Synthesis, characterization, and biological properties of small branched RNA fragments containing chiral (Rp and Sp) 2′,5′-phosphorothioate linkages

Article abstract of DOI:10.1080/15257770500447004

Synthetic branched RNA fragments were prepared to examine the stereochemical requirements for hydrolysis of RNA lariats by the yeast debranching enzyme (yDBR). Specifically, two branched trinucleoside diphosphates and a tetranucleoside triphosphate contai

Full text of DOI:10.1080/15257770500447004

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Synthesis, characterization, and
biological properties of small branched
RNA fragments containing chiral (Rp and
Sp) 2′,5′-phosphorothioate linkages
Rawan Mourani a & Masad J. Damha a
a Department of Chemistry , McGill University , Montreal, Quebec,
Canada
Published online: 07 Feb 2007.
To cite this article: Rawan Mourani & Masad J. Damha (2006) Synthesis, characterization,
and biological properties of small branched RNA fragments containing chiral (Rp and Sp) 2′,5′-
phosphorothioate linkages, Nucleosides, Nucleotides and Nucleic Acids, 25:2, 203-229, DOI:
10.1080/15257770500447004
To link to this article: http://dx.doi.org/10.1080/15257770500447004
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Nucleosides, Nucleotides, and Nucleic Acids, 25:203–229, 2006
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770500447004
SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL PROPERTIES
OF SMALL BRANCHED RNA FRAGMENTS CONTAINING CHIRAL
(RP AND SP) 2ꢀ,5ꢀ-PHOSPHOROTHIOATE LINKAGES
Rawan Mourani and Masad J. Damha
Department of Chemistry, McGill
2
University, Montreal, Quebec, Canada
Synthetic branched RNA fragments were prepared to examine the stereochemical requirements
2
for hydrolysis of RNA lariats by the yeast debranching enzyme (yDBR). Specifically, two branched
trinucleoside diphosphates and a tetranucleoside triphosphate containing a 2 ꢁ,5 ꢁ-linked phospho-
rothioate linkage of defined stereochemistry, namely Rp-A(2 ꢁps5 ꢁG)pC, Sp-A(2 ꢁps5 ꢁG)pC and Sp-
ApA(2 ꢁps5 ꢁG)pC, were prepared via solution-phase methods. Unlike the all-phosphodiester control,
A(2 ꢁp5 ꢁG)pC, the Rp-thioated trimer was not cleaved by yDBR, demonstrating that changing the
pro-Rp oxygen at the 2 ꢁ,5 ꢁ-phosphodiester bond averts hydrolysis by the enzyme. In contrast, the Sp
branched compounds (trimer and tetramer) were cleaved yDBR, albeit with reduced efficiency relative
to the corresponding all-phosphodiester branched compounds. Furthermore, the small branched RNAs
(5 nt) were not cleaved as efficiently as a 18-nt bRNA, suggesting that the enzyme appears to have a
stronger preference for larger bRNA substrates. The non-hydrolyzable branched RNA fragments pre-
pared during these studies may be promising candidates for the future co-crystallization and X-ray
analyses of DBR:bRNA complexes.
Keywords Branched RNA; Debranching enzyme
INTRODUCTION
The cellular transformation of a precursor messenger RNA (pre-mRNA)
into its mature or functional form, mRNA, proceeds by way of a splicing
reaction, in which “exon” sequences are joined together to form the ma-
ture linear RNA. Typically, the “introns” or intervening sequences are ex-
cised in the shape of small circles with tails, better known as RNA “lariats”
Received 26 May 2005; accepted 31 October 2005.
This work was supported by a grant from the National Science and Research Council of Canada
(NSERC). We thank J.D. Boeke for the generous gift of the yeast debranching enzyme, and M.M. Mangos
for helpful discussions and thorough proofreading of the manuscript. M.J.D. is recipient of a James McGill
professorship (McGill University).
Address correspondence to Masad J. Damha, Department of Chemistry, Otto Maass Chemistry
Building, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, H3A 2K6, Canada. E-mail:
masad.damha@mcgill.ca
203

204
R. Mourani and M. J. Damha
FIGURE 1 Cis splicing of pre-mRNA proceeds via lariat RNA intermediates.
(Figure1).[1] Thebranchpointofthemoleculecomprisesaconservedadeno-
sine residue containing vicinal 2ꢁ,5ꢁ and 3ꢁ,5ꢁ-phosphodiester linkages. In
many eukaryotic organisms, pre-mRNA splicing is not autocatalytic but oc-
curs in the “spliceosome,”[2−4] a multi-protein complex consisting of small
nucleolar ribonucleoprotein units (snRNPs), which themselves are intricate
assemblages of polypeptides and small nuclear RNAs (snRNAs). Assembly of
these factors on the pre-mRNA occurs in a multistep fashion that involves
binding and association of the snRNPs along the transcript to fold and orient
the strand while holding the reactive centers in close proximity to aid in the
subsequent transesterification steps.
Branched RNA molecules were first isolated from total nuclear RNA by
Wallace and Edmonds in 1983.[5] Their low abundance relative to the mature
mRNA suggested that the molecules were being hydrolyzed soon after their
formation, probably to yield the corresponding linearized RNA. Accordingly,
a novel 2ꢁ,5ꢁ-phosphodiesterase activity was observed by Ruskin and Green
in 1985 in HeLa cell extracts.[6] This “debranching” activity specifically hy-
drolyzes 2ꢁ,5ꢁ-phosphodiester linkages in branched molecules to generate
linear RNA species. S. cerevisiae Dbr1 (“DBR”) is a 405-amino acid RNA de-
branching enzyme first isolated and characterized by Boeke and Chapman in
1991.[7] The only role the lariat debranching enzyme was believed to have was
to provide a method for linearizing the lariats for further degradation. How-
ever, recent studies have shown that introns, along with DBR, appear to play
more essential and vital roles in vivo.[8−10] For example, small nucleolar RNA
introns (snoRNAs) participate in splicing through processing and modifica-
tion of pre-rRNA and rRNA nucleotides, repectively.[9] The active snoRNA
species are generated upon debranching of the circularized precursor RNA
lariat in S. cerevisiae; dbr1 deletion mutants produce the intronic snoRNA as

Small Branched RNA Fragments Containing Chiral Linkages
205
“trapped” lariat forms, with concomitant reductions in cellular levels of ma-
ture intronic snoRNAs.[8] The function of DBR may be more crucial in higher
eukaryotic cells, which contain a greater number of larger introns in most
genes. Accordingly, Boeke and Nam demonstrated that S. pombe mutants
with a defective dbr gene showed severe growth defects and altered cell mor-
phologies relative to the wild-type yeast strains, whereas S. cerevisiae displayed
no significant phenotypic defects, presumably owing to a relatively simpler
genomic organization (ca. 40% and 2.5% intron content, respectively).[10]
These lines of evidence suggest DBR to be crucial for cell viability in higher
eukaryotes (e.g., mammalian cells) that therefore merit further exploration
of the substrate specificity and properties of this unique phosphodiesterase.
We have previously shown that yDBR can digest various branched nu-
cleic acids (bNA) but exhibits a strong preference for purines over pyrim-
idines at the branchpoint as well as the 2ꢁ-position.[11−14] Conversely, the
nucleotide at the 5ꢁ-position has only a negligible influence on debranching
efficacy, given that branched oligonucleotides lacking the 5ꢁ-nucleotide were
still highly susceptible to efficient debranching by the enzyme.[13,14] In a sep-
arate study examining the role of substrate stereochemistry on debranching
activity, Padgett and coworkers[15] showed an inability for the HeLa cell RNA
debranching enzyme to hydrolyze an RNA lariat containing an Rp phospho-
rothioate at the 2ꢁ,5ꢁ-linkage. However, the susceptibility of an Sp 2ꢁ, 5ꢁ-linkage
towards debranching by the yeast or mammalian enzymes is unknown at
present.
With the recent studies and discoveries on DBR, more is known and
appreciated about its cellular importance, especially in higher organisms.
However, not much information currently exists regarding its precise mech-
anism of action, structure, or substrate specificity. It is thus the focus of this
work to explore the solution syntheses of the first branched RNA fragments
containing Rp and Sp stereochemistry at the 2ꢁ,5ꢁ-phosphorothioate linkage
(Figure 2). Both isomers of the branched phosphorothioate were used as
substrates to further elucidate the particular substrate specificity of this im-
portant enzyme.
MATERIALS AND METHODS
Materials
Pyridine was continuously refluxed over barium oxide under nitrogen
and distilled prior to use. Tetrahydrofuran (THF) was refluxed over sodium
and benzophenone under nitrogen until a purple color persisted. Acetoni-
trile (CH3CN), used for organic synthesis, was refluxed over phosphorus
pentoxide under inert atmosphere before use, while CH3CN used for nucle-
oside phosphoramidite coupling, in solution or on solid phase, was further
refluxed and distilled over calcium hydride (CaH2). Dichloromethane

206
R. Mourani and M. J. Damha
FIGURE 2 Structure of the stereoisomeric Rp- and Sp-branched trinucleoside diphosphates.
(DCM) was refluxed over CaH2. N,N-dimethylformamide (DMF), collidine
(2,3,6-trimethylpyridine), and diisopropylethylamine (DIPEA) were all dried
by refluxing over calcium hydride (CaH2), distilled under reduced pressure,
˚
and stored over 4A molecular sieves. Triethylamine (TEA) was distilled over
a
calcium hydride (CaH2) and was stored over 4A molecular sieves.
Methods
Gravity and flash silica gel column chromatography were performed on
Merck Kieselgel 60 (200–400 mesh) silica gel (VWR). Reactions were ana-
lyzed and monitored using Merck Kieselgel 60F 254 silica analytical TLC alu-
minum sheets (0.2 × 20 cm × 20 cm). Preparative TLC to purify compounds
was carried out using glass plates (20 cm × 20 cm) coated with a thick layer of
Kieselgel 60 GF 254 silica gel. Ammonium salts produced by the desilylation
reaction via TBAF were removed using Dowex 50W-X8 cation exchange resin
(Na+ form, 20–50 mesh). The resin was regenerated by washing with 0.1 N
HCl, followed by 0.1 N NaOH, followed by autoclaved H2O until the filtrate
was of neutral pH. It was then autoclaved and packed into a column (5 ×
1.5 cm) and equilibrated with autoclaved H2O prior to usage.[16]
NMR spectra were obtained at ambient temperature using Varian
Mercury-200, Mercury-300, Mercury-400, and UNITY-500 spectrophotome-
1
ters. H NMR spectra were referenced to the solvent deuterium signal,
whereas 85% H3PO4 was used as an external reference for 31P NMR
(δ 0 ppm). UV/VIS spectra were collected using a Varian CARY1 UV/VIS
spectrophotometer at ambient temperature (Varian: Mulgrave, Victoria,
Australia). The data was computed using Cary WinUV version 2 software
(Varian Ltd.). Spectra were obtained in 95% ethanol for protected nucleo-
side derivatives, and water for the completely deprotected oligonucleotides.
Circular dichroic spectra were recorded using a JASCO J-710 spectropo-
larimeter at ambient temperature.[17] The spectra were recorded using an

Small Branched RNA Fragments Containing Chiral Linkages
207
average of five scans between 180 and 350 nm, at a rate of 50 nm/min and
sampling wavelength of 0.2 nm. The buffer used was 50 mM Na2HPO4, 100
mM HCl, at pH 7.25.
FAB-MS spectra were collected using KRATOS MS25RFA high-resolution
spectrometer under Xenon gas with a beam energy of ∼7 KV. Nitrobenzyl
alcohol (NBA) matrix was used. ESI and APCI spectra were collected using
a Finnigan LCQ Duo, ThermoQuest.
MALDI-TOF was performed using a Kratos mass spectrometer. The
oligomers (0.5 OD dissolved in 5 μL H2O) were mixed with 5 μL of matrix,
and 1 μL of the solution was used per analysis. The matrix used contained
10 mg/mL 6-Aza-2-thiothymine in a solution of 1:1 CH3CN/ammonium
acetate (20 mM) as described elsewhere.[18]
HPLC analysis and purifications were carried out on a Waters HPLC
equipped with an M486 Tunable UV detector and a W600E multisolvent de-
livery system, dual pump heads (225 μL), a UK6 injector, and a temperature
controller. The system is interfaced to a PC running MillenniumꢀR software
version 3.20. Solvents were filtered through a 0.45-μm filter and degassed
with helium prior to use. For oligonucleotide purification, an anion ex-
change 7.5 × 75 mm Protein-PakTM DEAE 5PW HPLC column was used. The
mobile phases consisted of: Solvent A: H2O (doubly distilled and deionized
using the Millipore system), Solvent B: 1 M LiClO4. Chromatograms were
run using a gradient of 0–10% Solvent B over 60 min with a flow rate of 1.2
mL/min at ambient temperature. For analytical runs, 0.3–0.7 O.D. of sample
was injected; for preparative runs, 3–7 O.D. was injected. Enzymatic diges-
tion analyses were performed using a reverse-phase HP Hypersil ODS-C18
column (4.6 × 250 mm, 5 μm) from Waters with a flow rate of 1.2 mL/min.
The mobile phases comprised of: Solvent A: 50 mM TEAA (triethylammo-
nium acetate), Solvent B: CH3CN. Chromatograms were obtained utilizing
a gradient of 0–10% Solvent B over 10 min, followed by 10–35% Solvent
B over 15 min.[19] Under these conditions, the order of elution of nucleo-
sides was: rC (5.9 min), rI (12.3 min; resulting from deamination of rA), rG
(12.7), and rA (15.2 min). The deprotected isomers of the phosphorothioate
dimers were resolved on an HB Zorbax RX-C18 semi-preparative reverse-
phase column (9.4 × 250 mm, 5 μm) at r.t. with a flow rate of 4 mL/min.
The mobile phases consisted of, Solvent A: 50 mM TEAA, solvent B: CH3CN.
The gradient utilized 0–20% Solvent B over 60 min at a temperature of
22◦C.
Monomer Synthesis
N2-Isobutyrylguanosine (2a).
Guanosine (35.3 mmol, 10 g) was dried
by coevaporation (anhydrous pyridine) and suspended in anhydrous pyri-
dine (160 mL) to which chlorotrimethylsilane (TMSCl; 7.8 eq, 265 mmol,
33.6 mL) was added dropwise at 0◦C. The mixture was stirred at r.t. for 1.25 h,

208
R. Mourani and M. J. Damha
cooled to 0◦C, and isobutyric anhydride (5 eq, 176.5 mmol, 29 mL) was
added, after which stirring was resumed overnight at r.t. Cold H2O (150 mL)
was added to the reaction followed by 28% NH4OH (65 mL) at 0◦C and the
mixture was stirred at r.t. for 50 min, at which time it was concentrated to
an oil, dissolved in water, and washed with ethyl acetate. Purification was car-
ried out via crystallization from H2O, to yield a white powder in 75% isolated
1
ꢁ
yield. H NMR (DMSO-d6, 400 MHz): 8.24 (s, 1, H8), 5.77 (d, 1, H1 ), 4.40
ꢁ
ꢁ
ꢁ
ꢁ
ꢁꢁ
(m, 1, H2 ), 4.41 (m, 1, H3 ), 3.88 (dt, 1, H4 ), 3.59–3.52 (m, 2, H5 , H5 ), 2.75
(m, 1, CH-iBu), 0.99 (dd, 6, CH3-iBu).
N2-Isobutyryl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl) guanosine (2b).
N2-Isobutyr-
ylguanosine (2a) (24.4 mmol, 8.6 g) was dried by coevaporation with
anhydrous pyridine and then suspended in (80 mL) anhydrous pyridine.
The mixture was cooled to 0◦C before adding, dropwise, a mixture
of dimethoxytrityl (DMT) chloride (1.2 eq, 29 mmol, 9.9 g) and 4-
dimethylaminopyridine (DMAP, catalytic amount, 40 mg) in pyridine (25
mL) under a nitrogen atmosphere. The mixture was allowed to warm to
r.t. and was stirred overnight before it was stopped by adding 5% NaHCO3
(100 mL). The crude product was then extracted with ethyl acetate, and
the combined organic layers dried over anhydrous Na2SO4. The solvent was
removed in vacuo to afford a dark brown oil. The crude mixture was purified
via flash silica gel column chromatography CH2Cl2/MeOH/NEt3 (gradient
99.5:0:0.5 to 96.5:3:0.5), to recover N2-iBu-guanosine as a white foam in
80% yield (12.7 g). TLC: CH2Cl2/MeOH 9:1, Rf 0.36. MS (FAB-nitrobenzyl
alcohol, calc. M+: 655), found: MH+: 656, DMT+: 303. 1H NMR (DMSO-d6,
400 MHz): 8.11 (s, 1, H8), 7.31–7.20–6.80 (overlapping m, 13, DMT), 5.84
ꢁ
ꢁ
ꢁ
(d, 1, H1ꢁ), 5.62 (d, 1, OH2 ), 5.19 (d, 1, OH3 ), 4.51 (m, 1, H2 ), 4.19 (m, 1,
ꢁꢁ
ꢁ
ꢁ
ꢁ
H3 ), 4.01 (dt, 1, H4 ), 3.70 (s, 6, OCH3), 3.23–3.15 (m, 2, H5 , H5 ), 2.74
(m, 1, CH-iBu), 1.09 (dd, 6, CH3-iBu).
N2-Isobutyryl-2ꢁ,3ꢁ-O-dibenzoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)guanosine(2c).
Compound (2b) (3 mmol, 2 g) was dried by coevaporation with anhydrous
pyridine, suspended in anhydrous pyridine (40 mL), and cooled to 0◦C
before adding benzoyl chloride (2.4 eq, 7.3 mmol, 0.85 mL) dropwise.
The mixture was allowed to warm to r.t. and was stirred for 3.5 h before it
was stopped by adding 20 mL of H2O at 0◦C and stirring at r.t. for 5 min.
The crude product was dissolved in CH2Cl2 (100 mL) and washed with
H2O. The organic phase was dried over anhydrous Na2SO4, and the solvent
was removed under reduced pressure. The crude product was purified by
flash silica gel column chromatography using CH2Cl2/MeOH (gradient
0–3% MeOH), to yield the desired nucleoside (containing some of the
the corresponding N2-benzoyl nucleoside; see compound 2d below) as a
white foam (77%, 2 g). TLC: CH2Cl2/MeOH 9/:, Rf 0.54. MS (APCI-methyl

Small Branched RNA Fragments Containing Chiral Linkages
209
alcohol, calc. M+ for product with N2-iBu guanine: 863, calc. M+ for product
with N2-Bz: 897), found: M+: 863, 896 in ratio of 10:1, respectively, DMT+:
303.
N2-Isobutyryl-O6-p-nitrophenylethyl-2ꢁ,3ꢁ-O- dibenzoyl-5ꢁ-O-(4,4ꢁ-dimeth-
oxytrityl)guanosine (2d).
To a stirred solution of (2c) (0.69 mmol,
0.6 g), triphenylphosphine (Ph3P; 3 eq, 2.08 mmol, 0.53g) and 2 − p-
nitrophenylethanol (3 eq, 2.08 mmol, 0.34 g) in anhydrous THF, was added
diethyl azodicarboxylate (DEAD; 2.25 eq, 1.56 mmol, 0.24 mL) dropwise
and the reaction was stirred overnight at r.t. The reaction was concentrated
to an oil and extracted from 5% NaHCO3 with CH2Cl2. The organic
phase was dried over anhydrous Na2SO4, and the solvent removed under
reduced pressure. The crude product was purified by flash silica gel column
chromatography using 100% CH2Cl2 until the impurities were flushed out
before using a gradient containing 1–2% MeOH (yield: 0.48 g, 69%). TLC:
CH2Cl2/MeOH 19:1, Rf 0.77; CH2Cl2/MeOH 29:1, Rf 0.6; Toluene/EtOAc
1:1, Rf 0.58; Et2O/CH2Cl2/EtOH 21:76:3, Rf 0.79. MS (FAB-nitrobenzyl
alcohol, calc. M+ for product with N2-iBu guanine: 1013, calc. M+ for
product with N2-Bz guanine: 1047), found: MH+: 1014, 1048 in ratio of ca.
3:1, respectively.
N2-Isobutyryl-O6-p-nitrophenylethyl-2ꢁ,3ꢁ-O-dibenzoylguanosine (2e). To
compound (2d) (0.56 mmol, 0.48 g) was added p-toluenesulfonic acid-
MeOH/CH2Cl2 (12 mL; prepared by stirring 3 g of p-TSA, 30 mL MeOH
and 120 mL CH2Cl2) at r.t. After 15 min the reaction was diluted with
CH2Cl2 and washed with 5% NaHCO3. The organic phase was dried over
anhydrous Na2SO4, followed by solvent removal in vacuo to afford a dark yel-
low crude residue. The pure product was precipitated from stirred hexanes
and filtered to give 2e contaminated with N2-benzoyl-O6-p-nitrophenylethyl-
2ꢁ, 3ꢁ-O-di-benzoylguanosine as a white residue, ca. 3:1 ratio (85% combined
yield, 0.28 g). TLC: CH2Cl2/MeOH 9:1, Rf 0.59; CH2Cl2/acetone 3:1, Rf
0.4. MS (FAB-nitrobenzyl alcohol, calc. M+ for product with N2-iBu: 710,
calc. M+ for product with N2-Bz: 744), found: MH+: 711, 745 in ratio of 3:1,
respectively.
N4-Benzoyl-2ꢁ,3ꢁ-O-dibenzoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)cytidine
(3c).
N4-Benzoyl-5ꢁ-O-dimethoxytritylcytidine (5.0 mmol, 3.27 g) was dried by
coevaporation (anhydrous pyridine), suspended in anhydrous pyridine (60
mL) and the mixture was cooled to 0◦C prior to the dropwise addition of
BzCl (3.5 eq, 17.6 mmol, 2.04 mL). The mixture was allowed to warm to
r.t. and stirred for 3.5 h before it was quenched with H2O (30 mL) at 0◦C
and stirring continued at r.t. for 5 min. The crude product was dissolved in
CH2Cl2 (120 mL) and washed with H2O. The organic phase was dried over

210
R. Mourani and M. J. Damha
anhydrous Na2SO4, and the solvent was removed under reduced pressure.
The crude product was purified by flash silica gel column chromatography
using CH2Cl2/MeOH (gradient 0–3% MeOH), to give a pale yellow foam
(86% yield, 3.73g). TLC: CH2Cl2/MeOH 9:1, Rf 0.74. MS (FAB-nitrobenzyl
alcohol, calc. M+: 857), found: MH+: 858, DMT+: 303.
N4-Benzoyl-2ꢁ,3ꢁ-O-dibenzoylcytidine (3d).
To compound (3c) (3.76
mmol, 3.24 g) was added a solution consisting of p-toluenesulfonic
acid/MeOH/CH2Cl2 (76 mL; prepared by dissolving p-TSA (3 g) in MeOH
(30 mL) and CH2Cl2 (120 mL) at r.t. After 15 min, the reaction was diluted
with CH2Cl2 and washed with 5% NaHCO3. The organic phase was dried (an-
hydrous Na2SO4), filtered and the solvent removed in vacuo to afford a dark
yellow crude residue. The pure product was precipitated from a cold solution
of stirred hexanes and was filtered to yield a white solid (79% yield, 1.51 g).
TLC: CH2Cl2/MeOH 9.5:0.5, Rf 0.40; Hexanes/EtOAc/NEt32.7 : 7 : 0.5, Rf
0.19. MS (FAB-nitrobenzyl alcohol, calc. M+: 555), found: MH+: 556.
N4-Benzoyl-2ꢁ,3ꢁ-O-dibenzoylcytidine-5ꢁ-O-[β-cyanoethyl-N,N-diisopropyl]
phosphoramidite (3e).
To a stirred solution of (3d) (2.71 mmol, 1.5 g),
DIPEA (4 eq, 10.84 mmol, 1.86 mL) and DMAP (0.1 eq, 0.271 mmol,
33 mg) in anhydrous THF (24 mL) was added N,N -diisopropylamino-
β-cyanoethylphosphonamidic chloride (1.2 eq, 3.25 mmol, 0.72 mL)
dropwise at 0◦C. The reaction was allowed to go to r.t. and was stirred
for 2.5 h. The mixture was diluted with EtOAc (150 mL, prewashed with
5% NaHCO3) and the crude product was washed with saturated brine
solution (4 × 60 mL). The organic phase was dried over anhydrous Na2SO4
and the solvent removed under reduced pressure to afford a light yellow
oil. Purification proceeded by flash silica gel column chromatography
using EtOAc/Hexanes/NEt3 (gradient 4:5.5:0.5 to 5:4.5:0.5), to afford a
pure 3e as a white foam (86% yield, 1.76 g). TLC: EtOAc/hexanes/NEt3
7:2.5:0.5, Rf 0.38; CH2Cl2/MeOH 9.5:0.5, Rf 0.65. MS (FAB-nitrobenzyl
alcohol, calc. M+: 755), found: MH+: 656. 31P NMR (in acetone–d6): 150.4,
149.9 ppm.
2ꢁ,5ꢁ-Linked ApsG Triesters (Rp-4 and Sp-4).
To an Argon purged
glass hypovial with an oven-dried magnetic stirring bar was added guano-
sine 2e (0.128 mmol, 91 mg), N -Bz, 5ꢁ,3ꢁ-O-protected adenosine-2ꢁ-O-
phosphoramidite (1; 1.2 eq, 0.154 mmol, 0.15 g) and 1H-tetrazole (4.4 eq,
0.565 mmol, 40 mg). The reaction was started by injecting anhydrous acetoni-
trile (1.5 mL) under nitrogen, and stirred at r.t. until complete consumption
of the starting material was observed after 2 h. To this mixture was added a
solution of 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) (1.2 eq, 0.154 mmol,
25 mg) in anhydrous acetonitrile (1 mL) under an inert atmosphere to

Small Branched RNA Fragments Containing Chiral Linkages
211
TABLE 1 Rf Values of Oligoribonucleotides
Rf Value in CH2Cl2/MeOH, 9:1
Compound
Rp Isomer
Sp Isomer
4
5
6
7
8
0.47
0.4
0.63
0.42
0.33
0.45
0.31
0.27
0.29
0.35
0.44
0.28
0.26
0.25
9
10
sulfurize the phosphite triester intermediate. The mixture was stirred at r.t.
for 10 min and the solvent was evaporated. The crude product was dissolved
in CH2Cl2 and washed with 5% NaHCO3. The organic phase was dried over
anhydrous Na2SO4, and the solvent was removed under reduced pressure to
obtain a yellow solid. The crude dimer was purified by flash silica gel column
chromatography in order to separate the two isomers using CH2Cl2/Et2O
5:5 (79% total yield, 109 mg (53%) of the first isomer (Rp-4), 53 mg (26%) of
the second isomer (Sp-4). TLC: CH2Cl2/Et2O 9:1, Rf value for Sp-4 and Rp-4
dimers are 0.16, 0.34; CH2Cl2/MeOH 9:1, Rf 0.47, 0.63; CH2Cl2/Acetone
3:1, Rf 0.4, 0.53, respectively. Refer to Tables 1 and 2 for the Rf and 31P NMR
values. MS (FAB-nitrobenzyl alcohol, calc. M+ for product with N2 − iBu
guanine: 1629, calc. M+ for product with N2-Bz guanine: 1663), found for
the Rp isomer: MH+: 1630, 1664 in ratio of 2.3:1, respectively, and DMT+:
303; and for the Sp isomer: MH+: 1630, 1664 in ratio of 2.5:1, respectively,
and DMT+: 303.
2ꢁ,5ꢁ-Linked Diester Rp-ApsG (Rp-5).
Rp-2ꢁ,5ꢁ-ApG triester (Rp-4)
(0.062 mmol, 101 mg) was dissolved in 3.5 mL TEA/CH3CN (4:6 v/v) and
the reaction was stirred at r.t. for 4 h. The solvent was then evaporated and
the crude dimer redissolved in CH2Cl2 and washed with 5% NaHCO3. The
TABLE 2 31P-NMR Data of Oligoribonucleotides
31P NMR (ppm)
Oligomer # in
NMR solvent
Rp Isomer
Sp Isomer
4 in acetone–d6
5 in acetone–d6
6 in acetone–d6
7 in CH2Cl2-d2
8 in CH2Cl2-d2
69.4
59.4, 59.5
59.5, 59.8
68.8, 68.9
60.2, 60.5
—
—
60.2, 1.0
59.6, −0.3, −1.1
61.1, 1.1
∗Isolated as mixture of N-Bz and N-iBu guanine bases, which converge to
one compound upon N-deacylation (see Materials and Methods).

212
R. Mourani and M. J. Damha
organic phase was dried over anhydrous Na2SO4, and the solvent evaporated
under reduced pressure generating the crude phosphorothioate diester
which was used without further purification in the ensuing reaction (97%
yield, 94 mg). Refer to Tables 1 and 2 for the Rf and 31P NMR values. MS
(FAB-nitrobenzyl alcohol, calc. M+ for product with N2-iBu guanine: 1575,
calc. M+ for product with N2-Bz guanine: 1609), found: MH+: 1576, 1610
in ratio of 2.8:1, respectively. DMT+: 303.
2ꢁ,5ꢁ-Linked diester Sp-ApsG (Sp-5). Sp-2ꢁ,5ꢁ-ApsG triester (Sp-4) (0.031
mmol, 50 mg) was dissolved in 1.5 mL TEA/CH3CN (4:6 v/v) and the reac-
tion was stirred at r.t. for 3 h. The solvent was then evaporated and the crude
dimer redissolved in CH2Cl2 and washed with 5% NaHCO3. The organic
phase was dried over anhydrous Na2SO4 and the solvent evaporated under
reduced pressure generating the crude phosphorothioate diester which was
used without further purification in the ensuing reaction (98% yield, 47.3
mg). Refer to Tables 1 and 2 for the Rf and 31P NMR values. MS (FAB-
nitrobenzyl alcohol, calc. M+ for product with N2 − iBu guanine: 1575, calc.
M+ for product with N2-Bz guanine: 1609), found: MH+: 1576, 1610 in ratio
of 1.7:1, respectively. DMT+: 303.
2ꢁ,5ꢁ-Linked diester Rp-ApsG (Rp-6).
Rp-2ꢁ,5ꢁ-ApsG diester (Rp-5),
(0.049 mmol, 77 mg) was dissolved in excess TBAF solution (1.0 M in THF)
which had been pH adjusted to 7.0 by adding glacial acetic acid. The reac-
tion was stirred at 45◦C overnight before it was quenched with H2O and the
crude product extracted with CH2Cl2 and dried under reduced pressure.
The crude dimer was purified by flash silica gel column chromatography
using EtOAc/MeOH (gradient 0–2% MeOH) (70% yield, 50 mg). Refer
to Tables 1 and 2 for the Rf and 31P NMR values. MS (ESI-MeOH-negative
mode, calc. M+ for product with N2-iBu guanine: 1461, calc. M+ for product
with N2-Bz guanine: 1495), found: M-H−: 1460, 1494 in ratio of ca. 2.4:1,
respectively.
2ꢁ,5ꢁ-Linked diester Sp-ApsG (Sp-6).
Sp-2ꢁ,5ꢁ-ApsG diester (Sp-5) (0.03
mmol, 47.3 mg) was dissolved in excess TBAF solution (1.0 M in THF) that
had been pH adjusted to 7.0 by adding glacial acetic acid. The reaction was
stirred at 42◦C for 19 h before it was quenched with H2O and the crude
product extracted with CH2Cl2, and dried under reduced pressure. The
crude dimer was purified by flash silica gel column chromatography using
EtOAc/MeOH (gradient 0–2% MeOH) (82% yield, 36 mg). Refer to Tables
1 and 2 for the Rf and 31P NMR values. MS (ESI-MeOH-negative mode, calc.
M+ for product with N2-iBu guanine: 1461, calc. M+ for product with N2-Bz
guanine: 1495), found: M-H−: 1460, 1494 in ratio of ca. 1.3:1, respectively.

Small Branched RNA Fragments Containing Chiral Linkages
213
Branched trimer Rp-A(psG)pC (Rp-7). To an Ar-purged glass hypovial
with a magnetic stirring bar, was added Rp dimer (Rp-6) (0.027 mmol, 40 mg),
cytidine-5ꢁ-phosphoramidite (3e) (6 eq, 0.164 mmol, 127 mg) and tetrazole
(6 eq, 0.164 mmol, 12.6 mg). The reaction was initiated by injecting an-
hydrous acetonitrile (0.75 mL) and stirred at r.t. for 3 h in a dry nitrogen
box. 2,4,6-collidine (6 eq, 0.164 mmol, 21.7 μL) was added followed by ad-
dition of aqueous iodine solution (0.1 M in THF/water 3:1.5, excess) in
order to oxidize the phosphite triester intermediate. After 5 min, the re-
action was diluted with CH2Cl2 and washed with 5% sodium bisulfite. The
organic phase was washed with 5% NaHCO3, dried over anhydrous Na2SO4,
and the solvent was removed under reduced pressure to obtain a foam.
The crude trimer was purified by flash silica gel column chromatography
using CH2Cl2/MeOH/0.5%TEA (gradient 0–2% MeOH) (82% total yield,
47.5 mg). Refer to Tables 1 and 2 for the Rf and 31P NMR values.
Branched trimer Sp-A(psG)pC (Sp-7).
To an Argon purged glass hy-
povial with a magnetic stirring bar, was added Sp dimer (Sp-6) (0.021 mmol,
30 mg), cytidine-5ꢁ -phosphoramidite (3e) (6 eq, 0.123 mmol, 90 mg) and
tetrazole (6 eq, 0.123 mmol, 8.7 mg). The reaction was initiated by injecting
anhydrous acetonitrile (0.60 mL) and stirred at r.t. for 1.25 h in a dry nitro-
gen box. 2,4,6-collidine (6 eq, 0.123 mmol, 16.25 μL) was added followed
by addition of aqueous iodine solution (0.1 M in THF/water 3:1.5, excess)
in order to oxidize the phosphite triester intermediate. After 5 minutes the
reaction was diluted with CH2Cl2 and washed with 5% sodium bisulfite. The
organic phase was washed with 5% NaHCO3, dried over anhydrous Na2SO4,
and the solvent was removed under reduced pressure to obtain a foam. The
crude trimer was purified by flash silica gel column chromatography using
CH2Cl2/MeOH/0.5%TEA (gradient 0–2% MeOH) (75% total yield, 32.2
mg). Refer to Tables 1 and 2 for the Rf and 31P NMR values.
Branched trimers A(psG)pC Rp-8 and Sp-8.
These were obtained by
treatment of Rp-7 and Sp-7 with triethylamine/acetonitrile as described above
for the synthesis of dinucleoside phosphorothioates Rp-5 and Sp-5. Charac-
terization data (Rf and 31P NMR) are provided in Tables 1 and 2.
Branched trimer Rp-A(psG)pC (Rp-9).
Trimer Rp-8 (0.011 mmol,
23 mg) was dissolved in 2.5 mL CH2Cl2 and 2.5 mL (p-TSA/MeOH/CH2Cl2),
prepared by stirring 0.5 mg p-TSA in 5 mL MeOH and 20 mL CH2Cl2, and
the reaction was stirred at r.t. for 25 min. It was diluted with CH2Cl2, washed
with 5% NaHCO3, dried over anhydrous Na2SO4, and the solvent was dried
under reduced pressure. The crude trimer was purified by flash silica gel
column chromatography using CH2Cl2/MeOH (gradient 0–2% MeOH).
(>90% yield) Refer to Table 1 for the Rf values. MS (ESI-MeOH-negative

214
R. Mourani and M. J. Damha
mode, calc. M+ for product with N2-iBu guanine: 1775, calc. M+ for prod-
uct with N2-Bz guanine: 1809), found: M-H−: 1775, 1809 in ratio of 2.75:1,
respectively.
Branched trimer Sp-A(psG)pC (Sp-9).
Trimer Sp-8 (0.010 mmol,
22 mg) was dissolved in 3 mL CH2Cl2 and 0.6 mL (p-TSA/MeOH/CH2Cl2),
prepared by stirring 0.5 mg p-TSA in 5 mL MeOH and 20 mL CH2Cl2, and
the reaction was stirred at r.t. for 10 min. It was diluted with CH2Cl2, washed
with 5% NaHCO3, dried over anhydrous Na2SO4, and the solvent was dried
under reduced pressure. The crude trimer was purified by flash silica gel col-
umn chromatography using CH2Cl2/MeOH (gradient 0–2% MeOH). (93%
yield, 17.4 mg) Refer to Table 1 for the Rf values. MS (ESI-MeOH-negative
mode, calc. M+ for product with N2-iBu guanine: 1775, calc. M+ for prod-
uct with N2-Bz guanine: 1809), found: M−: 1775, 1809 in ratio of ca. 3:1,
respectively.
Branched tetramer Sp-ApA(psG)pC (Sp-10).
To an Argon purged glass
hypovial with a magnetic stirring bar was added Sp-trimer (Sp-9) (0.01 mmol,
17.4 mg),5ꢁ-O-dimethoxytrityl-2ꢁ-O-t-butyldimethylsilyladenosine-3ꢁ-O-[β-cy-
anoethyl-N,N-diisopropyl]phosphoramidite (6 eq, 0.059 mmol, 59 mg) and
tetrazole (18 eq, 0.176 mmol, 11 mg). The reaction was commenced by
injecting anhydrous acetonitrile (0.5 mL) and stirred at r.t. for 2.5 h in
a dry nitrogen box. 2,4,6-Collidine (6 eq, 0.059 mmol, 7.8 μL) was then
added followed by addition of aqueous iodine solution (0.1 M in THF/H2O
3:1.5, excess) to oxidize the phosphite triester intermediate. After 5 min,
the reaction was diluted with CH2Cl2 and washed with 5% sodium bisulfite.
The organic phase was washed with 5% NaHCO3, dried over anhydrous
Na2SO4, and the solvent removed under reduced pressure to attain a
foam. The crude tetramer was partially purified by flash silica gel column
chromatography using CH2Cl2/MeOH/0.5%TEA (gradient 0–2% MeOH)
(67% yield, 17.9 mg). Refer to Table 1 for the Rf values. MS (MALDI-TOF,
calc. M− for product without the cyanoethyl protecting group: 2626), found:
M-2H−: 2633 = 2626 + 7 (Li cation).
Deprotection of Branched Oligonucleotides
The procedure can be illustrated by the deprotection of the Sp-A(psG)pC
trimer Sp-7.
Detritylation.
Sp-trimer Sp-7 (5 mg) was dissolved CH2Cl2 (0.5 mL)
followed by the addition of trifluoroacetic acid (12 μL) at r.t. The solution
turned orange immediately (ditrityl cation), indicating the desired detrityla-
tion reaction. After 7 min, CH2Cl2 was added and the resulting mixture was

Small Branched RNA Fragments Containing Chiral Linkages
215
washed with 5% NaHCO3. The organic phase was collected and dried over
anhydrous Na2SO4 to afford, after evaporation under reduced pressure, a
dark yellow crude residue. In the case of the Sp-tetramer Sp-10, detritylation
was conducted by treatment with p-toluenesulfonic acid (p-TSA) as follows:
Sp-10 (6.5 mg) was dissolved in CH2Cl2 (0.3 mL) and p-toluenesulfonic acid-
MeOH/CH2Cl2(0.3 mL) was added at r.t.; the latter solution prepared by
stirring 0.52 g p-TSA, 5.2 mL MeOH and 20.8 mL CH2Cl2. Work-up was
carried out as described above for the detritylation of Sp-7.
Simultaneous removal of cyanoethyl, benzoyl, and isobutyryl groups.
The material obtained after step (a) above was dissolved in 1.5 mL conc.
aqueous ammonia (NH4OH) and 0.5 mL EtOH, and the reaction was stirred
at r.t. for 48 h. After cooling the sample in an acetone/dry ice bath, ammonia
was removed under low vacuum, followed by removal of ethanol and H2O
using a high vacuum Speed-VacꢁR concentrator.
Simultaneous removal of tert-butyldimethylsilyl and p-nitrophenylethyl
groups.
The residue obtained above was treated with 1.0 M TBAF/THF
(560 μL) for 4 h at r.t. Water was added and the resulting mixture passed
through a column filled with Dowex Na+ ion exchange resin. The eluent
(10 mL) was quantitated on a UV spectrometer to yield 42 A260 units (42
O.D.) of crude Sp-trimer [A(psG)pC].
Oligonucleotide Analysis, Purification, and Desalting
Rp- and Sp-ApsG phosphorothioates (and enzymatic digests) were an-
alyzed and resolved by reverse-phase HPLC. Fully deprotected branched
trimers were purified via anion exchange HPLC, whereas tetramer Sp-
ApA(psG)pC was purified via polyacrylamide gel electrophoresis (PAGE)
as described below.
Polyacrylamide Gel Electrophoresis (PAGE).
The tetranucleotide Sp-
ApA(psG)pC (32 O.D. units) was analyzed and purified by denaturing poly-
acrylamide gel electrophoresis (24%, 7 M urea).[20] The separate bands were
visualized under UV lamp (254 nm) and the desired (slowest moving) band
excised with a scalpel blade, and crushed in a sterile 10-mL polypropylene
tube. Autoclaved H2O (5 mL) was added to the tube and it was shaken at
r.t. for 24 h in order to extract the oligomer from the gel matrix. The tube
was centrifuged and the oligomer-containing supernatant evaporated under
reduced pressure on a Speed-VacꢁR concentrator.
Desalting.
The samples purified by anion exchange HPLC and PAGE
were desalted using Sep-Pak reverse-phase C18 cartridges (Waters). The

216
R. Mourani and M. J. Damha
cartridges were flushed with 10 mL MeOH then equilibrated with 10 mL
of doubly distilled and autoclaved H2O. The oligomers were dissolved in 1
mL of 50 mM triethylammonium acetate (TEAA) and were slowly loaded
into the cartridges to collect the first fraction in a 1-mL microtube. The car-
tridge was flushed further with 50 mM TEAA (3 mL) in order to elute the
salts. The oligomers were eluted from the column in 10 fractions of 1 mL
each using a solution of 50 mM TEAA:MeOH (7:3). The individual fractions
were analyzed by diluting the samples in 1 mL of sterile water, and measuring
the UV absorbance profile at 260 nm. Typically, the compound of interest
was found in the first two or three fractions.
In Vitro Enzymatic Studies
Enzymatic Digestion via Snake Venom Phosphodiesterase (SVPDE).
SVPDE from Crotalus adamanteus, a 3ꢁ-exonuclease (0.027 units/mg), was
dissolved in 2 mL buffer A (0.11 M Tris, 0.11 M NaCl, and 15 mM MgCl2 with
pH 8.9) to give a final concentration of 0.00135 U/μL. The residual nucleo-
side 5ꢁ-monophosphates were converted to their constituent nucleosides via
in situ treatment with alkaline phosphatase from calf intestinal mucosa (AP,
2000 units/mL), which was kept in the storage buffer provided (10 mM tris-
HCl (pH 8.0), 50 mM KCl, 1 mM MgCl2, 0.1 mM ZnCl2 and 50% glycerol).
AP (5 μL) was added to 10 μL of its 10X buffer (100 mM Tris-acetate, 100
mM magnesium acetate, and 500 mM potassium acetate) and 85 μL of H2O,
to give a 20-fold dilution of enzyme. 90 μL of this solution was added to each
assay. The oligomers (0.2 O.D. each) were dissolved in 30 μL buffer B (50 mM
Tris-HCl and 10 mM MgCl2 with pH 8). The SVPDE (0.002 U) and AP (9 U)
enzymes were added, and the reactions were incubated at 37◦C for the allot-
ted times before they were analyzed using HPLC as described above. Under
these conditions, the branched compounds and corresponding hydrolysis
products had the following retention times: ApG diester (15.5 min), Rp-
A(ps)G, (17.1 min), and Sp-A(ps)G (17.7 min), Rp-A(psG)pC (18.4 min),
and Sp-A(psG)pC (18.3 min), rC (5.9 min), rI (12.3 min; resulting from
deamination of rA), rG (12.7 min), and rA (15.2 min).
3ꢁ End32 P labeling.
Labeling the branched oligomers and linear con-
trol molecules at their 3ꢁ-termini was conducted in a similar method to Uh-
lenbeck et al. using the enzyme T4 RNA ligase and [5ꢁ-32P]cytidine 3ꢁ, 5ꢁ-
biphosphate.[21] The oligomers (0.01 OD units) were dissolved in 0.5 μL
of the 10 × T4 RNA Ligase buffer (500 mM Tris-HCl (pH 7.5), 100 mM
MgCl2, 100 mM DTT, and 10 mM ATP), 0.5 μL deionized DMSO-d6, 0.5
μL acetylated bovine serum albumin (BSA; 0.1 mg/mL), 0.5 μL (5 U) T4
RNA ligase (10 units/μL), 2 μL [5ꢁ-32P]cytidine 3ꢁ,5ꢁ-biphosphate (3,000
Ci/mmol), to a final volume of 5 μL with sterile water. The reaction mixtures
were incubated at 37◦C overnight, and the enzyme inactivated by heating

Small Branched RNA Fragments Containing Chiral Linkages
217
at 70◦C for 10 min. Their labeling reactions were taken up in gel load-
ing dye (5 μL), purified from any unlabeled material on a 24% denatur-
ing polyacrylamide gel, and visualized by autoradiography (Kodak X-Omat
AR film). The labeled oligonucleotides were excised from the gel and ex-
tracted by crushing and soaking the gel section in H2O at 37◦C overnight,
and desalted via size-exclusion chromatography on Sephadex G-5 columns.
The labeled oligonucleotides were stored as dry pellets at –20◦C until
needed.
Debranching of synthetic oligomers (purified yDBR).
Branched com-
pounds labeled in the above manner were hydrolyzed at the 2ꢁ, 5ꢁ-linkage
using purified His-tagged yeast lariat debranching enzyme (yDBR), a gen-
erous gift of Dr. J.D. Boeke (Johns Hopkins University). The3ꢁ end labeled
oligomers were dissolved in 2.5 μL yDBR debranching buffer (final concen-
tration of 20 mM HEPES, pH 7.6, 125 mM KCl, 0.5 mM MgCl2, 1 mM DTT,
10% glycerol), 0.5–1 μL yDBR (50–100 ng), and H2O to obtain a final volume
of 7.5 μL per reaction. The debranching reactions were incubated at 37◦C,
inactivated by heating at 70◦C for 10 min, and lyophilized before loading dye
was added and the mixtures were analyzed using 24% denaturing PAGE and
visualized by autoradiography.
RESULTS AND DISCUSSION
The general scheme for the synthesis of the branched 2ꢁ,5ꢁ-phosphoro-
thioate oligonucleotides commences with the coupling and sulfurization
of the adenosine 2ꢁ-amidite (1) with the properly protected guanosine
monomer (2e) to yield the 2ꢁ,5ꢁ-phosphorothioate dimer A(psG) (4). Sep-
aration of the two isomers at this stage was next accomplished by chro-
matographic resolution according to established protocols,[22] followed by
selective removal of the TBDMS protection from the adenosine 3ꢁ-hydroxyl
function. The cytidine 5ꢁ-amidite (3) was subsequently coupled to either
dimer (Rp and Sp isomers separately) at the 3ꢁ terminus, followed by oxi-
dation to give a 3ꢁ,5ꢁ-phosphodiester linkage, yielding the V-shaped trimers
A(psG)pC (7). The 5ꢁ-DMT was last removed from the adenosyl branching
residue, and the deprotected trimer coupled with an adenosine 3ꢁ-amidite to
extend the chain in the 5ꢁ-direction and yield the Y-tetramer (10) (Scheme 1).
Synthesis of Branched 2ꢀ,5ꢀ-Phosphorothioate RNA
Phosphorothioate-linked dimer (4) was synthesized by sulfurizing
the intermediary phosphite triester using 3-ethoxy-1,2,4-dithiazoline-5-
one (EDITH).[23−25] The Beaucage reagent (3H-1,2-benzodithiole-3-one
1,1-dioxide),[26,27] also afforded the dimer, although some oxidized

218
R. Mourani and M. J. Damha
SCHEME 1 Chemical synthesis of branched RNAs.
phosphotriester dimer formed as well (data not shown). A non-stereo-
selective method was employed for the synthesis of the phosphorothioate
dimer since both isomers were required for probing yDBR stereospecificity,
although Rp isomer(4) was produced to a greater extent than Sp isomer
(4) (ca. 2:1 respectively, reaction repeated three times). Stereochemistry was
enzymatically assigned using snake venom phosphodiesterase (SVPDE; see
below) and 31P-NMR, in which a relatively larger chemical shift of the Rp
versus the Sp isomer was observed (Figure 3). Additional evidence for the
presence of both chemical moieties in each of the two chiral samples was
provided via mass spectrometric analysis.
Desilylation of the dimers was first attempted by treating dimer (4) with
tetra-n-butylammonium fluoride (TBAF) as a solution in THF. However, this
led to the removal of the O6-p-nitrophenylethyl protecting group due to the
basic nature of the solution. Furthermore, the acidity of triethylamine
trihydrofluoride (TREAT-HF) promoted cleavage of the dimethoxytrityl-
protecting group. Therefore, an alternative method with neutral conditions

Small Branched RNA Fragments Containing Chiral Linkages
219
FIGURE 3 31P-NMR spectra of Rp-A(psG)pC (7 & 8) in CH2Cl2-d2, (A) before and (B) after the removal
of the cyanoethyl group at the 3ꢁ,5ꢁ-internucleotide linkage. The phosphorothiolate and phosphate link-
ages resonate at ca. 60 and 1 ppm, respectively.
was developed in order to remove the silyl group selectively. The method
of choice involved treatment of the dimer with a “preneutralized” solution
of 1.0 M TBAF in THF using AcOH. This method was very clean and
quantitative, and as confirmed by mass spectroscopy (ESI) led to the
selective removal of the TBDMS group.
After the removal of the silyl group the dimer was coupled to cytidine 5ꢁ-
phosphate monomer to yield the V-shaped trimer (7). In addition to NMR,
MS, and HPLC, UV profiles provided a good additional characterization
tool to differentiate between the A(psG) dimer and the A(psG)pC trimer,
where the trimer absorbs at an additional wavelength, ca. 312 nm, due to the
presence of cytidine.

220
R. Mourani and M. J. Damha
During the coupling of the trimer to the adenosine phosphoramidite,
and due to the very similar Rf values of the protected trimer and tetramer, it
was not possible to determine by TLC whether all of the V-trimer had been
consumed. However, HPLC analyses following deprotection of the products
confirmed the nearly complete consumption of Sp-V-trimer. The synthesized
tetramer was verified by mass spectroscopy (MALDI-TOF), HPLC, and CD
(Figure 4).
The CD spectra of the phosphorothioate oligonucleotides (V-trimers,
and Sp-Y-shaped tetramer), and some of the corresponding 2ꢁ, 5ꢁ and
3ꢁ, 5ꢁ dinucleoside monophosphates and trinucleoside diphosphates were
obtained and analyzed to gain further information on possible base
interactions.[28−30] The CD spectra of the two A(psG) isomers have simi-
lar peak positions to the corresponding A(pG) phosphodiester dinucleotide
reported by Damha,[31] but are less intense in magnitude. The CD spectra of
Rp and Sp phosphorothioate V-trimers show a non-conservative cotton effect
(Figure 4a, b). These types of non-conservative spectra are usually exhibited
by base-stacked dimers containing guanosine bases due to the presence of at
least two overlapping π → π* bonds from the guanosine monomers in the
spectral region ranging from 210 to 300 nm.[32,33] As a result, interpreting
CD spectra of oligomers containing guanosine can prove quite difficult. How-
ever, this observation indicates that the guanine of the 2ꢁ-nucleotide residue
in the phosphorothioate V-trimers are involved in base stacking interactions.
The low intensity band observed in the 2ꢁ,5ꢁ-A(psG) signature is retained
in the branched phosphorothioate trimers, which also possess similar CD
patterns to the phosphodiester V-trimers A(pG)pC reported by Damha,[33]
and in the phosphorothioate Sp-Y-tetramer’s case (Figure 4c). This similarity
in intensity between the branched phosphorothioate oligomers and 2ꢁ, 5ꢁ-
A(psG), is possibly a second indication to the involvement of the 2ꢁ-guanosine
residues in stacking interactions. The CD profiles of both phosphorothioate
V-trimers and phosphorothioate Sp-Y-tetramer appear to be intermediate
between the corresponding 2ꢁ, 5ꢁ- and 3ꢁ, 5ꢁ-linked dinucleotide monophos-
phates. This suggests that two relatively equal populations may exist for the
phosphorothioate V-trimers, in which the branched adenine base forms a
stack with the chromophore of the 2ꢁ-residues in one population, and with
the 3ꢁ-residues in the second. This characteristic is absent in the spectra of
normal phosphodiester V-shaped trimers, in which a predominant popula-
tion of stacked branchpoint adenines with the adjacent 2ꢁ-residue occurs.[30]
Stereochemical Assignment of the Phosphorothioate Isomers
Using Snake Venom Phosphodiesterase (SVPDE)/Alkaline
Phosphatase (AP)
Stereochemical assignment of the phosphorothioate isomers was accom-
plished via treatment with snake venom exonuclease (phosphodiesterase

Small Branched RNA Fragments Containing Chiral Linkages
221
I; SVPDE). This enzyme stereoselectively hydrolyzes only the Rp isomer
of either 3ꢁ, 5ꢁ- or 2ꢁ, 5ꢁ- phosphorothioates linkages.[21,34] Since little is
known about the behavior of SVPDE toward branched phosphorothioate
FIGURE 4 (a)–(c) CD spectra of unprotected di, tri and tetranucleotides in 50 mM Na2HPO4, 100 mM
HCl, at pH 7.25. (Continued )

222
R. Mourani and M. J. Damha
FIGURE 4 (Continued).
oligonucleotides, the enzymatic characterization was first carried out on the
two separated isomers of 2ꢁ, 5ꢁ-Ap(s)G 4.
The SVPDE/AP assays were carried out using as control an ApG di-
ester, and the two isomers of A(ps)G. The phosphodiester linkage of ApG
was fully hydrolyzed within 4 h. However, the faster eluting phosphoroth-
ioate dimer on reverse-phase HPLC showed more than 50% hydrolysis
after 4 h, while the slower eluting dimer showed no signs of hydrolysis
even after 24 h. Thus, the faster eluting dimer has an Rp configuration
and the slower eluting dimer has an Sp configuration (see Materials and
Methods).
The SVPDE/AP assays were also conducted using V-trimers [5ꢁ-
A(psG)pC] as substrates in order to confirm the stereochemistry of the
two isomers, and to verify the enzyme’s ability to hydrolyze branched
oligoribonucleotides. The Sp-V-trimer showed full hydrolysis of the 3ꢁ, 5ꢁ-
phosphodiester bond after 2 h, yielding deoxycytidine 5ꢁ-monophosphate
and Sp-configured ApsG dimer as products. Conversely, HPLC analysis of
the Rp-V-trimer digestion profiles with SVPDE showed a relatively broader
product distribution, corresponding to deoxycytidine 5ꢁ-monophosphate
and intact Rp dimer, as well as the constituent mononucleosides arising
from partial hydrolysis of the Rp-linkage. These results demonstrate the en-
zyme’s ability to stereoselectively hydrolyze Rp-branched phosphorothioate
oligonucleotides.

Small Branched RNA Fragments Containing Chiral Linkages
223
FIGURE 5 32pCp Labeling of branched trinucleotides. The (−)ve control contains 32pCp and ligase
without branched RNA substrate.
3ꢁ-End Labeling with 32
P
T4 PNK-mediated incorporation of a 5ꢁ-phosphorus radiolabel within
32
V-branched compounds using γ − P-[ATP] as a phosphate source is ex-
ceedingly difficult, owing to the absence of a 5ꢁ-nucleotide tract in these
substrates.[35,36] These oligonucleotides were therefore 3ꢁ end labeled using
T4 RNA ligase and 5ꢁ-32P-cytosine-3ꢁ, 5ꢁ-bisphosphate (i.e.,32pCp) (Figure 5).
This also proved to be advantageous for monitoring debranching efficacy by
DBR, as it allows one to follow the release of both 2ꢁ, 5ꢁ- and 3ꢁ, 5ꢁ-linked phos-
phodiester (or phosphorothioate) hydrolysis fragments generated by the
enzyme. However, the small size of the oligonucleotides, and consequently
greater steric hindrance of 2ꢁ and 3ꢁ end termini, caused them to be labeled
with low efficiency. As shown in Figure 5, the all-phosphodiester V-trimer was
more efficiently labeled by T4 RNA ligase relative to the corresponding Rp
and Sp thioated isomers.
Debranching Studies Using His-Tagged yDBR
Previous studies on DBR hydrolytic efficacy have only examined
as test substrates lariats with an isomerically pure Rp-configured 2ꢁ, 5ꢁ-
phosphorothioate linkage, which were not cleaved by the enzyme.[15] To
examine the effect of linkage chirality at the branchpoint on this reaction,
we therefore evaluated the ability of the newly synthesized Y-shaped com-
pound with Sp-configuration to undergo debranching by DBR (Figure 6). We
also determined the susceptibility of both V-shaped Rp- and Sp compounds

224
R. Mourani and M. J. Damha
FIGURE 6 Debranching of branched compounds by yDBR. The (−)ve lane contains only the oligomer
(no yDBR).
to debranching, in order to demonstrate the necessity of a residue at the
5ꢁ-position of the adenosine branchpoint for substrate recognition by the
enzyme.
Debranching assays were initially conducted using HeLa cell extracts,
however, significant degradation appeared to accompany the observed de-
branching of all oligonucleotide test substrates as verified using a linear con-
trol. We subsequently switched to a purified source of enzyme to eliminate the
occurrence of competing nuclease degradation reactions, and thereby sim-
plify both the characterization and quantitation of debranching efficiency
in these substrates. His-tagged purified yDBR was thus used to carry out de-
branching assays. When this enzyme was used to debranch a Y-shaped 18-mer
(i.e., 5ꢁ-UACUAA(pGUAUGU)pGUAUGU), over half of the starting material
was debranched at the earliest time point (30 min; data not shown). Con-
versely, linear oligomers were not hydrolyzed by the pure enzyme.
In principle, scission of the branchpoint 2ꢁ, 5ꢁ-bond of the 3ꢁ end labeled
V-shaped compounds should yield two products, consisting of the linearized
3ꢁ-tail trimer (i.e., ApC32pCp) and the 2ꢁ-tail fragment (e.g., psG32pCp). By
comparison, scission of the 2ꢁ, 5ꢁ-bond of the phosphorothioate Y-shaped
compound should yield similar products, which differ only in the nature of
the linear 3ꢁ-tail (e.g., ApApC32pCp and psG32pCp). Indeed, debranching
of the labeled Sp-V-shaped oligomer yielded two bands that migrated faster
than the starting material, one of which corresponded to the linear control
trimer ApC32pCp (Figure 6). The new species were evident after only 6 h of
incubation with the enzyme. The labeled Sp-Y-shaped oligomer was similarly
hydrolyzed by yDBR, as evidenced by the presence of a debranching product
co-migrating with one of the products of the V-shaped substrate, suggesting
this to be the linear 2ꢁ-tail dimer (psG32pCp; Figure 6). However, the presence
of a second product band could not clearly be visualized, owing to the low
amount of radioactivity used in the assay. The Rp-V-shaped compound, on

Small Branched RNA Fragments Containing Chiral Linkages
225
the other hand, showed no signs of debranching even after 24 h (Figure 6)
as expected from previous work.[15]
Debranching studies of the control phosphodiester A(pG32pCp)G32pCp
revealed partial debranching at 6 h, as indicated by the appearance of a new
faster moving band migrating close to the linear ApC32pCp control (data
not shown). Although the extent of debranching increased at 24 h, there
were significant amounts of branched molecule still intact, indicating that
it was not recognized as efficiently by the enzyme as with larger branched
molecules (e.g., Y-shaped 18mer).
Stereospecificity of yDBR-Rp Versus Sp
yDBR is a member of the metallophosphoesterase superfamily that in-
cludes bacteriophage phosphatase (a phosphomonoesterase) and Pyrococcus
furiosus Mre11 (a phosphodiesterase).[37–42] Structural studies of these en-
zymes showed that conserved His, Asp, and Asn side chains are involved
in binding of the two manganese ions, whereas conserved His and Asn
side chains contact the phosphate in the active site.[41,42] Recent structure-
function analysis of yDBR by Schwer and co workers revealed that the enzyme
requires a similar group of conserved amino acids for activity.[37] Specifi-
cally, they found that six of the seven metal-binding amino acids in Mre11
(His10, Asp49, Asn84, His173, His206, His208) are conserved in yDBR (as
His13, Asp40, Asn85, His179, His231, His233) and essential for debranching
in vivo. Their work strongly suggested that yDBR likely contains a binuclear
manganese cluster similar to that seen in Mre11.[37]
Figure 7 shows PfuMre11 structure with 5ꢁ-dAMP bound in the active site
(adapted from reference 37). As discussed by Schwer and coworkers,[37] the
bridging water between the two manganese ions is a good candidate for the
˚
attacking nucleophile, because it is located 3.1A from the phosphorus atom
and is almost perfectly apical to the phosphate 2ꢁ-oxygen atom of the leaving
group, which is a nucleoside 3ꢁ-O in the case of Mre11. The bridging water
ligand between the manganese ions is activated by the metals for attack on
the phosphate, which, in Mre11, is coordinated by Asn84 and His85 side
chains.[41] Since the corresponding Asn85 and His86 side chains of yDBR are
critical for debranching, Schwer and co-workers proposed that Asn85 and
His86 of yDBR contact the scissile 2ꢁ-5ꢁ phosphodiester of the branched RNA
in a manner analogous to that seen in the Mre11-dAMP complex.[37] Accord-
ing to this model, it can be appreciated that replacement of the prochiral
Rp-oxygen with the larger sulfur atom would lead to steric interactions with
both Asn85 and a manganese ion. Therefore binding of Rp-bRNA, if it occurs,
will likely result in non-optimal yDBR-bRNA interactions for catalysis. Such
steric effects are less evident for the Sp-bRNA isomer, where only binding to
the second metal ion would be perturbed (Figure 7). This may explain the
inability of yDBR to debranch 2ꢁ, 5ꢁ-linked Rp-phosphorothioate linkages.

226
R. Mourani and M. J. Damha
FIGURE 7 The figure, adapted from the work of Schwer and coworkers,[37] depicts the active site of
the manganese- and 5ꢁ-dAMP-bound P. furiosus phosphodiesterase Mre11 from the crystal structure (PDB
1II7).[42] The amino acid side chains coordinating the binuclear metal cluster and the nucleotide 5ꢁ
phosphate are shown. The corresponding amino acids of yDBR enzyme are indicated in parentheses.
The manganese ions and water nucleophile are also shown. For simplicity, only the phosphate and the
ribose C4ꢁ and C5ꢁ atoms of the nucleotide are shown. The departing O2ꢁ atom (O3ꢁ in the case of dAMP)
is shown apical to the water nucleophile.
It is intriguing to note that whereas yDBR carries out stereoselective hy-
drolysis of the Sp isomer (branched tri and tetranucleotides), SVPDE prefers
the Rp configuration of the substrate tested (2ꢁ,5ꢁ-linked dinucleotides). The
apparent biochemical differences between the two enzymes appear to ex-
tend to a mechanistic level, e.g., hydrolysis by SVPDE proceeds via a covalent
nucleotide enzyme intermediate (2-step process),43 whereas that of yDBR
is likely one step (e.g., in-line attack at phosphorus by a water molecule;
Figure 7).
CONCLUSIONS
Our results indicate that the size of the substrate plays a significant role
in efficient debranching, since the small substrates were not cleaved as effi-
ciently as the large branched 18-mer. Furthermore, the 2ꢁ, 5ꢁ-internucleotide
linkage of a branched Rp oligonucleotide was not cleaved by the yDBR en-
zyme, suggesting that the yDBR enzyme is sensitive to changes carried out
on the pro-Rp oxygen. The Sp phosphorothioate isomers, along with all

Small Branched RNA Fragments Containing Chiral Linkages
227
phosphodiester branched compounds [V-A(pGpCp)pGpCp and Y-shaped
18-mer], were all substrates of the yDBR enzyme, albeit with diverse efficacies.
Employing capillary electrophoresis as an alternative method to study the
debranching of these small branched molecules by yDBR[14] could prove a
good method to verify and corroborate the results obtained here in addition
to avoiding the problems that arise from radiolabeling these small branched
and sterically hindered molecules.
Future studies involving subjecting the Sp-branched RNA substrate to the
yDBR and studying the stereochemistry of the debranching products could
provide further insight into the mechanism of specific 2ꢁ, 5ꢁ-phosphodiester
linkage hydrolysis by the yDBR enzyme. Moreover, competitive inhibition
studies can be carried out in order to ascertain if a Rp phosphorothioate
branched RNA (the substrate which is not hydrolyzed) is nonetheless rec-
ognized by the enzyme and binds to its active site. Given that this isomer
(or other analogues under study in our laboratory) may potentially be an
inhibitor of debranching, it may find use in co-crystallization of yDBR:bRNA
complexes, thus possibly allowing for the attainment of a much desired X-ray
crystal structure.
REFERENCES
1. Padgett, R.A.; Konarska, M.M.; Grabowski, P.J.; Hardy, S.F.; Sharp, P.A. Lariat RNAs as inter-
mediates and products in the splicing of messenger RNA precursors. Science 1984, 225, 898–
903.
2. Grabowski, P.J.; Seiler, S.R.; Sharp, P.A. A multicomponent complex is involved in the splicing of
messenger RNA precursors. Cell 1985, 42, 345–353.
3. Brody E., Abelson J. The “spliceosome”: yeast pre-messenger RNA associates with a 40S complex in
a splicing-dependent reaction. Science 1985, 228, 963-967.
4. Frendewey, D.; Keller, W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and
specific intron sequences. Cell 1985, 42, 355–367.
5. Wallace, J.C., Edmonds, M. Polyadenylated nuclear RNA contains branches. Proc. Natl. Acad. Sci.
USA. 1983, 80, 950–954.
6. Ruskin, B.; Green, M.R. An RNA processing activity that debranches RNA lariats. Science 1985, 229,
135–140.
7. Chapman, K.B.; Boeke, J.D. Isolation and characterization of the gene encoding yeast debranching
enzyme. Cell 1991, 65, 483–491.
8. Ooi, S.L.; Samarsky, D.A.; Fournier, M.J.; Boeke, J.D. Intronic snoRNA Biosynthesis in S. cerevisiae
depends on the Lariat debranching enzyme: Intron length effects and activity of a precursor snoRNA.
RNA. 1998, 4, 1096–1110.
9. (a) Leader, D.J.; Clark, G.P.; Watters, J.; Beven, A.F.; Shaw, P.J.; Brown, J. W.S. Clusters of multiple
different small nucleolar RNA genes in plants are expressed as and processed from polycistronic
pre-snoRNAs. EMBO J. 1997, 16, 5742–5751.
(b) Leader, D.J.; Sanders, J.F.; Waugh, R.; Shaw, P.; Brown, J.W.S. Molecular characterization of plant
U14 small nucleolar RNA genes closely linked genes are transcribed as polycistronic U14 transcripts.
Nucleic Acids Res. 1994, 22, 5196–5203.
(c) Maxwell, E.S.; Fournier, M.J. The small nucleolar RNAs. Annu. Rev. Biochem. 1995, 64, 897–
934.
10. Nam, K.; Lee, G.; Trambley, J.; Devine, S.E.; Boeke, J.D. Severe growth defect in a Schizosac-
charomyces pombe mutant defective in intron lariat degradation. Mol. Cell. Biol. 1997, 17, 809–
818.

228
R. Mourani and M. J. Damha
11. Nam, K.; Hudson, R.H.E.; Ganeshan, K.; Chapman, K.B.; Damha, M.J.; Boeke, J.D. Yeast lariat de-
branching enzyme: substrate and sequence specificity. J. Biol. Chem. 1994, 269, 20613–20621.
12. Ooi, S.L.; Dann, C., III; Nam, K.; Leahy, D.; Damha, M.J.; Boeke, J.D. The yeast lariat debranching
enzyme. Methods Enzymol. 2001, 342, 233–250.
13. Hudson, R.H.E. Synthesis and studies of branched oligonucleotides. Ph.D. Dissertation, University
of Toronto, 1995.
14. Ganeshan, K.; Tadey, T.; Nam, K.; Braich, R.; Purdy, W.C.; Boeke, J.D.; Damha, M.J. Novel approaches
to the synthesis and analysis of branched RNA. Nucleos. Nucleot. 1995, 14, 1009–1013.
15. Maschhoff, K.L.; Padgett, R.A. The stereochemical course of the first step of pre-mRNA splicing.
Nucleic Acids Res. 1993, 21, 5456–5462.
16. Damha, M.J.; Ogilvie, K.K. Oligoribonucleotide synthesis. In Methods in Molecular Biology; Protocols for
Oligonucleotides and Analogs; Agrawal, S., Ed.; Humana Press Inc., Totowa, NJ., 1993, 20, 81–114.
17. Wasner, M.; Arion, D.; Borkow, G.; Noronha, A.; Uddin, A.H.; Parniak, M.A.; Damha, M.J. Physico-
chemical and biochemical properties of 2ꢁ,5ꢁ-linked RNA and 2ꢁ,5ꢁ- RNA:3ꢁ,5ꢁ-RNA “hybrid” duplexes.
Biochem. 1998, 37, 7478–7486.
18. Lecchi, P.; Le, H.M.T.; Pannell, L.K. 6-Aza-2-thiothymine: A matrix for MALDI spectra of oligonu-
cleotides. Nucleic Acids Res. 1995, 23, 1276–1277.
19. Damha, M.J.; Ganeshan, K.; Hudson, R.H.E.; Zabarylo, S.V. Solid-phase synthesis of branched olig-
oribonucleotides related to messenger RNA splicing intermediates. Nucleic Acids Res. 1992, 20,
6565–6573.
20. Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor
Laboratory Press, Plainview, NY, 1989.
21. Uhlenbeck, O.C.; Gumport, R.I. T4 RNA ligase. In The Enzymes; Boyer, P.D., Ed.; Academic Press,
Inc.; New York, 1982; 15B, 31–60.
22. Nelson, P.S.; Bach, C.T.; Verheyden, J.P.H. Synthesis of P-thioadenylyl- (2ꢁ–5ꢁ)-adenosine and P-
thioadenylyl-(2ꢁ–5ꢁ)-P-thioadenylyl-(2ꢁ–5ꢁ)-adenosine. J. Org. Chem. 1984, 49, 2314–2317.
23. Ma, M.Y.X.; Dignam, J.C.; Fong, G.W.; Li, L.; Gray, S.H.; Jacob-Samuel, B.; George, S.T. Evaluation of
3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as a new sulfurising reagent in combination with labile
exocyclic amino protecting groups for solid-phase oligonucleotide synthesis. Nucleic Acids Res. 1997,
25, 3590–3593.
24. Xu, Q.; Musier-Forsyth, K.; Hammer, R.P.; Barany, G. Use of 1,2,3- dithiazolidine-3,5-dione (DtsNH)
and 3-ethoxy-1,2,4-dithiazoline-5-one for synthesis of phosphorothioate-containing oligodeoxyri-
bonucleotides. Nucleic Acids Res. 1986, 24, 1602–1607.
25. Xu, Q.; Barany, G.; Hammer, R.P.; Musier-Forsyth, K. Efficient introduction of phosphorothioates
into RNA oligonucleotides by 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH). Nucleic Acids Res. 1996,
24, 3643–3644.
26. Iyer, R.P.; Egan, W.; Regan, J.B.; Beaucage, S.L. 3H-1,2-Benzodithiole-3-one 1,1-dioxide as an im-
proved sulfurizing reagent in the solid-phase synthesis of oligodeoxyribonucleoside phosphoroth-
ioates. J. Am. Chem. Soc. 1990, 112, 1253–1254.
27. Iyer, R.P.; Phillips, L.R.; Egan, W.; Regan, J.B.; Beaucage, S.L. The automated synthesis of sulfur-
containing oligodeoxyribonucleotides using 3H-1,2- benzodithiol-3-one 1,1-dioxide as a sulfur-
transfer reagent J. Org. Chem. 1990, 55, 4693–4699.
28. Cantor, C.R.; Schimmel, P.R.; Biophysical Chemistry—Part II: Techniques for the Study of Biological Structure
and Function; W. H. Freeman and Co., San Francisco., 1990, 349–472.
29. Bush, C.A. Basic Principles in Nucleic Acid Chemistry, Ts’o, P.O.P. Ed., Academic Press, New York, 1974,
2, 92–169.
30. Harada, N.; Nakanishi, K. In Circular Dichroic Spectroscopy; Exciton Coupling in Organic Stereochemistry;
University Science Books, New York, 1983.
31. Damha, M.J. Developments in the chemical synthesis of oligonucleotides and their analogs. Ph.D.
Dissertation, McGill University, 1987.
32. Warsaw, M.M.; Cantor, C.R. Oligonucleotide interactions. IV. Conformational differences between
deoxy- and ribodinucleoside phosphates; Biopolymers 1970, 9, 1079–1103.
33. Brahms, J.; Maurizot, J.C.; Michelson, A.M. Conformational stability of dinucleotides in solution. J.
Mol. Biol. 1967, 25, 481–495.
34. Burgers, P.M.J.; Eckstein, F. A study of the mechanism of DNA polymerase I from Escherichia coli
with diastereomeric phosphorothioate analogs of deoxyadenosine triphosphate J. Biol. Chem. 1979,
254, 6889–6893.

Small Branched RNA Fragments Containing Chiral Linkages
229
35. Braich, R.S. Branched nucleic acids: Novel probes for studying pre-m RNA processing and triple
helical DNA. Ph.D. Dissertation, McGill University, 1999.
36. Kierzek, R.; Kopp, D.W.; Edmonds, M.; Caruthers, M.H. Chemical synthesis of branched RNA. Nu-
cleic Acids Res. 1986, 14, 4751–4764.
37. Khalid, M.F.; Damha, M.J.; Shuman, S.; Schwer, B. Structure-function analysis of yeast RNA debranch-
ing enzyme (Dbr1), a manganese-dependent phosphodiesterase, Nucleic Acids Research, 2005, 33,
6349–6360.
38. Koonin, E.V. Conserved sequence pattern in a wide variety of phosphoesterases. Protein Sci. 1994;
3, 356–358.
39. Zhuo, S.; Clemens, J.C.; Stone, R.L.; Dixon, J.E. Mutational analysis of a Ser/Thr phosphatase:
identification of residues important in phosphoesterase substrate binding and catalysis. J. Biol. Chem.
1994, 269, 26234–26238.
40. Goldberg, J.; Huang, H.; Kwon, Y.; Greengard, P.; Nairn, A.C.; Kuriyan, J. Three-dimensional structure
of the catalytic subunit of protein serine/threonine phosphatase-1. Natuure 1995, 376, 745–753.
41. Voegtli, W.C.; White, D.J.; Reiter, N.J.; Rusnak, F.; Rosenzweig, A.C. Structure of the bacteriophage
lambda Ser/Thr protein phosphatase with sulfate ion bound in two coordination modes. Biochem.
2000, 39, 15365–15374.
42. Hopfner, K.P.; Karcher, A.; Craig, L.; Woo, T.T.; Carney, J.P.; Tainer, J.A. Structural biochemistry and
interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.
Cell 2001, 105, 473–485.
43. Burgers, P.M.J.; Eckstein, F.; Hunneman, D.H. Stereochemistry of hydrolysis by snake venom phos-
phodiesterase. J. Biol. Chem. 1979, 254, 7476–7478.