

Efficient process technologies for the preparation of oligonucleotides.

Efficient process technologies for the preparation of 2'-substituted nucleoside monomers, as well as for oligonucleotide preparation, are introduced. A novel method for efficient preparation of 2'-substituted uridines is presented. This method employs the 3'-hydroxyl group of 2,2'-anhydrouridine as a tether for the facile intramolecular introduction of nucleophiles to the 2'-position. It allows access to 2'-alkoxy substituents from their alcohol precursors and to substituted 2'-amino substituents, such as the novel O-substituted 2'-hydroxylaminouridines. A novel process for large-scale oligonucleotide synthesis is discussed, which allows solution phase coupling of the monomer to the growing oligonucleotide chain. This is followed by selective isolation of productive coupling product by anchoring to a resin. Release from this resin completes a coupling cycle.

Potent 2'-amino-2'-deoxypyrimidine RNA inhibitors of basic fibroblast growth factor.

Screening of random oligonucleotide libraries with SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505-510] has emerged as a powerful method for identifying high-affinity nucleic acid ligands for a wide range of molecular targets. Nuclease sensitivity of unmodified RNA and DNA, however, imposes considerable restrictions on their use as therapeutics or diagnostics. Modified RNA in which pyrimidine 2'-hydroxy groups have been substituted with 2'-amino groups (2'-aminopyrimidine RNA) is known to be substantially more resistant to serum nucleases. We report here on the use of SELEX to identify high-affinity 2'-aminopyrimidine RNA ligands to a potent angiogenic factor, basic fibroblast growth factor (bFGF). High-affinity ligands with the same consensus primary structure have been isolated from two independent libraries of approximately 6 x 10(14) molecules containing 30 or 50 randomized positions. Compared to unmodified RNA with the same sequence, 2'-aminopyrimidine ligands are at least 1000-fold more stable in 90% human serum. The sequence information required for high-affinity binding to bFGF is contained within 24-26 nucleotides. The minimal ligand m21A (5'-GGUGUGUGGAAGACAGCGGGUGGUUC-3'; G = guanosine, A = adenosine, C = 2'-amino-2'-deoxycytidine, U = 2'-amino-2'-deoxyuridine, and C = 2'-amino-2'-deoxycytidine or deoxycytidine) binds to bFGF with an apparent dissociation constant (Kd) of 3.5 +/- 0.3) x 10(-10) M at 37 degrees C in phosphate-buffered saline (pH 7.4). Disassociation of m21A from bFGF is adequately described with a first-order rate constant of (1.96 +/- 0.08) x 10(-3) s-1 (t1/2 = 5.9 min). The calculated value for the association rate constant (kon = k(off)/Kd) was 5.6 x 10(6) M-1 s-1. Highly specific binding of m21A to bFGF was observed: binding to denatured bFGF, five proteins from the FGF family (acidic FGF, FGF-4, FGF-5, FGF-6, and FGF-7), and four other heparin binding proteins is substantially weaker under the same conditions with KdbFGF/Kdprotein values ranging from (4.1 +/- 1.4) x 10(-2) to > 10(-6). Heparin but not chondroitin sulfate competed for binding of m21A to bFGF. In cell culture, m21A inhibited [125I]bFGF binding to both low-affinity sites (ED50 approximately 1 nM) and high-affinity sites (ED50 approximately 3 nM) on CHO cells expressing transfected FGF receptor-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Ribonucleosides and RNA.

Landmark discoveries such as the autocatalytic cleavage activity of certain RNA molecules, as well as small oligoribonucletide ribozymes and later the in vitro evolution of novel bioactive oligoribonucleotides (SELEX), have created entire new fields of biochemical research. The discovery of SELEX has provided a method for producing high-affinity nucleic acid ligands with high binding specificity to important medicinal targets. Including modified nucleotides into RNA ligands derived from SELEX may yield improved RNA therapeutics. The chemistry of oligoribonucleotides in comparison to oligodeoxyribonucleotides has led to resurgent attention on the role of modified nucleotides in RNA structure and function. Such modifications are also employed to impart stability towards endonuclease degradation on oligoribonucleotides.

Cis,cis-muconate lactonizing enzyme from Trichosporon cutaneum: evidence for a novel class of cycloisomerases in eucaryotes.

The absolute stereochemical courses of cis,cis-muconate lactonizing enzyme (MLE;EC 5.5.1.1) from Trichosporon cutaneum (TcMLE) and chloromuconate cycloisomerase (MLE II; EC 5.5.1.7) from Pseudomonas sp B13 have been determined from 1H NMR measurements. Both cycloisomerases convert cis,cis-muconate to (4S)-muconolactone by a syn lactonization, the absolute stereochemical outcome of which is identical to that observed with MLE from Pseudomonas putida. The regiochemical courses of cyclization of 3-halo-cis,cis-muconates by TcMLE and MLE II have been characterized and shown to differ in a halogen substituent dependent manner, suggesting at least a different active site architecture of the two MLEs. Moreover, the regiochemical preferences of MLE II and TcMLE parallel results previously observed for the nonenzymatic lactonization of the 3-halomuconates at pH 1-6 and in concentrated HCl, respectively, in which alternate mechanisms of cyclization were proposed [Pieken, W. A., & Kozarich, J. W. (1990) J. Org. Chem. 55, 3029-3035]. Complementary DNA clones encoding TcMLE have been isolated from phenol induced T. cutaneum cDNA using the polymerase chain reaction. The deduced amino acid sequence does not exhibit any similarity to that of MLE from P. putida. It does however, exhibit moderate sequence similarity (21% residue identity, 14 gaps) with 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa, which catalyzes a regiochemically analogous and stereochemically identical lactonization reaction with 3-carboxymuconate. The limited data available suggest that the fungal CMLE and yeast MLE are representative of a unique class of eucaryotic cycloisomerases which have evolved convergently with the bacterial MLEs.

Importance of exocyclic base functional groups of central core guanosines for hammerhead ribozyme activity.

The three guanosines of the central core of a hammerhead ribozyme were replaced by 2-aminopurine ribonucleoside, xanthosine, isoguanosine, inosine, and deoxyguanosine. These analogues were incorporated by automated solid-phase synthesis, with the exception of isoguanosine. This was introduced by ligating a donor, which carried the isoguanosine at its 5'-end, and an acceptor oligoribonucleotide by a T4 DNA ligase-catalyzed reaction. Most of these modifications lowered the rate constant of cleavage by the hammerhead ribozyme drastically. Inspection of the possible hydrogen-bonding interactions disturbed by these modifications suggests that there is no G12A9 or A13G8 mismatched base pair in the central region. Increasing the Mg2+ concentration from 10 to 50 mM did not enhance these rates appreciably. This makes it improbable that the guanosines, including their 2'-hydroxyl groups, are involved in the binding of the catalytically active Mg2+. Transition-state destabilizing energies of 0.6-4.7 kcal mol-1 suggest that essentially all guanosines are involved in a hydrogen-bonding network.

Chemically modified RNA: approaches and applications.

The modification of phosphate into phosphorothioate internucleotidic linkages in various RNAs and their usefulness in identifying phosphate positions essential for function are described. Several modifications of the 2'-hydroxyl group of the ribose, particularly the replacement by fluorine atoms and amino groups, is discussed. These studies have been concentrated on hammerhead ribozymes in order to determine hydroxyl groups important for the catalytic activity. In addition these derivatives have been instrumental in rendering ribozymes more stable toward nucleases.

Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications.

The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed.

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.

Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes.

The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.

Structure-function relationship of hammerhead ribozymes as probed by 2'-modifications.

Hammerhead ribozymes containing 2'-fluoro- or 2'-aminonucleotides were prepared by automated chemical synthesis. Incorporation of 2'-fluorouridines, 2'-fluorocytidines or 2'-aminouridines did not appreciably decrease catalytic activity. The presence of 2'-aminocytidines, however, reduced the activity about 20-fold. No catalytic activity could be measured for ribozymes in which all adenosines were replaced by the 2'-fluoro analogue in presence of MgCl2. No single position could be found responsible for this loss of activity. In an attempt to construct ribozymes to hydrolyse HIV-RNA in the 5'-LTR region several constructs were tested on synthetic substrate as well as on run-off transcripts of about 1000 nucleotides length.