New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes

Article abstract of DOI:10.1073/pnas.1006447107

The CuAAC reaction (click chemistry) has been used in conjunction with solid-phase synthesis to produce catalytically active hairpin ribozymes around 100 nucleotides in length. Cross-strand ligation through neighboring nucleobases was successful in covale

Full text of DOI:10.1073/pnas.1006447107

New strategy for the synthesis of chemically
modified RNA constructs exemplified by
hairpin and hammerhead ribozymes
Afaf H. El-Sagheera,b and Tom Browna,1
aSchool of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, United Kingdom; and bChemistry Branch, Department of Science
and Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University, Suez 43721, Egypt
Edited by P. H. Seeberger, Max Planck Institute of Colloids and Interfaces, Germany, and accepted by the Editorial Board July 15, 2010 (received for review May
9, 2010)
The CuAAC reaction (click chemistry) has been used in conjunction
with solid-phase synthesis to produce catalytically active hairpin
ribozymes around 100 nucleotides in length. Cross-strand ligation
through neighboring nucleobases was successful in covalently
linking presynthesized RNA strands with high efficiency (trans-
ligation). In an alternative strategy, intrastrand click ligation was
employed to produce a functional hammerhead ribozyme contain-
ing a novel nucleic acid backbone mimic at the catalytic site (cis-
ligation). The ability to synthesize long RNA strands by a combina-
tion of solid-phase synthesis and click ligation is an important
addition to RNA chemistry. It is compatible with a plethora of site-
specific modifications and is applicable to the synthesis of many
biologically important RNA molecules.
objectives (12). T4 RNA ligase can be used to join nucleotides in
single-stranded regions such as RNA loops, whereas T4 DNA
ligase functions on double-stranded RNA. However, neither en-
zyme can be used to link together RNA strands at other positions,
such as across the bases or sugars, or to create unnatural linkages.
The use of ligases has other drawbacks; they are often contami-
nated with RNases that can partially degrade the ligation prod-
ucts, and the ligation protocols are quite complicated, requiring
phenol extraction to remove the protein. Moreover, enzymatic
ligation methods are not suitable for the large scale synthesis of
RNA, and the yields of enzymatic RNA ligation are often low. This
is partly due to the tendency of T4 RNA ligase to cyclize phos-
phorylated RNA strands and to join together incorrect strands.
Here we describe click RNA ligation, a strategy that overcomes
the above limitations and could lead to a step-change in the synth-
esis of biologically active RNA constructs. The CuAAC reaction
(13, 14) was chosen for RNA ligation owing to its very high speed,
efficiency, orthogonality with functional groups present in nucleic
acids and compatibility with aqueous media. In this procedure,
individual RNA oligonucleotides are assembled by automated
solid-phase synthesis, purified by HPLC then chemically ligated
by click chemistry (15) to produce much larger molecules. In this
study we demonstrate the power of click ligation by synthesising a
series of chemically modified hairpin and hammerhead ribo-
zymes. The hairpin ribozyme belongs to a family of catalytic
RNAs that cleave their RNA substrates in a reversible reaction
to generate 2′,3′-cyclic phosphate and 5′-hydroxyl termini. It was
discovered in tobacco ringspot virus satellite RNA where ribo-
zyme-mediated self-cleavage and ligation reactions participate
in processing RNA replication intermediates (16). It was chosen
for the current study for three reasons: Its size is such that direct
chemical synthesis is problematic and therefore click ligation be-
comes a valuable technique, its biophysical and catalytic proper-
ties have been extensively studied and are well understood (17),
and its functionality can be extended by the incorporation of che-
mical modifications such as fluorescent tags (18). In this study
three analogues of the hairpin ribozyme were synthesized from
individual oligonucleotides by crosslinking the side-chains of
modified uracil bases in opposite strands of the “unclicked” seg-
ments (trans-ligation). An RNA ribozyme (98-RNA) was synthe-
sized and a DNA/RNA chimera (98-DNA/RNA) was made in
which segments a and b contain tracts of DNA and segment c
is entirely DNA (Fig. 1, Left). Ribozymes are known to tolerate
chemical ligation ∣ click RNA ligation ∣ CuAAC ∣ triazole backbone ∣
biocompatible
ligonucleotide chemistry is central to the advancement of
O
core technologies such as DNA sequencing and genetic
analysis and has impacted greatly on the discipline of molecular
biology (1, 2). Oligonucleotides and their analogues are essential
tools in these areas. They are produced by automated solid-phase
phosphoramidite synthesis, a highly efficient process that can be
used to assemble DNA strands over 100 bases in length (3).
Synthesis of RNA is less efficient owing to problems caused by
the presence of the 2′-hydroxyl group of ribose, which requires
selective protection during oligonucleotide assembly. This
reduces the coupling efficiency of RNA phosphoramidite mono-
mers due to steric hindrance. In addition, side-reactions that oc-
cur during the removal (or premature loss) of the 2′-protecting
groups cause phosphodiester backbone cleavage and 3′ to 2′
phosphate migration (4). Although several ingenious strategies
have been developed to minimize these problems (5) and to im-
prove the synthesis of long RNA (6), the chemical complexity of
solid-phase RNA synthesis dictates that constructs longer than 50
nucleotides in length remain difficult to prepare. Most biologi-
cally important RNA molecules such as ribozymes (7), aptamers
(8), and riboswitches (9, 10) are significantly longer than this, so
new approaches to the synthesis of RNA analogues are urgently
required. Although RNA synthesis by transcription might seem a
viable alternative, it does not permit the site-specific incorpora-
tion of multiple modifications at sugars, bases, or phosphates. In
contrast, automated solid-phase RNA synthesis is compatible
with the introduction of fluorescent tags, isotopic labels (for
NMR studies) and other groups to improve biological activity
and resistance to enzymatic degradation. The scope and utility
of important RNA constructs can be significantly extended by
such chemical modifications, and the scale of chemical synthesis
is potentially unlimited (11). These are important factors when
planning structural studies on RNA or the development of ther-
apeutic oligoribonucleotides analogues, and should be consid-
ered when devising new methods of RNA assembly. Enzymatic
ligation might appear to be a method of achieving some of these
Author contributions: A.H.E. and T.B. designed research, performed research, analyzed
data, and wrote the paper.
The authors declare no conflict of interest.
This article is
Editorial Board.
a PNAS Direct Submission. P.H.S. is a guest editor invited by the
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: tb2@soton.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1006447107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1006447107
PNAS ∣ August 31, 2010 ∣ vol. 107 ∣ no. 35 ∣ 15329–15334

Fig. 1. Assembly of hairpin ribozymes by the CuAAC reaction. The substrate cleavage site (þ1 − 1) is indicated by an arrow. 8% polyacrylamide gels: Right-
hand lanes show CuAAC crude reaction mixtures and all other lanes show reactants (oligonucleotide segments). The solid arrows point to full-length ribozyme
products in all three reactions, and the dashed arrow points to DNA splints in the 77-RNA reaction. Gels visualized by UV shadowing. (Left) The 98-mer RNA
hairpin ribozyme 98-RNA and DNA–RNA hybrid 98-DNA/RNA. The sequences are the same but 98-DNA/RNA contains deoxyribose sugars at the positions
marked with asterisks* and has a different pattern of dye-labeling (segment a ¼ 50-cy5, segment b ¼ 50-cy3, segment c ¼ 50-FAM). The ES− mass spectrum
of the 98-DNA/RNA ribozyme is shown (found: 33169.4 Da, requires: 33170 Da). (Right) The truncated RNA 77-mer hairpin ribozyme 77-RNA. ES− mass spectrum
(found: 26983.6 Da, requires: 26983 Da).
deoxyribonucleosides outside the catalytic site and the incorpora-
tion of DNA offers the possibility of synthesising longer fragments
for click ligation. Therefore we were anxious to confirm that
the CuAAC reaction is compatible with DNA–RNA hybrids. A
shorter 77-nucleotide version of the hairpin ribozyme was also
prepared (77-RNA, Fig. 1, Right) to evaluate a simple splint-
mediated modification of the above click ligation strategy. In a
different approach, a hammerhead ribozyme (19, 20) was made
by the splint-mediated cis-ligation of two RNA strands (Fig. 2).
The hammerhead is a small catalytic RNA motif that is found in
plant RNA viruses, satellite RNA, viroids, and repetitive satellite
DNA (21) where it catalyzes cleavage and ligation of RNA during
rolling circle replication (22, 23). It was chosen as a model to in-
vestigate the chemistry of a new triazole nucleic acid backbone
mimic in click ligation, and to explore its biocompatibility.
analogues (25). Oligonucleotide sequences are shown in Figs. 1
and 2 and in Table S1. For the trans-click ligation reactions, the
individual oligonucleotide segments require an alkyne or azide
group at the 5-position of a uracil base at the appropriate locus.
Results and Discussion
Hairpin Ribozymes. The oligonucleotide building blocks required
in the assembly of the hairpin ribozyme analogues (segments
a, b, and c in Fig. 1) were prepared by automated solid-phase
phosphoramidite synthesis using 2′-t-butyldimethylsilyl (TBS)
protected monomers for the RNA tracts. The TBS method of
RNA synthesis (24) was chosen as it is widely used, the requisite
reagents are readily available, and it has been shown to be effec-
tive in the synthesis of a wide range of chemically modified RNA
Fig. 2. Structure and sequence of the hammerhead ribozyme (green) and
substrate (blue) with cleavage site in red. The triazole linkage lies in the cat-
alytic pocket opposite to the cleavage site between C3 and U4. Nucleotides
C3 and G8 form a base pair in the active conformation of the ribozyme–
substrate complex (33).
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The reaction scheme and the structures of the alkyne and azide-
labeled nucleoside components 3 and 4 are shown in Fig. 3.
Azides are unstable to PIII, and do not survive the conditions
of phosphoramidite oligonucleotide synthesis. Therefore, the
azide functionalities were introduced after oligonucleotide as-
sembly by selective reaction of the N-hydroxysuccinimide ester
of azidohexanoic acid 2 with the primary amino groups of the
modified uracil bases (26). All oligonucleotides were purified
by reversed-phase HPLC and analyzed by mass spectrometry
prior to click ligation. For construction of the all-RNA and
DNA/RNA 98-mer ribozymes the individual segments were an-
nealed and covalently linked in a self-templated CuAAC reaction
(27). The CuI catalyst was generated in situ from CuII sulfate and
aqueous sodium ascorbate, and a water-soluble CuI-binding
ligand 5 was added to prevent RNA degradation (28). During
the reaction segment a was crosslinked to segment b near termi-
nus B, and segment b to segment c near terminus C across the
major grooves of the ribozyme stems. The crosslinking positions
are indicated in Fig. 1, and the chemical structure of the resultant
triazole linkage 6 is shown in Fig. 3. The two simultaneous click
ligation reactions proceeded efficiently in the assembly of both
ribozymes, and full-length constructs were purified by polyacry-
lamide gel-electrophoresis (Fig. 1, Left). Despite the self-templat-
ing, a minor side-reaction occurred due to cyclization of segment
b, which contains both alkyne and azide. This impurity was easily
removed during ribozyme purification. A shorter 77 nucleotide
version of the hairpin ribozyme was also prepared (77-RNA,
Fig. 1, Right), but in this case segment b was synthesized with
two azide groups, one near the 5′- terminus and the other near
the 3′-end, rather than alkyne and azide. Segments a and c were
synthesized with an alkyne near their 3′- and 5′-ends respectively
to ensure chemical complementarity with segment b. This facile
change in strategy eliminated the possibility of unwanted cycliza-
tion of segment b and is an example of the flexibility offered by
chemical ligation. Segments a, b, and c were labeled with cy5, cy3,
and FAM respectively to facilitate fluorescence resonance energy
transfer (FRET) studies (7, 29). There are many cases when RNA
assembly cannot be self-templated, so an alternative strategy is
necessary. For the smaller hairpin ribozyme 77-RNA we elected
to explore the use of two complementary 24-mer DNA splints to
hold the short arms of the ribozyme segments in place during
click ligation. This method gave a clean reaction and the resultant
short hairpin ribozyme was purified by polyacrylamide gel-
electrophoresis.
The catalytic activity of the three hairpin ribozyme analogues
was investigated by running cleavage reactions at 55 °C in the
presence of five equivalents of the RNA substrate (Fig. 4). In
all cases five cycles of denaturation (95 °C) followed by cleavage
(55 °C) consumed the substrate. It was cleaved at the expected
site (indicated in Fig. 1), as confirmed by mass spectrometry
of the two fragments (ES−, found: 5944 Da, 2154 Da, requires:
5943 Da, 2155 Da). The ribozymes also cleaved the substrate
without temperature cycling (Fig. S3). The clicked ribozymes
were robust and could be isolated from the cleavage gels and re-
used in subsequent reactions with no significant loss of activity.
The fact that the triazole hairpin ribozyme cleaves its substrate
rapidly and produces the same products as the natural ribozyme
is not surprising as the click linkages are located remotely from
the active site. This design feature can be conveniently incorpo-
rated into most large RNA constructs. This click linkage stretches
across the major groove where it does not perturb the duplex or
change its conformation (26). Cross-strand linking between nu-
cleobases in RNA is unique to chemical ligation and cannot
be achieved by enzymatic methods.
The Hammerhead Ribozyme. A minimal hammerhead ribozyme
was synthesized by splint-mediated intrastrand click ligation
(cis- ligation) of 3′-alkyne and 5′-azide oligoribonucleotides. This
alternative method was chosen to investigate the suitability of
click chemistry for linking RNA strands through their backbone
rather than between the nucleobases. The success of this strategy
in producing an active ribozyme requires the design of a triazole
linkage that closely resembles a phosphodiester group. The struc-
ture of this linkage and the synthetic strategy to produce it are
shown in Fig. 5. The key building block for the synthesis of
the 3′-alkyne labeled RNA strand is 5′-DMT-3′-propargyl-5-
methyldeoxycytidine 9. This was made in two steps, the first
involving 3′-alkylation of 5′-DMT thymidine 7 with propargyl
bromide in the presence of sodium hydride in THF to give inter-
mediate 8 in 82% yield, a considerable improvement on the pub-
lished method (30). In the second step, conversion of thymine to
5-methyl cytosine was carried out using phosphorus oxychloride,
N-methylimidazole and aqueous ammonia. Compound 9 was
coupled to succinylated aminoalkyl solid support to give deriva-
tized support 10, which was used in the solid-phase synthesis of
3′-propargyl oligonucleotide 11 (Fig. 5). This solid support will be
useful in the synthesis of oligonucleotides for various click liga-
tion or labeling applications. The 5′-azide RNA strand 13 was
made from the corresponding unmodified 5′-hydroxyl RNA con-
gener 12 by treatment of the support-bound oligonucleotide with
methyltriphenoxyphosphonium iodide followed by sodium azide.
This 5′-azide labeling method has been used previously on DNA
Fig. 4. Hairpin ribozyme substrate cleavage reactions. Lanes 1, 3, 5: cleavage
reactions of 98-RNA, 98-DNA/RNA and 77-RNA ribozymes respectively with 5
equivalents of substrate. Lanes 2, 4, 6: control—uncleaved substrate. 20%
polyacrylamide gels visualized by fluorescence.
Fig. 3. The synthetic procedure used to covalently link the arms of the hair-
pin ribozyme across the major groove of the duplex.
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Fig. 5. Synthesis of the hammerhead ribozyme. Synthesis of alkyne and azide RNA strands, construction hammerhead, and structure of triazole backbone.
(31) and here we demonstrate its compatibility with RNA. The
two components of the hammerhead ribozyme (11 and 13) were
designed with extra thymidine nucleotides at the termini to aid
gel-filtration, whereas the 5′-FAM dye on the DNA splint was
included to differentiate it from the clicked ribozyme during re-
versed-phase HPLC purification. Splint-mediated ligation of the
two RNA strands 11 and 13 proceeded smoothly to give ribozyme
14 with a triazole linkage in the backbone (Fig. 6A, lane 4). The
modified hammerhead ribozyme was characterized by mass spec-
trometry (ES-, found: 7585 Da, requires: 7585 Da) and was shown
to cleave its substrate, generating an identical product to that
formed by the normal unmodified ribozyme (Fig. 6B, lanes 1
and 2). Cleavage occurred at the predicted scissile phosphodiester
bond between cytidine and adenosine as confirmed by mass spec-
trometry of the two fragments (ES-, found: 6490 Da, 1495 Da;
requires: 6490 Da, 1496 Da). Parallel experiments on a 5′-fluor-
escein labeled substrate demonstrated complete cleavage in
30 min at 37 °C, similar to the native ribozyme (Fig. 6C). This is
a satisfying result, particularly as the unnatural triazole linkage
is located at the active site between residues C3 and U4 of the
ribozyme (Fig. 2). X-ray structures of the minimal (19, 20, 32)
and full-length hammerhead ribozymes (33) confirm the critical
positioning of C3. It is postulated that a base pair between C3
and G8 holds the ribozyme in its catalytically competent confor-
mation (33). The observed activity of the triazole ribozyme con-
firms that in this structure the designed linkage is a suitable
analogue of a phosphodiester group.
To further demonstrate that the triazole linkage is a viable
substitute for a phosphodiester group we carried out UV-melting
Fig. 6. (A). CuAAC reaction to synthesize the hammerhead ribozyme, 20%
polyacrylamide gel: Lanes 1, 2: starting oligonucleotides (alkyne and azide),
lane 3: DNA splint, lane 4: crude reaction mixture. (B). Hammerhead ribozyme
cleavage reaction with nonfluorescent substrate. Lane 1: S þ TR, lane 2:
S þ NR, lane 3: TR, lane:
4
NR, lane 5: S. (S ¼ uncleaved substrate,
NR ¼ normal ribozyme control, TR ¼ triazole ribozyme). Gels A and B visua-
lized by UV shadowing. TR (24-mer) is longer than NR (16-mer) due to the
additional thymidines at the termini. (C). Hammerhead ribozyme cleavage
reaction with 5′-fluorescein labeled substrate. Lane 1: FS, lane 2: FS þ NR
30 min, lane 3: FS þ TR 30 min, lane 4: FS, lane 5: FS þ NR 1 h, lane 6:
FS þ TR 1 h. (FS ¼ fluorescent substrate, gel imaged by fluorescence).
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Sterile plastic/glassware and buffers were used for all procedures involving
RNA analogues.
studies on the natural and triazole ribozymes with their RNA sub-
strate in the absence of magnesium ions (to prevent substrate
cleavage). We obtained melting curves that were very similar
(Fig. S4), with a slight lowering in melting temperature in the
triazole case (ΔTm ¼ 1.1 °C). We then hybridized the native
and triazole hammerhead ribozymes to their DNA complement
to produce DNA–RNA hybrid duplexes. In this case the triazole
analogue was almost as stable as its natural counterpart
(ΔTm ¼ 3.8 °C) (Fig. S4). The CD spectra of the above constructs
indicate that the triazole modification does not change the duplex
conformation (Fig. S5). Although it would not generally be
necessary to place the triazole modification at a critical catalytic
position when designing RNA constructs we have shown here
that such a substitution can lead to stable biologically active
ribozymes, thereby demonstrating the biocompatibility of the
triazole linkage.
Assembly of hairpin ribozymes 98-RNA, 98-DNA/RNA and 77-RNA. The three
segments of each ribozyme (Fig. 1) (10 nmol of each) in 0.2 M NaCl
(950 μL) were annealed by heating at 80 °C for 5 min, cooling slowly to room
temperature (1 h) then maintaining the temperature at 0 °C for 15 min. A
solution of CuI click catalyst was prepared from trishydroxypropyltriazole
ligand 5 (Fig. 3) (28) (3.5 μmol in 0.2 M NaCl, 50.0 μL), sodium ascorbate
(50.0 μmol in 0.2 M NaCl, 50.0 μL), and CuSO4 · 5H2O (0.5 μmol in 0.2 M NaCl,
5.0 μL). This solution was added to the annealed ribozyme segments and the
reaction mixture was kept at 0 °C for 15 min, then at room temperature for
1 h. A NAP-10 gel-filtration column was used to remove reagents. The clicked
hairpin ribozymes were analyzed and purified by denaturing 8% polyacryla-
mide gel electrophoresis. To synthesize the short hairpin ribozyme (77-RNA)
two complementary 24-mer DNA splints (10 nmol of each) (Table S1), were
added and the reaction was carried out under the above conditions.
Conclusion
Templated assembly of triazole hammerhead ribozyme (Fig. 6A). Alkyne ham-
merhead segment, azide hammerhead segment and hammerhead splint
(Fig. 2 and Table S1), (10 nmol of each) in 0.2 M NaCl (200 μL) were annealed
by heating at 80 °C for 5 min and cooling slowly to room temperature (1 h). To
a solution of trishydroxypropyltriazole ligand 5 (28) (0.7 μmol) in 0.2 M NaCl
(50.0 μL) was added sodium ascorbate (1.0 μmol in 0.2 M NaCl, 2.0 μL)
followed by CuSO4 · 5H2O (0.1 μmol in 0.2 M NaCl, 1.0 μL) under argon. This
mixture was added to the annealed oligonucleotides and the reaction was
left under argon at room temperature for 1 h, made up to 1 mL with water
and gel-filtered to remove reagents (NAP-10). The ligated oligonucleotides
were analyzed using denaturing 20% polyacrylamide gel electrophoresis and
purified by reversed-phase HPLC.
In this study click chemistry has been used to ligate presynthe-
sized oligonucleotides to construct RNA molecules and DNA/
RNA chimeras up to 100 nucleotides in length. The method is
compatible with site-specific modifications, mixed backbones
and various reporter groups. The reaction is quick, simple, amen-
able to large scale synthesis, and highly efficient. It is a very flex-
ible procedure that can be used with a diverse range of alkyne and
azide-labeled oligonucleotides, which are accessible from com-
mercial sources. It creates novel chemical linkages that cannot
be produced by enzymatic methods. Click ligation is therefore
an important addition to RNA chemistry and biochemistry.
Two different strategies were employed in this study, although
others could also have been used (27, 34, 35). One variant of the
click reaction (trans-ligation) was carried out between the nucleo-
bases with self-templating, and also by splint-mediated ligation.
The resultant synthetic hairpin ribozymes contain multiple fluor-
escent labels which can be used to monitor RNA folding and
melting. The hammerhead ribozyme was constructed by an alter-
native strategy, templated cis-ligation, to produce a novel triazole
nucleic acid backbone mimic that is compatible with catalytic
activity. The chemistry described here could be applied to the
synthesis of many important RNA molecules such as riboswitches
(9, 10, 36), siRNA delivery systems (37), multivalent aptamers
(38), and components of ribosomes (39). Using click ligation,
several variants of segments of such RNA constructs could be
prepared on a large scale, purified and ligated in different com-
binations to provide a number of full-length RNA analogues
for structural and biochemical studies. It is also interesting to
contemplate the possible advantages of using chemical and enzy-
matic RNA ligation in tandem for the synthesis of RNA con-
structs beyond the size limit of either individual method. This
could be achieved using oligonucleotide building blocks functio-
nalized with 5′-phosphates and alkynes/azides. The enzymatic
ligations could be carried out first, then subsequent introduction
of Cu(I) and additional alkyne/azide oligonucleotides to the mix-
ture would trigger a set of orthogonal click ligation reactions. This
strategy could be used to place native and modified (nonhydro-
lyzable) linkages at specific positions in the final construct.
Cleavage of substrate with native and clicked hammerhead ribozymes (Fig. 6 B
and C). The native and clicked hammerhead ribozymes (0.2 nmole of each)
were dissolved in 30 μL Tris-HCl buffer (50 mM Tris-HCl, 10 mM MgCl2, pH
7.6) and 0.1 nmole of the fluorescent hammerhead RNA substrate in
30 μL of the same buffer was added to each ribozyme. The reaction mixtures
were incubated at 37 °C for 30 min after which time half the sample (30 μL)
was removed, mixed with formamide (30 μL) and frozen in liquid nitrogen.
After a further 30 min the remaining 30 μL of the reaction mixture was trea-
ted in the same way. The samples were then analyzed by 20% denaturing
polyacrylamide gel electrophoresis with fluorescent detection. Cleavage of
the nonfluorescent hammerhead substrate was carried out in the same
way using 1.25 nmole of the ribozymes and 1.0 nmole of the substrate.
The reaction was analyzed after 1 h and the gel was visualized by UV
shadowing.
Cleavage of substrate with 98-RNA, 98-DNA/RNA and 77-RNA hairpin ribozymes.
The clicked hairpin ribozymes (0.15 nmol of each) and substrate (0.75 nmol)
were dissolved in 40 μL Tris-HCl buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 7.6),
vortexed, and heated in a thermal cycler (PCR machine) at 55 °C for 30 min
then 5 cycles of 95 °C for 2 min and 55 °C for 30 min. Formamide (40 μL) was
added and the reaction mixture was heated at 80 °C for 5 min to denature
the complex, cooled on ice and analyzed by 20% denaturing polyacrylamide
gel electrophoresis. To cleave the substrate for analysis by mass spectrometry
the clicked hairpin ribozyme and substrate (1.0 nmol of each) were dissolved
in 80 μL of Tris-HCl buffer and heated at 55 °C for 1 h, after which the solution
was made up to 1 mL with water and desalted by NAP-10 gel-filtration.
ACKNOWLEDGMENTS. We thank Professor D.M.J. Lilley for helpful discussions.
The research leading to these results has received funding from the European
Community’s Seventh Framework Programme (FP7/2007-2013) under Grant
HEALTH-F4-2008-201418) entitled READNA.
Materials and Methods
Mass spectra of ribozymes were recorded on a Bruker micrOTOF™ II focus
ESI-TOF MS instrument in ES− mode and data were processed using MaxEnt.
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