Synthesis and evaluation of RNA transesterification efficiency using stereospecific serinol-terpyridine conjugates

Article abstract of DOI:10.1080/15257770500230426

Six novel artificial ribonucleases were synthesized employing a stereochemically pure abasic serinol backbone residue for attachment of the RNA transesterification agent copper(II) terpyridine. These stereochemically pure abasic residues were synthesized

Full text of DOI:10.1080/15257770500230426

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Synthesis and Evaluation of RNA Transesterification
Efficiency Using Stereospecific Serinol-Terpyridine
Conjugates
William C. Putnam a & James K. Bashkin a b
a Department of Chemistry , Washington University , St. Louis, Missouri, USA
b Department of Chemistry and Biochemistry , University of Missouri—St. Louis , St. Louis,
MO, 63121, USA
Published online: 31 Aug 2006.
To cite this article: William C. Putnam & James K. Bashkin (2005) Synthesis and Evaluation of RNA Transesterification
Efficiency Using Stereospecific Serinol-Terpyridine Conjugates, Nucleosides, Nucleotides and Nucleic Acids, 24:9, 1309-1323,
DOI: 10.1080/15257770500230426
To link to this article: http://dx.doi.org/10.1080/15257770500230426
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (9):1309–1323, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1080/15257770500230426
SYNTHESIS AND EVALUATION OF RNA TRANSESTERIFICATION
EFFICIENCY USING STEREOSPECIFIC
SERINOL-TERPYRIDINE CONJUGATES
5
William C. Putnam and James K. Bashkin
Department of Chemistry, Washington
University, St. Louis, Missouri, USA
5
Six novel artificial ribonucleases were synthesized employing a stereochemically pure abasic serinol
backbone residue for attachment of the RNA transesterification agent copper(II) terpyridine. These
stereochemically pure abasic residues were synthesized as phosphoramidite building blocks from the
parent L-serine and D-serine starting building blocks and incorporated into oligonucleotides via solid-
phase DNA synthesis. These artificial ribonucleases were constructed to determine if the stereochemistry
of the alpha carbon of an abasic serinol residue has influence over RNA transesterification through
selective placement of a pendant transesterification agent in either the major or minor groove. The novel
artificial ribonucleases and previously synthesized artificial ribonucleases were challenged with a
28-mer and 159-mer RNA substrate. It was determined that the stereochemistry of the carbon atom
derived from the a-carbon of serine did not influence the extent of cleavage in these studies using
copper(II) terpyridine conjugated artificial ribonucleases.
Keywords Antisense, Artificial Ribonuclease, RNA Cleavage, Transesterification, Abasic
Backbone, Serinol
Received 5 November 2004.
Acknowledgment for partial support of this research is made by to the donors of the Petroleum Research
Fund, administered by the ACS and the National Science Foundation (CHE-9802660). Mass spectrometry was
provided by the Washington University Mass Spectrometry Resource, an NIH Research Resource (Grant No.
P41RR0954). We thank Dr. Lee Ratner of the Washington University Medical School (St. Louis, Missouri) for the
plasmid containing the HIV gag-gene fragment.
Current address for James K. Bashkin: Department of Chemistry and Biochemistry, University of Missouri—
St. Louis, St. Louis, MO 63121, USA.
Address correspondence to William C. Putnam, School of Pharmacy, Texas Tech University, 4500
S. Lancaster Road, Dallas, TX 75216, USA; Fax: (214)-372-5020; E-mail: trey.putnam@ttuhsc.edu
1309
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1310
W. C. Putnam and J. K. Bashkin
INTRODUCTION
The optimization of artificial ribonucleases for therapeutic applications has
been a goal for over 15 years.[1–17] Artificial ribonucleases are synthetic analogues
to ribonucleases. These artificial ribonucleases are conjugates of a nucleic acid (for
molecular recognition) and an RNA transesterification catalyst (for RNA cleavage).
They are also synthetic mimics of ribozymes (like the hammerhead) with the large
catalytic domain replaced by a small molecule catalyst; thus, they are often termed
ribozyme mimics. The use of a small molecule catalyst confers many advantages to
these synthetic analogues that their natural counterparts do not possess. Two such
advantages are that artificial ribonucleases can easily be modified by synthetic
techniques to optimize activity and their small molecular weight may expedite in
vivo delivery.[18–24]
Initially, our group synthesized and evaluated modified building blocks that
were designed not to interfere with normal Watson-Crick base pairing (Figure 1a).[9]
These initial artificial ribonucleases were not tremendously efficient at cleaving
RNA.[11,12] In order to increase the transesterification efficiency, a number of
optimization strategies were used. One of the optimization strategies taken
employed the attachment of a transesterification catalyst to a serinol-based abasic
backbone structure.[13,15,25] The use of the abasic structure directly removed the
hydrogen bonds of one of the base pairs, resulting in increased local flexibility
proximal to the catalyst. This molecular design was employed in response to
reports that the rigidity of the RNA–DNA duplex suppresses transesterification of
the RNA strand when compared to single-stranded RNA. This suppression is
believed to result from the hindered formation of the 5-coordinate phosphorane
that serves as the transition state for transesterification.[26] Serinol, the reduced form
of serine, was chosen because the three-carbon diol structure also mimics the
spacing of natural nucleotides. Using a copper(II)-terpyridine (Cu-terpy) RNA
transesterification catalyst, Daniher and coworkers demonstrated a three-fold
increase in transesterification efficiency using a artificial ribonuclease containing the
FIGURE 1 Active sites of serinol Cu(II)-terpy artificial ribonucleases: (a) Attachment at C5 position preserve
hydrogen bonding sites (arrows). (b) Abasic residue attachment three carbon diol structure of serinol is shown
in bold.

RNA Transesterification Efficiency Evaluation
1311
FIGURE 2 Phosphoramidite building blocks serinol-terpyridine ribozyme mimics: Phosphoramidite 1 and
Probe 11.ST.
serinol residue (Figure 1b) when compared to a analogous fully duplexed ribozyme
mimic.[13]
The use of serinol also greatly simplified the synthesis over previously modified
native-nucleoside residues. This artificial ribonuclease with a serinol-terpy residue at
the 11 position of a 17-mer was constructed using solid-phase automated DNA
synthesis and phosphoramidite 1 (Figure 2).
The synthesis of the phosphoramidite building block 1 (Scheme 1) yielded two
diastereomers, because the meso compound serinol was coupled to terpy acid 3 in
the synthesis (step b) to yield the diol 4. During the subsequent protection of one of
the primary alcohols with a DMT (dimethoxytrityl) protecting group, the
stereochemistry at the a-carbon was set (Figure 3). The carbon bearing the amino
group of serinol is termed the a-carbon because it is derived from the a-carbon
of serine.
One mechanism for RNA transesterification involves the general base
deprotonation of the 2’-OH of RNA, thus, the 2’-OH of RNA is an essential
moiety for RNA transesterification. The 2’-OH of RNA resides in the minor groove
of the A-form RNA-DNA duplex. In these serinol-terpyridine oligonucleotides it is
SCHEME 1 Synthesis of the serinol-terpy building block 1. a) 4-hydroxybutyric acid, KOH, DMSO; b) serinol,
EDC-HCl, DMF; c) 1) DMT-Cl, Py; 2) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, methylene chloride,
triethylamine.

1312
W. C. Putnam and J. K. Bashkin
FIGURE 3 DMT protection of serinol-terpyridine (R = CH2CH2CH2-O-Terpy): Production of enantiomers.
possible that only one of the stereoisomers geometrically allows the pendant
transesterification agent to reach in the minor groove. In this case, only one of the
two isomers generated from the phosphoramidite building block 1 would be active
at RNA transesterification. To evaluate if the stereochemistry around the alpha
carbon of a serinol based nonnucleoside backbone affects the transesterification of
RNA, the two isomers were independently synthesized and tested.
Groove discrimination by pendant ligands attached to abasic nucleic acid
backbone residues was previously investigated by Fukui and coworkers.[27–29] In
this study of intercalation, acridine was attached to the non-nucleoside residue
threoninol and placed internally in a strand of DNA. It was determined that the
(R,R)-isomer (R conformation at the a carbon) of threoninol places pendant ligands
in the major groove.
MATERIALS AND METHODS
All synthetic reagents were purchased in the highest quality available from
Sigma-Aldrich Chemical Co. (St. Louis, MO) and used as received unless otherwise
stated. All synthetic starting materials were co-evaporated from either toluene or
pyridine to remove excess water. All solvents were dried over the appropriate agent
(usually CaH2). All synthetic reactions were carried out under an inert atmosphere
of argon (Ar) or nitrogen (N2). All DNA synthesis reagents were purchased from
Oligos Etc. (Wilsonville, OR). All other reagents were purchased from the
indicated vendors and used as received.
Synthesis of R-Serinol-Terpy Phosphoramidite Building
Block (10R)
The R-serinol-terpy phosphoramidite building block 10R was synthesized
according to Schemes 2 and 3. L-serine ethyl ester 5R was initially N-TFA protected
by ethyl-trifluoroacetate. DMT-Cl was then used to protect the free hydroxyl 7R
(step b). A one-step deprotection-reduction sodium borohydride reaction was
applied to give the DMT-protected stereo-specific serinol building block 8R in a
75% yield over the 3 steps. The DMT-protected (R)-serinol 8R was coupled to the
terpyridine acid derivative 3 in the presence of the water-soluble coupling agent
EDC and 1-hydroxybenzotriazole (HOBt) to yield the DMT-(R)-ser-terpy 9R in a
60% yield. Reaction with the phosphoramidite reagent Cl-PA yielded the final
building block 10R in 82% yield. All yields presented as isolated yields.

RNA Transesterification Efficiency Evaluation
1313
SCHEME 2 Synthesis of DMT protected stereospecific serinol. (a) TFA-OEt, Et3N, MeOH, 12 h, RT; (b) DMT-
Cl, Py, 12 h, RT; (c) NaBH4, LiCl, EtOH, 12 h, 0°C-RT.
Synthesis of S-Serinol-Terpy Phosphoramidite Building
Block (10S)
Synthesis of the phosphoramidite building block based on (S)-serinol stereo-
chemistry was constructed in a directly analogous fashion, with the coupling to the
terpy acid derivative proceeding in a 63% yield and the phosphytilation yield of 85%.
Racemic Serinol-Terpy Phosphoramidite Building Block (1)
Phosphoramidite 1 was prepared and used in a similar fashion to previous
studies (Scheme 1).[13,17,30]
Control Phosphoramidite Building Block (11)
The three carbon diol phosphoramidite (control) was purchased from Oligo’s
Etc. and used as received (Figure 2).[13,17,30]
Synthesis of Artificial Ribonucleases Containing Stereospecific
Serinol-Terpy Active Sites
Standard automated DNA synthesis was used for the production of DNA
conjugates containing an abasic internal residue of stereospecific serinol terpy.
Oligonucleotides containing the L-serinol-terpy, X = L-serinol-terpy
Probe 6.LST—5’-CTA CAX AGT CTC TAA AG-3’
Probe 9.LST—5’-CTA CAT AGX CTC TAA AG-3’
Probe 11.LST—5’-CTA CAT AGT CXC TAA AG-3’
Oligonucleotides containing the D-serinol-terpy, X = D-serinol-terpy

1314
W. C. Putnam and J. K. Bashkin
Probe 6.DST—5’-CTA CAX AGT CTC TAA AG-3’
Probe 9.DST—5’-CTA CAT AGX CTC TAA AG-3’
Probe 11.DST—5’-CTA CAT AGT CXC TAA AG-3’
These oligonucleotides were synthesized along with the racemic a-carbon
serinol-terpy derived oligonucleotides (Probe 6.ST, Probe 9.ST, and Probe 11.ST)
and the control oligonucleotides (Probe 6.CL, Probe 9.CL, and Probe 11.CL).
These modified oligonucleotides were then gel purified and characterized. These
procedures were conducted in a similar fashion to previous work.[13,17,30] Coupling
yields were greater than 98% for the unmodified positions and greater than 90% for
our modified building blocks. Both matrix-assisted laser desorption ionization time-
of-flight (MALDI-TOF) mass spectrometry and direct-infusion electrospray
ionization quadrupole mass spectrometry were used in the characterization and
gave similar results. The masses found agreed well with the calculated values
( 2 amu).
Preparation of the 159-mer Substrate
The 159-mer RNA substrate was synthesized by runoff transcription using
Ambion’s MEGAshortscriptTM T7 kit. In a total volume of 20 mL, the following were
added at RT (the kit is stored at À20°C and the reagents were allowed to warm to
RT for 45 min before use): 5 mL of 179-mer DNA template (from HIVHXB2r) (ca.
0.035 nmoles), 3 mL of nuclease-free water, 2 mL of 10X Buffer, 2 mL of each NTP
(10 mM stock), and 2 mL of enzyme mix. The mixture was incubated at 37°C for
4 h (longer incubation times were also employed and good yields were also
observed). 3 mL of DNase One was added to degrade the DNA template. The
RNA was diluted by the addition of 115 mL of nuclease-free water and 15 mL of
Ammonium Acetate (NH4OAc) solution. The resultant solution was extracted
twice with 150 mL of (24:25:1) phenol/chloroform/iso-amylalcohol. The aqueous
layer was retained, spun through a S200 (Sephadex) column for desalting, and
precipitated by the addition of 300 mL of EtOH (À70°C overnight). The ethanolic
supernatant was removed after centrifugation (14,000 rpm for 15 min) and the pellet
was dried by vacuum centrifugation (10 min).
The 5’-end was dephosphorylated using Ambion’s KinaseMAXTM kit. The
RNA was resuspended in 16 mL of nuclease-free water and 2 mL of 10 Â
dephosphorlation buffer was added, followed by the addition of 2 mL of calf-
intestine-alkaline phosphatase (CIAP). The solution was mixed thoroughly and
incubated in a heating block for 1 h at 37°C. The solution was extracted as before
(with a third extraction of chloroform above). The RNA containing a 5’-OH was
then ethanol-precipitated and dried as before.
The RNA was 5’-end labeled with Ambion’s KinaseMAXTM kit. The RNA was
reconstituted in 9 mL of nuclease-free water, 5 mL of g-32P ATP (Amersham), 4 mL
of 5 Â reaction buffer, and 2 mL of T4 polynucleotide kinase (T4 PNK) was added

RNA Transesterification Efficiency Evaluation
1315
to give a total solution volume of 20 mL. The reaction was incubated at 37°C for
1 h. The RNA was extracted, precipitated using 3 M NaOAc (pH 5.2), and dried
as before.
The RNA was resuspended in 15 mL of nuclease-free water and 15 mL of 1X
loading buffer (LB). It was then loaded on a 6% polyacrylamide gel and run for 3.5 h
at 2000 V. This separated the desired product, the 159-mer, from failure sequence
oligos (of shorter length) in the mixture. A film autoradiogram was taken of the gel.
Using the film as a guide, the desired RNA band, the topmost band in the lane, was
excised from the gel and the gel fragment transferred to a 1.5 mL Eppendorf tube.
The gel fragment was crushed in the tube, and 200 mL of gel elution buffer (0.5 M
ammonium acetate, 10 mM magnesium sulfate, 1 mM EDTA, 0.1% sodium dodecyl
sulfate) were added to the tube. The solution was sealed with Parafilm and stirred
or shaken overnight to elute the RNA from the gel.
After this overnight incubation, the RNA was desalted by EtOH precipitation
as before. The RNA was resuspended in nuclease-free water and the UV
absorbance at 260 nM was measured to evaluate the concentration.
Preparation of the 28-mer RNA Substrate
The 28-mer substrate was purchased (Oligos Etc.) and 5’-end labeled in a similar
fashion to the 159-mer. The RNA was resuspended in 15 mL of nuclease-free water
and 15 mL of 1X loading buffer (LB). It was then loaded on a 20% polyacrylamide gel
and run for 3.5 h at 1400 V. This separated the desired product, the 28-mer, from
failure sequence oligos (of shorter length) in the mixture. A film autoradiogram was
taken of the gel. Using the film as a guide, the desired RNA band, the top-most band
in the lane, was excised from the gel and the gel fragment transferred to a 1.5 mL
Eppendorf tube. The gel fragment was crushed in the tube, and 200 mL of gel elution
buffer (0.5 M ammonium acetate, 10 mM magnesium sulfate, 1 mM EDTA, 0.1%
sodium dodecyl sulfate) was added to the tube. The solution was sealed with
Parafilm and stirred or shaken overnight to elute the RNA from the gel.
After this overnight incubation, the RNA was purified by EtOH precipitation as
before. The RNA was resuspended in nuclease-free water and the UV absorbance
at 260 nM was taken to evaluate the concentration.
RNA Cleavage Reactions
RNA cleavage reactions were carried out in 10 mM (N-[2-Hydroxyethyl]piper-
azine-N’-[2-ethanesulfonic acid]) (HEPES) buffer (pH = 7.4), with 0.1 M NaClO4,
the respective RNA-mer [0.1 mM RNA (28-mer) and 0.01 mM RNA (159-mer)],
5 mM probe, and 5 mM metal (total reaction volume = 10 mL). The reactions were
heated to the desired temperature and time. The reactions were quenched by the
addition of 5 mL loading buffer and loaded on a denaturing polyacrylamide gel
(20% gel for the 28-mer and 6% gel for the 159-mer). The gel image was quantified

1316
W. C. Putnam and J. K. Bashkin
using a Molecular Dynamics PhosphorImager and the ImageQuantD (Version 3.3)
software package.
RESULTS AND DISCUSSION
Synthesis of Stereospecific Serinol-Terpy Phosphoramidite
Building Blocks
The stereospecific serinol-terpy phosphoramidite building blocks were
synthesized according to Scheme 2. The production of the modified stereospecific
serinol phosphoramidites was based on the chemistry of Benhida et al.[31] who
studied the effects of abasic residues on duplex and triplex stability by melting
temperature methods. Our synthetic strategy employed Benhida and coworkers’[31]
stereospecific synthesis of the TFA-protected serinol 8. That building block was the
conjugated to the terpyridine-acid derivative 3 prior to phosphoramidite production
and incorporation into an oligonucleotide.
The stereochemistry of the a-carbon of serinol in the stereospecific
phosphoramidites is derived from the chemistry of the parent amino acid serine.
Following trifluoroacetate (TFA) protection of the amine of serine ethyl ester, the
free hydroxyl of the R-group is protected with a DMT group. This protection
strategy avoids the direct use of the meso compound serinol, where there would be
no direct stereoselectivity between the hydroxyls. In a subsequent deprotection-
reduction step with lithium borohydride, the second hydroxyl is generated from the
acid moiety and the amine is deprotected. This is functionally a double
SCHEME 3 Synthesis of the stereo-specific serinol-terpy phosphoramidite building block. (a) EDC-HCl, HOBt,
DMF, 12 h, RT, (b) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, methylene chloride, triethyl amine,
1 h, RT.

RNA Transesterification Efficiency Evaluation
1317
deprotection step because we are using the acid moiety as a protecting group for
hydroxyl. The amine is then coupled via an amide bond to the ligand. In the final
step, the free hydroxyl is reacted with 2-cyanoethyl-N,N-diisopropylchloro-
phosphoramidite to give the appropriate stereospecific serinol phosphoramidite
building block.
Nomenclature Note: the numbering scheme will denote the compound and
the isomer of serine from which it came. The DMT-protected (R)-serinol will
be called compound 8R and the DMT-protected (S)-serinol will be called
compound 8S so there will be a series of compounds labeled as derived from
the L-isomer of serine and R series of compounds labeled S that are derived
from the D-isomer.
The DMT protected stereo-specific serinol was synthesized as detailed in
Scheme 2 where the ethyl ester of the amino acid L-serine is reacted with ethyl
trifluoroacetate (TFA-OEt) in methanol with triethylamine as a base to give the N-
TFA protected L-serine ethyl ester 6R. After overnight reaction, TLC (silica;
staining with p-anisaldehyde) illustrated that all of the starting material had been
consumed and a single spot of higher mobility (Rf) had been formed. After removal
of the MeOH, the N-TFA protected intermediate was reacted with DMT-Cl in
pyridine to give 7R. TLC (UV silica) after stirring overnight showed that all of the
starting material had been consumed to give predominately one spot of higher
mobility. After removal of pyridine, the reaction mixture was subjected to the
deprotection-reduction conditions of sodium borohydride in the presence of
ethanolic lithium chloride. TLC once again revealed a single spot, this time of lower
mobility. This product, 8R, was isolated by column chromatography (silica; 10%
methanol-methylene chloride). Spectral characterization of the product proved that
it was the desired 8R DMT-protected (R)-serinol. A similar route with the ethyl
ester of D-serine yielded 8S the DMT-protected (S)-serinol.
To determine if two isolated DMT-protected intermediates (8R and 8S) were
enatiomers with opposing stereochemistry, each of the two intermediates was
coupled to a stereochemically-pure amino acid FMOC-L-histidine(BOC)-OH and
subjected to TLC. The racemic DMT intermediate, 8RS, was also synthesized and
coupled to the same L-histidine building block (Figure 4).
Two DMT active peaks were formed with the L-His-derivative of the racemic-
building block 8RS, demonstrating that the enantiomers could be separated by
TLC. The TLC of the DMT-(S)-serinol had one DMT active spot that
corresponded to the higher Rf, and the DMT-(R)-serinol contained a single
FIGURE 4 Racemic DMT-serinol.

1318
W. C. Putnam and J. K. Bashkin
DMT active spot that corresponded to the lower Rf. The spots were then isolated
by column chromatography to determine their structure and they were
demonstrated to be the coupled products of the DMT intermediates and the
L-His building block. This proved the stereochemistry of the DMT protected
intermediates was opposite in nature, and the stereochemistry was derived from the
a-carbon of serinol.
Scheme 3 details the synthesis of the stereospecific serinol-terpy phosphor-
amidite building blocks from the DMT-protected intermediates 8R and 8S. In
order to obtain comparable RNA cleavage results, the synthesis of the
phosphoramidites was conducted in a similar fashion to the original serinol-terpy
building block. These conserved molecular design principles include the 4-ether
substituent on terpy (keeping stereoelectronic effects on the metal constant) and the
length of the tether to the metal complex.
Evaluation of the Cleavage Efficiencies of Stereospecific Serinol-
Terpyridine Based Ribozyme Mimics
The cleavage efficiencies of our ribozyme mimics were evaluated with two
RNA substrates. The first target is a 159-mer substrate that is derived from the
mRNA of the HIV gag-gene, and the second substrate is a 28-mer, both of which
contain the same 17-mer recognition sequence. The 28-mer RNA target that
allowed ready identification of the cleavage products as true transesterification
products, and a 159-mer fragment of the HIV gag-gene mRNA that would indicate
the precise specificity of cleavage in the presence of many competing sites. Because
of the shared recognition sequence, cleavage differences between the two substrates
should predominately derive from either the differences in 1) molecular weight,
2) charge, or 3) the presence of secondary/tertiary structure.
RNA Transesterification 28-mer Substrate
Figure 5 is a typical audioradiogram of the sequence-specific cleavage of
the 5’-end labeled 28-mer RNA substrate by ribozyme mimics modified at the
11 position. In the presence of EDTA, no sequence-specific RNA cleavage was
observed with any of the probes. In accordance with previous reports, EDTA
treatment is necessary in the case of oligonucleotides with pendant terpy ligands
because of terpy’s high binding constant for copper, which allows it to scavenge
trace amounts of Cu(II) from commercial buffer salts.[13] For every RNA cleavage
reaction, background cleavage was found at all of the 5’-UA-3’ sites, in keeping
with their high natural propensity for scission.[32–34]
In the presence of copper, both 11.LST and 11.DST form active ribozyme
mimics. The products of all of the cleavage reactions co-migrate with alkaline
hydrolysis and Ribonuclease T1 (3’-G specific) digestion products. This
demonstrates that, in the presence of copper, these ribozyme mimics react via a

RNA Transesterification Efficiency Evaluation
1319
FIGURE 5 Audioradiogram of a 20% polyacrylamide gel (8 M Urea) of the site-specific cleavage of the 5’-end
labeled 28-mer RNA ([0.1 mM]) by ribozyme mimics (reaction conditions: [CuSO4] = 5 mM, [RNA] ꢀ60 nM,
[NaClO4] = 0.1 M, [HEPES] = 10 mM pH 7.4, [EDTA] = 50 mM, 15 h, 37°C). First lane: unreacted RNA starting
material. Second lane: Ribonuclease T1 digest (G specific). Third lane: NaOH digest. Lanes 4–11: Treatment with
stereospecific serinol-terpy artificial ribonculeases ([5 mM]) both in the absence and presence of copper and EDTA
(EDTA treatment to remove trace contamination of copper).
biomimetic pathway of transesterification and hydrolysis and not via oxidative
cleavage pathways.
Reactions were conducted in triplicate. The extent of sequence-specific RNA
cleavage was statistically equivalent for both 11.LST and 11.DST (Table 1). The
TABLE 1 Sequence-Specific Cleavage of 28-mer and 159-mer RNA by Cu(II)-Terpy Artificial Ribonucleases
Cleavage %
28-mer 68 h, 45°C
Cleavage site
28-mer
Cleavage %
159-mer 68 h, 45°C
Cleavage site
159-mer
Artificial ribonuclease
11.CL
11.ST
11.DST
11.LST
0
65
63
n/a
A16
A16
A16
0
n/a
5
7
79
80
80
8
4
6
A108
A108
A108
64 10
Reaction conditions for reactions of 28- and 159-mer substrates can be found in Figures 5 and 6. Note differences
in time and temperature. Percentages are given with standard deviations from the triplicate analyses.

1320
W. C. Putnam and J. K. Bashkin
FIGURE 6 Audioradiogram of 6% polyacrylamide gel (8 M Urea) of the site-specific cleavage of the 159-mer
RNA ([0.01 mM]) by artificial ribonucleases (reaction conditions: [Probe] = 5 mM, [CuSO4] = 5 mM,
[NaClO4] = 0.1 M, [HEPES] = 10 mM pH 7.4, [EDTA] = 50 mM, 30 h, 37°C). Last lane: unreacted RNA starting
material. Second to-last lane: Ribonuclease T1 digest (G specific). Third to-last lane: NaOH digest. Lanes 1–6:
Treatment with stereospecific serinol-terpy artificial ribonculeases ([5 mM]) both in the absence and presence of
copper and EDTA (EDTA treatment to remove trace contamination of copper).
amount of sequence-specific RNA cleavage also was equivalent to the amount of
cleavage observed when a mix of 11.LST and 11.DST was used as well as the
amount of cleavage observed when 11.ST was used. Effort was made to ensure that
there are not minor differences between the two isomers and their ability to cleave
RNA. Different concentrations of artificial ribonuclease and metal were examined.
Under all conditions attempted no statistical difference was observed. The results are
similar for the ribozyme mimics that are modified at the 6 and 9 positions, although
the overall extent of cleavage is less in accordance with previous results (data not
shown). Probe 11.CL, the control oligonucleotide containing the abasic site and no
pendant ligand, is completely inactive for RNA cleavage in the presence of Cu(II);
therefore, no site-specific cleavage is derived solely from the presence of an abasic
site. Similar results were obtained in the artificial ribonucleases modified in the 6 and
9 positions.

RNA Transesterification Efficiency Evaluation
1321
The equivalence of the extent of sequence-specific cleavage demonstrates that
the cleavage of RNA by serinol-terpy derived ribozyme mimics is independent of
the absolute conformation of the a-carbon of serinol under these conditions.
RNA Transesterification 159-mer Substrate
A representative image of an audioradiogram of the cleavage of the 159-mer
substrate is depicted in Figure 5. The extent of cleavage by the different isomers of
serinol-terpy containing oligonucleotides (11.LST or 11.DST) is equivalent.
Furthermore, the amount of cleavage by any one isomer (11.LST or 11.DST) is
equivalent to the racemic serinol-terpy containing oligonucleotide (11.ST). The
equivalence of the extent of sequence-specific cleavage demonstrates that the
cleavage of high-molecular-weight RNA substrates by serinol-terpy derived
ribozyme mimics is independent of the absolute conformation of the a-carbon of
serinol, as it was with the low-molecular weight 28-mer substrate.
CONCLUSIONS
Two novel phosphoramidites were synthesized based on stereo-specific serinol-
terpyridine chemistry. They were designed to investigate the possibility of groove
control at an abasic site to optimize the cleavage of RNA by ribozyme mimics. In
the presence of copper(II) these terpyridine containing oligonucleotides derived
from the chemistry of L-serine and D-serine transesterified RNA in equivalent
amounts. The stereochemistry of the a-carbon of serine does not influence the
amount of cleavage when using serinol-terpy-derived ribozyme mimics. We
conclude that the flexibility of the serinol-based abasic site allows both
conformations of serinol in Cu(II)-serinol-terpy ribozyme mimics to equally reach
the 2’-OH of RNA; therefore, the extent of cleavage is equivalent.
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