Synthesis of RNA having cap structure.

RNA consisting 43 nucleotides bearing cap structure was synthesized (Figure). In the first place, 9 mer of a leader sequence with the cap structure (F-1) was synthesized by the phosphotriester method and followed by the capping reaction. Next, 32 mer of a cistron was divided into two fragments and each was synthesized by the phosphoramidite method. The 3'-end nucleotide of the RNA, a modified guanosine 5'-phosphate, was introduced to F-3 by use of P1-2',3'-O-methoxymethylene guanosine-5'-yl P2-adenosine-5'-yl diphosphate (A5' ppGmM) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated with T4 RNA ligase to afford the desired RNA.     

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.     

Syntheses and Properties of Dansylated Deoxycytidine 3′,5′-Bisphosphate Derivatives Useful for 3′-Labeling of Oligoribonucleotides

Abstract

For non-RI labeling of RNAs with fluorescence markers, deoxycytidine 3′,5′-bisphosphate derivatives (1 and 2) were synthesized as dansyl donors which could be linked to the 3′-terminus of RNAs by T4 RNA ligase catalized joining reactions. Ligations of GpApC with these dansyl donors in the presence of T4 RNA ligase were studied.     

Model substrates of enzymes modifying ribonucleic acids. Synthesis of deca- and undecanucleotid-fragments of 21-member oligoribonucleotide simulating T psi C-branch of yeast valine tRNA

Decanucleotide (Ap)6GpTpUpC and undecanucleotide GpApUpCpCp (Up)5U have been synthesised. They constitute 5'- and 3'-parts of a 21-mer which imitates T psi C-arm of yeast tRNA(Val1) and is a potential substrate for m1A-methylases and pseudouridine synthetase. The oligonucleotide blocks, synthesised enzymatically by means of ribonucleases of various substrate specificity and polynucleotide phosphorylases (TpUpC, ApUpCpC, pGpTpUpC, GpApUpCpC) or obtained by hydrolysis of poly(U) and poly(A) with Serratia marcescens endonuclease (hexauridilate and hexaadenilate), were joined by T4 RNA ligase.     

Efficient RNA 5'-adenylation by T4 DNA ligase to facilitate practical applications

We describe a simple procedure for RNA 5'-adenylation using T4 DNA ligase. The 5'-monophosphorylated terminus of an RNA substrate is annealed to a complementary DNA strand that has a 3'-overhang of 10 nucleotides. Then, T4 DNA ligase and ATP are used to synthesize 5'-adenylated RNA (5'-AppRNA), which should find use in a variety of practical applications. In the absence of an acceptor nucleic acid strand, the two-step T4 DNA ligase mechanism is successfully interrupted after the adenylation step, providing 40%-80% yield of 5'-AppRNA after PAGE purification with few side products (the yield varies with RNA sequence). Optimized reaction conditions are described for 5'-adenylating RNA substrates of essentially any length including long and structured RNAs, without need for sequestration of the RNA 3'-terminus to avoid circularization. The new procedure is applicable on the preparative nanomole scale. This 5'-adenylation strategy using T4 DNA ligase is a substantial improvement over our recently reported adenylation method that uses T4 RNA ligase, which often leads to substantial amounts of side products and requires careful optimization for each RNA substrate. Efficient synthetic access to 5'-adenylated RNA will facilitate a range of applications by providing substrates for in vitro selection; by establishing a new protocol for RNA 5'-capping; and by providing an alternative approach for labeling RNA with (32)P or biophysical probes at the 5'-terminus.     

Enzymatic synthesis of a segment of bacteriophage Qbeta coat protein gene.

The oligoribonucleotide, A-A-A-C-U-U-U-Gp, constituting a segment of RNA bacteriophage Qbeta coat protein gene was efficiently synthesized at a milligram scale by a combination of enzymatic methods using bacteriophage T4 RNA ligase and the thermophilic polynucleotide phosphorylase. A-A-A-Cp was synthesized from A-A-A and pCp by the newly developed mononucleotide addition method using T4 RNA ligase in a yield of 83%, followed by dephosphorylation with bacterial alkaline phosphatase to obtain A-A-A-C. pU-U-U-Gp was synthesized from pU-U-U and GDP by the simultaneous action of polynucleotide phosphorylase and RNase T1 in a yield of 32%. finally, the two oligonucleotides (A-A-A-C and pU-U-U-Gp) were ligated with T4 RNA ligase and the octanucleotide, A-A-A-C-U-U-U-Gp, was obtained in a yield of 85%.     

Structure and mechanism of RNA ligase

T4 RNA ligase 2 (Rnl2) exemplifies an RNA ligase family that includes the RNA editing ligases (RELs) of Trypanosoma and Leishmania. The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3'-OH and 5'-PO4 RNA ends by Rnl2, but not for ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA ligase domain. We report the 1.9 A crystal structure of the ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA ligases, DNA ligases, and mRNA capping enzymes.     

Biotin and fluorescent labeling of RNA using T4 RNA ligase.

Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules.     

Periodicity in the length of 3'-poly(A) tails from native globin mRNA of rabbit.

Globin mRNA from rabbit reticulocytes was labelled at the 3'-end with [5'-32P]pCp by T4 RNA ligase. The 3'-poly(A) tail was released by digestion of mRNA with T1 ribonuclease and its size distribution determined by gel electrophoresis and autoradiography. The length of the 3'-poly(A) tails varied from about 15-150 residues, but the size distribution exhibited peaks in the abundance of poly(A) species at intervals of approx. 25 residues. This periodicity appears to reflect the manner in which proteins bind to the 3'-poly(A) tail. The function of such regular interactions may be to control mRNA breakdown in the cytoplasm.     

Polyribosome binding of rabbit globin messenger RNA and messenger ribonucleoprotein labelled with bacteriophage-T4 RNA ligase and 5''-[32P] phosphocytidine 3''-phosphate

Rabbit polyribosomal globin messenger RNA (mRNA) and messenger ribonucleoprotein (mRNP) were labelled at the 3′ poly(A) tail to high specific activity with T4 RNA ligase and [5′-³²P]pCp without consequent loss of functional activity. Labelled message was translated in both micrococcal nuclease treated and untreated rabbit reticulocyte lysates, as shown by the formation of labelled polyribosomes. The utilisation of labelled messenger was abolished by T2 toxin or sodium fluoride which are known to inhibit protein synthesis.     

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.     

T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates.

T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [alpha-32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, microm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [alpha-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2'p.     

A new strategy for introducing photoactivatable 4-thiouridine ((4S)U) into specific positions in a long RNA molecule.

We describe a new protocol, which does not require (4S)UpG, for introducing (4S)U into specific sites in a pre-mRNA substrate. A 5'-half and a full-length RNA are first synthesized by phage RNA polymerase. p(4S)Up, which is derived from (4S)UpU and can therefore be 32P-labeled, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3' phosphate of the ligated product is removed subsequently by CIP (calf intestinal alkaline phosphatase) to produce a 3'-OH group. The 3'-half RNA with a 5' phosphate is produced by site-specific RNase H cleavage of the full-length pre-mRNA directed by a 2'-O-methyl RNA-DNA chimera. The two half RNAs are then aligned with a bridging oligonucleotide and ligated with T4 DNA ligase. Our results show that 32P-p(4S)Up ligation to the 3' end of the 5'-half RNA is comparable to 32P-pCp ligation. Also, the efficiency of the bridging oligonucleotide-mediated two-piece ligation is quite high, approximately 30-50%. This strategy has been applied to the P120 pre-mRNA containing an AT-AC intron, but should be applicable to many other RNAs.     

Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase.

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.     

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

Plant and fungal tRNA ligases are trifunctional enzymes that repair RNA breaks with 2′,3′-cyclic-PO₄ and 5′-OH ends. They are composed of cyclic phosphodiesterase (CPDase) and polynucleotide kinase domains that heal the broken ends to generate the 3′-OH, 2′-PO₄, and 5′-PO₄ required for sealing by a ligase domain. Here, we use short _HORNA>p substrates to determine, in a one-pot assay format under single-turnover conditions, the order and rates of the CPDase, kinase and ligase steps. The observed reaction sequence for the plant tRNA ligase AtRNL, independent of RNA length, is that the CPDase engages first, converting _HORNA>p to _HORNA_2′p, which is then phosphorylated to pRNA_2′p by the kinase. Whereas the rates of the AtRNL CPDase and kinase reactions are insensitive to RNA length, the rate of the ligase reaction is slowed by a factor of 16 in the transition from 10-mer RNA to 8-mer and further by eightfold in the transition from 8-mer RNA to 6-mer. We report that a single ribonucleoside-2′,3′-cyclic-PO₄ moiety enables AtRNL to efficiently splice an otherwise all-DNA strand. Our characterization of a fungal tRNA ligase (KlaTrl1) highlights important functional distinctions vis à vis the plant homolog. We find that (1) the KlaTrl1 kinase is 300-fold faster than the AtRNL kinase; and (2) the KlaTrl1 kinase is highly specific for GTP or dGTP as the phosphate donor. Our findings recommend tRNA ligase as a tool to map ribonucleotides embedded in DNA and as a target for antifungal drug discovery.     

Characterization of a baculovirus enzyme with RNA ligase, polynucleotide 5'-kinase, and polynucleotide 3'-phosphatase activities

The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1-385), and a C-terminal kinase-phosphatase domain, Rnl1-(394-694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1-270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO(4) end to form RNA-adenylate. The end-healing domain Rnl1-(394-694) consists of a proximal 5'-kinase module with an essential P-loop motif ((404)GSGKS(408)) and a distal 3'-phosphatase module with an essential acylphosphatase motif ((560)DLDGT(564)). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.     

Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61--77 from Escherichia coli tRNAfMet.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.