A new method for 3''-labelling of polyribonucleotides by phosphorylation with RNA ligase and its application to the 3''-modification for joining reactions.

P1-Adenosine 5'-P2-o-nitrobenzyl pyrophosphate (nbzlppA) has been synthesized as a substrate for T4 RNA ligase catalyzed 3'-phosphorylation. Incubation of oligoribonucleotides and nbzlppA with RNA ligase yielded oligoribonucleotides having a 3'-o-(o-nitrobenzyl) phosphate. Photochemical removal of the o-nitrobenzyl group provided the free 3'-phosphate. Using [P2-32P] nbzlppA, 3'-termini of oligoribonucleotides could be labelled with 32P. This reaction was applied to modify the 3'-end of donor molecules in joining reaction with RNA ligase. A trinucleotide U-A-G was converted to U-A-Gpnbzl and phosphorylated with polynucleotide kinase. pU-A-Gpnbzl was then joined to an acceptor trinucleotide A-U-G to yield A-U-G-U-A-Gp.     

Synthesis and applications of oligoribonucleotides with selected 2''-O-methylation using the 2''-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] protecting group.

The synthesis of base protected 5'-O-dimethoxytrityl-2'-O-[1-(2- fluorophenyl)-4-methoxypiperidin-4-yl]-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) is described, using phenoxyacetyl protection for the exocyclic amino groups of guanosine and adenosine and acetyl protection of the amino group of cytidine. High yield assembly of these building blocks into oligoribonucleotides on aminopropyl controlled pore glass was achieved using 5-(4-nitrophenyl)-1H-tetrazole as activator. Mixed sequences containing selected 2'-O-methylation were also synthesised and their significance for the study of RNA biochemistry is discussed.     

Crosslinking of hnRNP proteins to pre-mRNA requires U1 and U2 snRNPs.

Proteins interacting with pre-mRNAs during early stages of spliceosome formation in a HeLa nuclear extract were investigated by photochemical RNA-protein crosslinking. The level of protein crosslinking to a beta-globin pre-mRNA was positively correlated with the presence of an intron. Proteins of 110,000, 59,000 and 39,000 mol. wt. were crosslinked to the beta-globin pre-mRNA, the latter of which was identified as the A1 hnRNP protein. Comparable experiments with an adenovirus pre-mRNA revealed crosslinked proteins of 110,000, 56,000 and 45,000 mol. wt., with the latter identified as belonging to the C group hnRNP proteins. Crosslinking of hnRNP proteins to both the beta-globin and adenovirus pre-mRNAs was eliminated by oligodeoxynucleotide-directed RNase H excision of an internal region (nt 28-42) of U2 RNA, but was not affected by oligo/RNase H cleavage of the 5'-terminal 15 nucleotides of U2 RNA. Cleavage of the 5'-terminal 15 nucleotides of U1 RNA preferentially eliminated crosslinking of the hnRNP A1 protein to both pre-mRNAs. The requirement of intact U1 snRNP for A1 protein crosslinking was further demonstrated by the fact that although micrococcal nuclease-treated extracts did not support crosslinking of A1 hnRNP protein to beta-globin pre-mRNA, crosslinking was restored by addition of a U1 snRNP-enriched fraction.     

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.     

Evaluation of 2'-hydroxyl protection in RNA-synthesis using the H-phosphonate approach.

A number of different protecting groups were compared with respect to their usefulness for protection of 2'-hydroxyl functions during synthesis of oligoribonucleotides using the H-phosphonate approach. The comparison was between the t-butyldimethylsilyl (t-BDMSi), the o-chlorobenzoyl (o-CIBz), the tetrahydropyranyl (THP), the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp), the 1-(2-chloro-4-methylphenyl)-4-methoxypiperidin-4-yl (Ctmp), and the 1-(2-chloroethoxy)ethyl (Cee) protecting groups. All these groups were tested in synthesis of dodecamers, (Up)11U and (Up)11A, using 5'-O-(4-monomethoxytrityl) or (4,4'-dimethoxytrityl) uridine H-phosphonate building blocks carrying the respective 2'-protection. The performance of the t-BDMSi and o-CIBz derivatives were also compared in synthesis of (Up)19U. The most successful syntheses were clearly those where the t-butyldimethylsilyl group was used. The o-chlorobenzoyl group also gave satisfactory results but seems somewhat limited with respect to synthesis of longer oligomers. The results with all tested acetal derivatives (Fpmp, Ctmp, Cee, THP) were much less successful due to some accompanying cleavage of internucleotidic H-phosphonate functions during removal of 5'-O-protection (DMT).     

Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), beta-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic 'Hammerhead Ribozyme'. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.     

Human placenta DNA primase: purification of enzyme and analysis of RNA primer synthesis.

The immunoaffinity purification of human placenta DNA primase devoid of DNA polymerase alpha activity is described. Primase consists of 52 and 59 kDa polypeptides. They form a single protein of 330 kDa under native conditions. The polypeptide structure of primase is believed to be (52 + 59)3. Primase synthesizes the oligoribonucleotides 2-9 monomers long and multimeric oligoribonucleotides of a modal length of about 10 monomers. The following model of RNA primer synthesis is proposed: 1) primase, being in free state or in complex with Pol alpha, forms a protein trimer or another structure that includes several primases; 2) primase synthesizes de novo only the oligonucleotides 2-10 monomers in length; 3) the newly synthesized oligonucleotides dissociate in solution or translocate to either Pol alpha or a neighbouring primase unit to be further elongated with the next 7-10 mononucleotides.     

A novel crosslinking reagent and its application for the detection and isolation of heparin-binding protein(s) on the platelet surface.

A new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl)-4-(4-azabicyclo-[2,2, 2]hexylammonio)-6-morpholino-1,3,5-triazine chloride, was designed to detect and isolate heparin-binding protein(s) that may act as heparin-receptor(s) on the platelet surface. In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alcoholic hydroxy group under mild conditions and its crosslinking photoreactivity was high. The reagent effectively formed similar covalent bonds with heparin, while preserving its anticoagulant anti-Xa activity. [3H]Heparin labeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BAS, Fuji Photo Film, Tokyo). Affinity crosslinking of [3H]heparin was then used to detect heparin-binding proteins on the surface of intact platelets. Several discrete protein bands were detected by the BAS-imaging of SDS-PAGE.     

Phosphoryl migration during the chemical synthesis of RNA.

By the use of high sensitivity assay systems, we have measured the occurrence of strand scission and phosphoryl migration that accompany the deblocking of chemically synthesized oligoribonucleotides. Substantial phosphoryl migration was observed for both enzymatically derived poly(uridylic acid) and synthetic uridine oligoribonucleotides 2'-O-protected with the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) group, when these species were subjected to the acidic conditions suggested for Fpmp deprotection. Strand scission occurred in parallel and could be demonstrated readily by 5'-32P end labeling, but not by 3'-32P end labeling, of the acid-treated oligoribonucleotides. Increasing the pH of the deprotection solution and decreasing the temperature at which the deprotection was accomplished diminished both phosphoryl migration and strand scission. A mechanism that can rationalize these results is discussed.     

Selective 2''-benzoylation at the cis 2'',3''-diols of protected ribonucleosides. New solid phase synthesis of RNA and DNA-RNA mixtures.

5'-0-(Dimethoxytrityl)-2'-0-(benzoyl or 3,4,5-trimethoxybenzoyl)-base protected ribonucleosides have been prepared by selective benzoylation of the 2'-hydroxyl group. The isomerization of the 2'-benzoates to the 3'-benzoates was studied. The protected ribonucleosides have been converted to either methylphosphochloridites or methylphosphoamidites and used to synthesize oligoribonucleotides on silica gel solid support. The synthetic RNA were deprotected and isolated using conditions that minimize internucleotide cleavage. The use of 2'-benzoates as protecting groups for ribonucleosides has made it possible to easily prepare and isolate mixtures of DNA and RNA.     

An RNA microchip containing immobilized oligoribonucleotides with protective groups at 2'-O-positions

To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.     

Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA.

The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.     

The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation.

We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases. All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (11 bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the non-methylated strand of the same DNA substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity. An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA.     

A 62,000 molecular weight spliceosome protein crosslinks to the intron polypyrimidine tract

Incubation in HeLa nuclear extract of a 32P-labeled 61 nucleotide-long RNA corresponding to the lariat branch site/polypyrimidine tract/3' splice site of the first intron of human beta-globin pre-mRNA led to the crosslinking of a single protein of approximately 62,000 mol. wt. (p62). p62 corresponds to a polypyrimidine tract-binding protein recently described by Garcia-Blanco et al. (Genes & Dev. 3: 1874-1886, 1989). Crosslinking of p62 to the 61 nt RNA was highly sequence specific. No p62 crosslinking was observed with a 60 nt pGEM vector RNA, a 63 nt RNA antisense to the 61-mer or a 72 nt U2 RNA sequence. p62 crosslinking to the 61 nt RNA was competed by unlabeled 61 nt RNA, by beta-globin pre-mRNA containing intron 1, and by poly(U) and poly(C), but was competed to a lesser extent or not at all by pGEM RNA, a beta-globin RNA lacking intron 1, or poly(A). Experiments with mutated RNAs revealed that neither the lariat branch site adenosine nor the 3' splice site were required for p62 crosslinking to polypyrimidine tract-containing RNA. Elimination of the polypyrimidine tract reduced p62 crosslinking, as did mutation of a polypyrimidine tract UU dinucleotide to GA. However, replacement of a pyrimidine-rich tract immediately adjacent (3') to the lariat branch site with a 57% A + G pGEM vector RNA sequence also significantly reduced p62 crosslinking, indicating the involvement of both this pyrimidine-rich region and the classical polypyrimidine tract adjacent to the 3' splice site. The sites of protein interaction were further defined by RNase H protection experiments, the results of which confirmed the patterns of p62 crosslinking to mutant RNAs. p62 crosslinking was efficiently competed by a DNA oligonucleotide having the same sequence as the 61 nt RNA, showing that p62 requires neither ribose 2' OH groups nor uracil bases for its interaction with the polypyrimidine tract. p62 was not crosslinked to double-stranded 61 nt RNA. Q-Sepharose chromatography of HeLa nuclear extract yielded an unbound fraction (QU) in which p62 was the only polypyrimidine tract-crosslinkable protein and a bound fraction (QB) in which, surprisingly, several non-p62 proteins were crosslinkable to the polypyrimidine tract RNA. Yet, when the two Q-Sepharose fractions were combined, p62 strongly out-competed the otherwise-crosslinkable QB proteins for polypyrimidine tract RNA crosslinking. This indicates that p62 may have the highest affinity and/or crosslinking efficiency for the intron polypyrimidine tract of any HeLa nuclear protein.     

The mRNA codon environment at the P and E sites of human ribosomes deduced from photo crosslinking with pUUUGUU

Three mRNA analogs--derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position--were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA(Phe) was used, while tRNA(Val) was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions -3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with soft UV light (lambda > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions -1 to +3 binds to G1207, while that in positions -2 or -3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the -3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the -3 position, this crosslinking was not observed in the absence of tRNA.     

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.     

The Chemical Synthesis of Biochemically Active Oligoribonucleotides Using Dimethylaminomethylene Protected Purine H-Phosphonates

Abstract

Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations.