Nan-Sheng Li1, Nicole Tuttle, Jonathan P Staley, Joseph A Piccirilli. 1. Department of Biochemistry & Molecular Biology, ‡Department of Chemistry, and §Department of Molecular Genetics & Cell Biology, University of Chicago , Chicago, Illinois 60637, United States.
Abstract
Oligoribonucleotides containing 3'-S-phosphorothiolate linkages possess properties that can reveal deep mechanistic insights into ribozyme-catalyzed reactions. "Photocaged" 3'-S- RNAs could provide a strategy to stall reactions at the chemical stage and release them after assembly steps have occurred. Toward this end, we describe here an approach for the synthesis of 2'-O-(o-nitrobenzyl)-3'-thioguanosine phosphoramidite starting from N(2)-isobutyrylguanosine in nine steps with 10.2% overall yield. Oligonucleotides containing the 2'-O-(o-nitrobenzyl)-3'-S-guanosine nucleotide were then constructed, characterized, and used in a nuclear pre-mRNA splicing reaction.
Oligoribonucleotides containing 3'-S-phosphorothiolate linkages possess properties that can reveal deep mechanistic insights into ribozyme-catalyzed reactions. "Photocaged" 3'-S- RNAs could provide a strategy to stall reactions at the chemical stage and release them after assembly steps have occurred. Toward this end, we describe here an approach for the synthesis of 2'-O-(o-nitrobenzyl)-3'-thioguanosine phosphoramidite starting from N(2)-isobutyrylguanosine in nine steps with 10.2% overall yield. Oligonucleotides containing the 2'-O-(o-nitrobenzyl)-3'-S-guanosine nucleotide were then constructed, characterized, and used in a nuclear pre-mRNA splicing reaction.
Oligonucleotides
containing
phosphorothiolate linkages, in which sulfur replaces the
3′- or 5′-bridging oxygen connected to the ribofuranose
ring (Figure 1, II and III), serve as powerful biochemical probes to investigate fundamental
features of enzyme catalysis.[1−4] RNAs containing a 3′-S-phosphorothiolate
linkage have been used in metal ion rescue experiments to elucidate
the mechanism of metalloribozymes such as the group I
intron, the group II intron, and the spliceosome.[5−8] These studies rely on careful comparison of the 3′-O and 3′-S substrates and must include
assays that monitor the chemical step. Despite broad utility of these
substrates in the investigation of phosphoryl transfer reactions,
complex assembly processes and conformational changes that accompany
biological catalysis frequently mask analysis of the chemical step.
Photocaging provides a well-established strategy to protect functional
groups from chemical reactions until released by UV irradiation.[9−11] In this respect, having access to “photocaged” 3′-S-modified RNAs could provide a strategy to stall reactions
at the chemical stage and release them only after assembly steps have
occurred. Moreover, photocaging provides the added advantage of increasing
the stability of these phosphorothiolate-modified oligonucleotides
by eliminating side reactions involving 2′-O-transphosphorylation.[1]
Figure 1
Structures
of wild type, 3′-S-modified,
and 5′-S-modified oligonucleotides.
Structures
of wild type, 3′-S-modified,
and 5′-S-modified oligonucleotides.The 2′-O-(o-nitrobenzyl)
derivatives of uridine,[12] adenosine,[13] cytidine,[13] and guanosine[14] have been synthesized and applied to the synthesis
of oligoribonucleotides containing one or multiple
2′-O-photoliable groups (Figure 1, I*).[12,13,15−18] Dinucleotides and trinucleotides containing a 2′-O-(o-nitrobenzyl) group were usually
synthesized via a solution method involving condensation between the
3′-phosphate or 5′-phosphate of one nucleoside and the
5′-OH or 3′-OH group of another nucleoside.[12,13,17] Longer oligoribonucleotides
(>3-mer) containing 2′-O-(o-nitrobenzyl) groups could be efficiently synthesized via solid-phase
synthesis using 3′-phosphoramidites[15,16] or 3′-H-phosphonates of 2′-O-(o-nitrobenzyl) nucleosides.[18] After removal of the 2′-O-(o-nitrobenzyl) group by photolysis, these
RNAs could initiate efficient and accurate ribozyme-catalyzed reactions.[15,16] This RNA-caging approach has also been used to investigate spliceosome
assembly during pre-mRNA splicing through introducing a 2′-O-(o-nitrobenzyl) group into the
branch adenosine nucleotide, which blocks the first step of splicing,
thereby allowing the study of the precatalytic stages during assembly.[19]The synthesis of oligonucleotides
containing a 2′-O-photoliable group and 3′-S-phosphorothiolate
linkage (Figure 1, II*) has not
yet been reported. Here we report the synthesis of the phosphoramidite
derivative of the 2′-O-(o-nitrobenzyl)-3′-thioguanosine and its application
for the construction of oligonucleotides containing a
2′-O-photocaged 3′-S-phosphorothiolate linkage via solid-phase synthesis.For the synthesis of 2′-O-(o-nitrobenzyl)-3′-thioguanosine phosphoramidite 8 (Scheme 1), we adapted previous approaches
for the synthesis of 2′-O-TBS-3′-thioguanosine
phosphoramidite[20] and 2′-O-methyl-3′-thioguanosine phosphoramidite.[21] We chose the o-nitrobenzyl
group in this study because the starting material o-nitrobenzyl bromide is commercially available and relatively
inexpensive. Additionally, use of the o-nitrobenzyl
group is well-established for the synthesis of 2′-O-photocaged RNAs. N2-Isobutyryl-2′-O-(o-nitrobenzyl)guanosine (2) was prepared from N2-isobutyrylguanosine
(1) in 47% yield as described previously, except that
we quenched the reaction with dilute aqueous HCl.[22] Reaction of 2 with (tert-butyl)diphenylsilyl
chloride gave the corresponding 5′-silyl derivative in 94%
yield. In the presence of 1.4 molar equiv of DMAP, the 5′-silyl
derivative reacted with 1.05 molar equiv of trifluoromethanesulfonyl
chloride at 0 °C to give the corresponding 3′-triflate
derivative 3 in 62% yield. When 2.0 molar equiv of DMAP
and 1.5 molar equiv of trifluoromethanesulfonyl
chloride were used, the reaction afforded 3′-triflate derivative 3 in 50% yield along with the guanosine 3′,O6-ditriflate derivative (∼13% yield).
Subsequent SN2 substitution with 4 molar equiv of NaBr
in refluxing acetone afforded 3′-β-bromo derivative 4 in 95% yield. Reaction of 4 with KSAc in DMF
at 60 °C gave a mixture of the 3′-S-acetyl
and 3′,4′-unsaturated derivatives in a 1.3/1 ratio.
Desilylation of the mixture with TBAF·xH2O/AcOH in THF and purification by silica gel chromatography
gave the desired pure 5′-deprotected derivative 5 in 54% yield over the two steps. Although the 3′-β-iodo
derivative could be prepared by SN2 substitution of 3 with NaI in 96% yield, the subsequent reaction with KSAc,
followed by desilylation of the reaction mixture, produced predominantly
the 3′,4′-unsaturated derivative and gave the desired
5′-deprotected derivative 5 only in 19% yield.
Protection of 5 with DMTCl in pyridine for 24 h afforded
the 5′-O-DMT ether 6 in 95% yield.
The 3′-S-acetyl group of 6 was
selectively removed by treatment with a 5:1 mixture of guanidine hydrochloride
and guanidine[23] to give 3′-SH derivative 7 in 90% yield. Phosphitylation of compound 7 with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
yielded the corresponding 2′-O-(o-nitrobenzyl)-3′-thioguanosine phosphoramidite 8 in 85% yield.
Scheme 1
Incorporation of phosphoramidite 8 into
an 18-mer oligonucleotide was then accomplished by manual coupling
using our previously described protocol.[24] The modified oligonucleotide, 5′-UUU AG3′ A GGU UGC UGC UUU-3′ (2′-O-photocaged-3′-S-RNA), was obtained
in 1.5% yield after standard deprotection and reverse-phase HPLC purification.
The presence of the modification was confirmed by MALDI-TOF MS (Figure
S1 in Supporting Information). Additionally,
the oligonucleotide was 5′-radiolabeled with γ32-ATP and further characterized by UV deprotection, alkaline
hydrolysis, silver ion cleavage, and RNase T1 cleavage (Figure 2). For comparison, we prepared the corresponding
2′-photocaged wild-type RNA, 5′-UUU AG3′ A GGU UGC UGC UUU-3′ (2′-O-photocaged-3′-O-RNA), by solid-phase synthesis
using 2′-O-(o-nitrobenzyl)guanosinephosphoramidite. The UV-deprotected 3′-S-RNA was readily cleaved under alkaline conditions (pH
9) to form predominantly three 5-nucleotide products. Three cleavage
products from top to bottom on line 8 of Figure 2 were assigned as (A) 5′-pUUUAG2′-OH,3′-SH-3′, (B) 5′-pUUUAG2′--3′ (2′-O,3′-S-cyclic phosphosphate 5-mer), and (C)
5′-pUUUAG2′-OH,3′-SP-3′ (3′-S-phosphate 5-mer).[25,26] These product assignments correspond to those identified for alkaline
cleavage of the dinucleotide pIspU.[26] As
anticipated, the UV-deprotected 3′-S-oligonucleotide
was more susceptible toward hydroxide-catalyzed strand scission than
the 2′-photocaged 3′-S-oligo (Figure 2, compare lane 7 to 8). Exposure to silver ion of
both 2′-O-photocaged 3′-S-RNA and UV-deprotected 3′-S-RNA resulted
in the cleavage products assigned as 5′-pUUUAG2′--3′ and 5′-pUUUAG2′–OH,3′-SH-3′, respectively (Figure 2, lanes
11 and 12).[26] These results confirm that
the RNA contains a 3′-S-phosphorothiolate
linkage, and the ratio of the D and A product band indicates that
UV irradiation converted ∼90% of the 2′-O-photocaged 3′-S-RNA to 3′-S-RNA (lane 12). Moreover, neither 2′-O-photocaged guanosine 3′-O-RNA nor 2′-O-photocaged guanosine 3′-S-RNA
was cleaved by RNase T1 at the photocaged residue until after UV deprotection
(Figure 2, compare lanes 13 to 14 and 15 to
16). These results confirm that the RNA contains a 2′-photocaged
3′-phosphorothiolate linkage at the designated
position and shows that the photocage can be removed efficiently.
Figure 2
Alkaline
hydrolysis, silver ion cleavage, and RNase T1 cleavage
of 2′-photocaged 3′-O-substrate (5′-UUU
AG3′ A GGU UGC UGC UUU-3′)
and 2′-photocaged 3′-S-substrate (5′-UUU
AG3′ A GGU UGC UGC UUU-3′):
(A) 5′-pUUUAG2′-OH,3′-SH-3′, (B) 5′-pUUUAG2′--3′ (2′-O,3′-S-cyclic phosphoryl 5-mer), (C) 5′-pUUUAG2′-OH,3′-SP-3′ (3′-S-phosphoryl 5-mer), and (D) 5′-pUUUAG2′--3′. The numbers in the picture are assigned for the ladders
of alkaline hydrolysis and T1 treatment of these two 18-mer RNAs.
Alkaline
hydrolysis, silver ion cleavage, and RNase T1 cleavage
of 2′-photocaged 3′-O-substrate (5′-UUU
AG3′ A GGU UGC UGC UUU-3′)
and 2′-photocaged 3′-S-substrate (5′-UUU
AG3′ A GGU UGC UGC UUU-3′):
(A) 5′-pUUUAG2′-OH,3′-SH-3′, (B) 5′-pUUUAG2′--3′ (2′-O,3′-S-cyclic phosphoryl 5-mer), (C) 5′-pUUUAG2′-OH,3′-SP-3′ (3′-S-phosphoryl 5-mer), and (D) 5′-pUUUAG2′--3′. The numbers in the picture are assigned for the ladders
of alkaline hydrolysis and T1 treatment of these two 18-mer RNAs.We next tested whether the 2′-O-photocaged
3′-S-oligonucleotide could be used to
stage nuclear pre-mRNA splicing reactions at the chemical step. Nuclear
pre-mRNA splicing is catalyzed by the spliceosome, a complex ribonucleoprotein
particle that undergoes assembly steps and rearrangements en route
to the chemical steps.[27] We incorporated
the photocaged 3′-S-oligonucleotide
into a widely used ACT1yeast splicing substrate
and used it in metal rescue experiments with the spliceosome
(Figure 3a).[28] The
oligonucleotide was designed to contain the photocaged residue at
the intron terminus, such that the phosphorothiolate was
in the leaving group position for the second step of splicing. To
assemble the full-length substrate, we 32P-radiolabeled
the O- and S-containing oligonucleotides
and then ligated them to two RNA oligonucleotides generated
by in vitro transcription (Figure 3a). Next
we incubated these RNA substrates in splicing-competent Saccharomyces cerevisiae extract so that spliceosomes
could assemble on them and catalyze branching (Figure 3b). The lariat intermediate product of the branching step
runs aberrantly slowly and can be seen as the top band of the splicing
gel (Figure 3c).
Figure 3
(a) Schematic for synthesis
of ACT1 yeast splicing
substrates. RNA is shown as black lines and boxes; the DNA splint
is shown in gray. The boxes represent the exons, and the black line
represents the intron. The red star indicates the position of the
radiolabel. The modified 2′-O-photocaged residue
is the last nucleotide of the intron. (b) Schematic of the two steps
of splicing. (c) Exon ligation of the 3′-O-pc and 3′-S-pc substrates in an in vitro
splicing assay (60 mM K2PO4 (pH 7), 3% PEG 8000,
2.5 mM MgCl2, 2 mM ATP, 40% yeast extract in the initial
incubation); “metal” indicates the addition of 0.5 mM
MgCl2, MnCl2, or nothing as indicated in the
main text.
(a) Schematic for synthesis
of ACT1yeast splicing
substrates. RNA is shown as black lines and boxes; the DNA splint
is shown in gray. The boxes represent the exons, and the black line
represents the intron. The red star indicates the position of the
radiolabel. The modified 2′-O-photocaged residue
is the last nucleotide of the intron. (b) Schematic of the two steps
of splicing. (c) Exon ligation of the 3′-O-pc and 3′-S-pc substrates in an in vitro
splicing assay (60 mM K2PO4 (pH 7), 3% PEG 8000,
2.5 mM MgCl2, 2 mM ATP, 40% yeast extract in the initial
incubation); “metal” indicates the addition of 0.5 mM
MgCl2, MnCl2, or nothing as indicated in the
main text.With the photocage in place, the
spliceosomes stalled prior
to exon ligation regardless of the identity of the atom (O or S) in
the leaving group position (Figure 3c, lanes
1 and 7). To remove the photocage and permit exon ligation (Figure 3b), we irradiated spliceosomes for 5 min with
365 nm UV light (lanes 4–6 and 10–12). To initiate exon
ligation, we then added additional MgCl2, MnCl2, or nothing and assayed for exon ligation. As expected, appearance
of the product of exon ligation of the 3′-O-pc substrate proceeded efficiently in the UV-treated spliceosomes
(lanes 4–6) but not without irradiation (lanes 1–3).
Exon ligation of the 3′-S-pc substrate only
proceeded efficiently with UV treatment and in the presence of MnCl2 (compare lane 12 with lanes 7–11). These results demonstrate
that the photocage comprises an effective block to splicing chemistry
and that, upon removal, splicing occurs in the presence of an added
metal ion. The dependence of the 3′-S-pc substrate
reaction on the “thiophilic” manganese(II) ion reflects
the role of the metal ion in leaving group stabilization.[8]In summary, we have developed an efficient
synthesis of the phosphoramidite
derivative of 2′-O-(o-nitrobenzyl)-3′-thioguanosine
starting from N2-isobutyrylguanosine
in nine steps. From this phosphoramidite, an 18-nucleotide
RNA containing a 2′-O-photoliable group adjacent
to a 3′-S-phosphorothiolate linkage
was successfully synthesized by the solid-phase synthesis. Irradiation
with UV light released the photocaging group without affecting the
integrity of the 3′-S modification. This capability
can enable effective use of the 3′-sulfur modification to perform
metal rescue experiments even in systems that undergo complex assembly
and conformational changes en route to the chemical step. As proof-of-principle,
we have incorporated this modified RNA into a model yeast pre-mRNA
splicing substrate and used it to probe for catalytic metal ion interactions
in the spliceosome.[8]
Experimental Section
N2-Isobutyryl-2′-O-(o-nitrobenzyl)guanosine (2)
Under argon, N2-isobutyrylguanosine 1 (1.72 g, 4.86 mmol) was treated with NaH (307 mg, 95%, 12.15
mmol) in DMF (40 mL) at 0 °C. After hydrogen gas generation ceased
(45 min), o-nitrobenzyl bromide (1.58 g, 7.30
mmol) was added, and the mixture was stirred at rt for 5 h. The reaction
was neutralized with 1 N HCl. The mixture was evaporated, and the
residue was purified by silica gel chromatography, eluting with 4–6%
methanol in dichloromethane to give the product 2a(14,22) as a yellow foam (1.12 g, 47% yield).
Under argon to a solution of 4 (2.99 g, 3.78
mmol) in dry DMF (25 mL) was added potassium thioacetate (1.30 g,
11.4 mmol), and the mixture was stirred at 60 °C for 7 h. After
the solvent was removed under reduced pressure, the residue was partitioned
between a saturated aqueous NaHCO3 solution/brine (v/v,
1:1) and CH2Cl2. The organic layer was dried
over MgSO4. After filtration and removal of the solvent,
the residue was dissolved in THF (45 mL), and AcOH (1.23 mL, 21.7
mmol) and n-Bu4NF·3H2O
(2.74 g, 8.69 mmol) were added. The mixture was stirred at rt for
14 h, then diluted with CH2Cl2, washed with
saturated aqueous NaHCO3 and brine, and dried over MgSO4. After filtration and removal of solvent, the residue was
purified by silica gel chromatography, eluting with 3% MeOH in CH2Cl2 to give 5 as a white foam: 1.12
g (54% yield); 1H NMR (CD3OD/TMS) δ 8.37
(s, 1H), 8.00 (dd, 1H, J = 1.2, 8.4 Hz), 7.71 (d,
1H, J = 6.8 Hz), 7.66 (dt, 1H, J = 1.2, 7.6 Hz), 7.51 (dt, 1H, J = 1.2, 7.8 Hz),
6.19 (d, 1H, J = 2.0 Hz), 5.25 (d, 1H, J = 13.6 Hz), 5.03 (d, 1H, J = 13.6 Hz), 4.55–4.44
(m, 2H), 4.25–4.18 (m, 1H), 3.92 (dd, 1H, J = 2.4, 12.8 Hz), 3.72 (dd, 1H, J = 2.8, 12.8 Hz),
2.75 (m, 1H), 2.36 (s, 3H), 1.25 (d, 3H, J = 4.4
Hz), 1.23 (d, 3H, J = 4.4 Hz); 13C NMR
(CDCl3) δ 196.0, 181.6, 157.4, 149.7, 149.1, 139.2,
134.7, 134.4, 130.8, 130.0, 125.8, 121.5, 89.2, 86.4, 85.2, 70.8,
61.4, 44.6, 37.0, 30.4, 19.4 19.3 ppm; HRMS calcd for C23H27N6O8S [MH+] 547.1606,
found 547.1606.
Under argon to a solution of 7 (137
mg, 0.17 mmol) in dry dichloromethane (10 mL) were added N,N-diisopropylethylamine (0.15
mL, 0.86 mmol), 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (96
μL, 0.43 mmol), and 1-methylimidazole (6.2 μL, 0.078
mmol). The mixture was stirred at rt for 1 h, quenched with MeOH (1
mL), and stirred for an additional 5 min. The solvent was removed,
and the residue was purified by silica gel chromatography, eluting
with 0–5% acetone in CH2Cl2 containing
0.5% Et3N to give product as a white foam: 0.146 g (85%
yield); 31P NMR (CD3CN) δ 164.6, 159.2
ppm; HRMS calcd for C51H60N8O10PS [MH+] 1007.3885, found 1007.3884.
To a solution of 5′-O-(dimethoxytrityl)-N2-isobutyryl-2′-O-(o-nitrobenzyl)guanosine[22] (47 mg, 0.059 mmol) in dry CH2Cl2 (5.0 mL) under Ar were added N,N-diisopropylethylamine (52 μL, 0.30 mmol),
2-cyanoethyl N,N-diisopropylchlorophosphoramidite
(42 mg, 0.18 mmol), and 1-methylimidazole (5.0 μL, 0.059
mmol). The mixture was stirred at rt until all starting material was
consumed (1 h). The reaction was quenched with MeOH (1 mL) and stirred
for 5 min. After the solvent was removed, the crude product was purified
by silica gel chromatography, eluting with 1% MeOH in CH2Cl2 containing 0.5% Et3N, to give the corresponding
phosphoramidite as a yellow foam: 56 mg (95% yield); 31P NMR (CD3CN) δ 152.8, 152.6; HRMS calcd
for C51H60N8O11P [MH+] 991.4119, found 991.4110.
Oligonucleotide Synthesis
The oligonucletide 5′-UUU
AG3′A GGU UGC UGC UUU-3′ (ACT1-3'-S)
was synthesized on an Expedite 8900 DNA synthesizer by manual coupling
of phosphoramidite 8 through a modified protocol
as previously described.[24] Following the
standard oligonucleotide deprotections[29] (a, concentrated NH4OH/EtOH, 3:1 (v/v), 55 °C, 17
h; b, TEA-3HF/TEA/NMP, 65 °C, 1.5 h), the oligonucleotide (1.5%
yield) was obtained by reverse-phase HPLC purification (C18 column,
0–30% aceonitrile/100–70% 0.1 M TEAA pH 7.0 over 30
min) and confirmed by the MALDI-TOF MS: calcd for (M + NH4) 5854.8, found 5853.1.
Oligonucleotide Characterization
Alkaline hydrolysis:
4k cpm of the 5′-radiolabeled oligonucleotide
(1 μL) with or without UV deprotection (UVP-B1000, 365 nm, 4
min) was treated with NaHCO3 (pH 9, 50 mM, 2 μL)
in a total volume of 10 μL solution at 90 °C for 10 min.
Formamide loading dye (2×, 10 μL) was added, and the mixture
was run on a 20% dPAGE gel.Silver ion cleavage: 4k cpm of the
5′-radiolabeled oligonucleotides (2 μL)
with or without UV deprotection (UVP-B1000, 365 nm, 4 min) was treated
with AgNO3 (100 mM, 0.4 μL) in a total volume of
20 μL solution in the dark at rt for 60 min. DTT (100 mM, 0.6
μL) was then added, and the mixture was spun at full speed for
5 min. A 15 μL aliquot of solution was withdrawn, added to 2×
formamide loading dye (15 μL), and run on a 20% dPAGE gel.Rnase T1 treatment: 4k cpm of the 5′-radiolabeled
oligonucleotides (1 μL) with or without UV deprotection
(UVP-B1000, 365 nm, 4 min) was combined with 8 M urea (pH 9, 6 μL),
200 mM sodium citrate (pH 5, 1 μL), and 1 unit of RNase T1 in
a final reaction volume of 9 μL. After a 10 min incubation at
37 °C, 2× formamide loading dye (8 μL) was added and
the sample was run on a 20% dPAGE gel.
Synthesis of ACT1 Yeast Splicing Substrates[8]
The
ACT1-1-373 (nucleotides 1–373)
was synthesized by in vitro transcription from a plasmid template
linearized with HindIII restriction and containing
ACT1-1-373 followed by an HDV ribozyme sequence. In cases where HDV
cleavage was inefficient during transcription, the RNA was resuspended
in Tris (10 mM, pH 7.5) and MgCl2 (20 mM). Ribozyme cleavage
was induced via 2–4 cycles of 90 °C for 1 min, rt for
15 min, and 37 °C for 15 min. The buffer conditions were then
adjusted for T4 PNK treatment of the transcript to remove the 2′,3′-cyclic
phosphate left by the ribozyme. The ACT1-392-590 (nucleotides 392–590)
was synthesized by in vitro transcription using a PCR-derived template
generated using plasmid bJPS149. As the subsequent ligation requires
a 5′-monophosphate group, a 4-fold excess of GMP over
GTP was included in the transcription reaction.ACT1 ligation
reactions consisted of 500 pmol of ACT1-1-373, 50 pmol of oligonucleotideACT1-3′-O or ACT1-3′-S, and 500 pmol of ACT1-392-590. The RNA was hybridized to 50 pmol
of ACT1 splint in buffer TEN50 (10 mM Tris-HCl, pH 7.5; 1 mM EDTA;
50 mM NaCl) on a thermal cycle by heating to 90 °C for 2 min
followed by reduction of the temperature by 1 °C for 1 min to
24 °C, then cooling to 4 °C for 5 min. T4 DNA ligase (∼100
pmol, synthesized in-house) or T4 RNA ligase 2 (2 units, New England
Biolabs) was then added, and reactions were incubated at 37 °C
for 4 h. The ligation reactions were DNase-treated (RNase-free DNase)
for 15 min to remove splint, phenol–chloroform extracted, and
ethanol precipitated before purification on 6% denaturing polyacrylamide
gel. Bands containing full-length ACT1 pre-mRNA were excised and recovered
by passive elution in TEN250 buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA,
250 mM NaCl) overnight at 4 °C. Yields typically ranged from
200 to 600 fmol, enough for 50–150 splicing reactions.
Splicing
Reactions
Yeast splicing-competent extracts
were prepared using the liquid nitrogen method, as described in the
literature.[30] In vitro splicing reactions
consisted of 32P-labeled substrates (0.2–0.4 nM),
40% yeast extract that was pretreated with 1 mM EDTA, 3% PEG 8000,
60 mM K2PO4 (pH 7), 3.5 mM MgCl2,
and 2 mM ATP. Reactions were incubated at 20 °C for 20 min, then
on ice for 5 min for 365 nm UV light treatment. Metal ion concentrations
were adjusted to 4 mM with either MgCl2 or MnCl2, and the reactions were incubated at 20 °C for another 20 min.
The reactions were then quenched and analyzed by 6% dPAGE gel as described
previously.[8]
Figure 1
Scheme 1
Figure 2
Figure 3