Small Molecule Recognition Triggers Secondary and Tertiary Interactions in DNA Folding and Hammerhead Ribozyme Catalysis

Article abstract of DOI:10.1021/jacs.7b05448

We have identified tris(2-aminoethyl)amine (tren)-derived scaffolds with two (t2M) or four (t4M) melamine rings that can target oligo T/U domains in DNA/RNA. Unstructured T-rich DNAs cooperatively fold with the tren derivatives to form hairpin-like structures. Both t2M and t4M act as functional switches in a family of hammerhead ribozymes deactivated by stem or loop replacement with a U-rich sequence. Catalysis of bond scission in these hammerhead ribozymes could be restored by putative t2M/t4M refolding of stem secondary structure or tertiary bridging interactions between loop and stem. The simplicity of the t2M/t4M binding site enables programming of allostery in RNAs, recoding oligo-U domains as potential sites for secondary structure or tertiary contact. In combination with a facile and general method for installation of the t2M motif on primary amines, the method described herein streamlines design of synthetic allosteric riboswitches and small molecule-nucleic acid complexes.

Full text of DOI:10.1021/jacs.7b05448

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Communication
Small molecule recognition triggers secondary and tertiary
interactions in DNA folding and hammerhead ribozyme catalysis
Jie Mao, Chris DeSantis, and Dennis Bong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05448 • Publication Date (Web): 10 Jul 2017
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Small molecule recognition triggers secondary and tertiary interactions
in DNA folding and hammerhead ribozyme catalysis
Jie Mao, Chris DeSantis and Dennis Bong*
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Department of Chemistry and Biochemistry, The Ohio State University, 100 W. 18th Avenue, Columbus, OH 43210.
Supporting Information Placeholder
Abstract: We have identified tris(2-aminoethyl)amine
(tren)-derived scaffolds with two (t2M) or four (t4M)
melamine rings that can target oligo T/U domains in
DNA/RNA. Unstructured T-rich DNAs cooperatively fold
with the tren derivatives to form hairpin-like structures.
Both t2M and t4M act as functional switches in a family of
hammerhead ribozymes deactivated by stem or loop
replacement with a U-rich sequence. Catalysis of bond
scission in these hammerhead ribozymes could be restored
by putative t2M/t4M refolding of stem secondary structure
or tertiary bridging interactions between loop and stem. The
simplicity of the t2M/t4M binding site enables programming
of allostery in RNAs, recoding oligo-U domains as potential
sites for secondary structure or tertiary contact. In
combination with a facile and general method for installation
of the t2M motif on primary amines, the method described
herein streamlines design of synthetic allosteric riboswitches
and small molecule-nucleic acid complexes.
Most nucleic acid binding small molecules are natively
derived,1 obtained via library selection,2 designed to target
pre-structured3,4 DNA and RNA or operate primarily via
intercalation.5 We report herein design and synthesis of
small molecule tren derivatives that trigger the global folding
of unstructured nucleic acid; further, these motifs mediate
tertiary interactions between oligo-U domains in partially
folded RNA. Binding is brokered by the same melamine base
previously reported in bPNA, a family of peptoid,6 peptide7–11
and polyacrylates12,13 that triplex hybridize with DNA/RNA
via base tripling triazine recognition14–18 with
thymine/uracil. In contrast to artificial base pairs,19–22 there
are fewer examples of artificial base triples,23 despite
considerable interest in triplex structures.24–26 Melamine
base-triple docking to single TT/UU mismatches in
structured nucleic acid is known to require a flexible, 4-atom
linkage to an acridine intercalator.27,28 Inspired by this
design, we hypothesized that replacement of the non-specific
intercalator with a second melamine ring would enable
binding and folding of oligo T/U domains. Herein are
described melamine-containing derivatives of tren, an open
chain, sp3-hybridized scaffold, that can bind and trigger
Figure 1. (Top) Melamine-thymine/uracil triple in nucleic acid (NA)
and tren scaffolds t2M, t3M and t4M. (a) Reductive alkylation of
Boc-tren with melamine acetaldehyde: 1) NaCNBH3, DMSO; 2) TFA.
M=melamine. (Below) Schematic of minimal hammerhead ribozyme
(HHmin) with tertiary AU contact indicated between enzyme (bold) and
substrate (grey) strands (cleavage site=blue dashed). U-Stem (S-U4n)
and U-loop (L-U4) enzymes were studied with “wild type” (wt) and U
enriched (5U) substrates. Oligo-U / t4M binding sites are indicated.
cooperative folding of unstructured oligo-T DNAs. Further,
these tren derivatives selectively bind oligo T/U bulges in
partially folded DNA and RNA, functioning as allosteric triggers
for hammerhead ribozyme catalysis via restoration of secondary
and tertiary RNA structures.29 While synthetic allosteric
switches are well-established,30–33 to our knowledge, this is the
first report of de novo designed RNA-small molecule allostery
that drives both secondary structure folding and formation of
tertiary RNA interactions, effectively re-coding oligo-U domains
as potential sites for structural turn-on.
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A minimal oligo-T binding motif, t3M (Figure 1), is
readily accessed in one step by triple arylation of tren with
chloro-diaminotriazine. Incubation of t3M with dT10C4T10
DNA yielded a cooperatively melting complex (Tm=47°C), as
observed by UV. This thermal signature only appeared with
the complex, and disappeared upon methylation of the
exocyclic melamine amines (t3MMe), supportive of specific
melamine recognition. Differential scanning calorimetry
(DSC) indicated strongly exothermic t3M-DNA binding
(-203 kcal/mole), consistent with prior studies on
melamine-thymine8 and native19,34–36 base-stacking (Figure
2). Binding occurs upon mixing, as evidenced by isothermal
titration calorimetry (ITC, S.I.) and circular dichroism,
which revealed significant DNA folding upon addition of
t3M (Figure 2).
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Figure 3. (Left) Fluorescence quenching in Fl-dT10C4T10-Dabcyl DNA is
induced by t2M(●)/t4M(■). Formation of the higher order complex
with t2M is shown schematically. (Right) Anisotropy-derived 1:1
binding isotherms of t2M-Fl* and t4M-Fl* to Tn-bulges (red) in DNA
duplexes [12-T2-12] (●) and [12-T4-12] (■), respectively. Fits to a 1:1
binding model are shown (—) for t2M and t4M, with Kd’s ~4.2 µM and
44 nM, respectively.
and robust affinity to an octathymidine bulge in duplex DNA
12-T4-12 (Kd=50 nM). Methylation of the melamine rings in
t2M and t4M abolished all DNA binding, indicating that
electrostatic interactions do not drive affinity.
To test the extent to which these molecules could be used to
complement native nucleic acid function, we designed
deactivated type I hammerhead U-ribozymes in which critical
secondary and tertiary structural elements were ablated by
replacement with oligo-U loops. Tertiary interactions between
loop II and stem I are known to be essential in catalysis, with
Figure 2. (Top) Illustration of dT10C4T10 DNA complexation with 5
equivalents of t3M. (Left) CD of 5 µM dT10C4T10 DNA (
)
witht3M at 10, 15, 20, 25, 50 (┄) and 100 µM (—) at 25°C (1 mm recent reports37 identifying a single essential A□○U Hoogsteen
cuvette). (Right) Thermal denaturation of the dT10C4T10●t3M
complex at 1:5 mole ratio followed by UV260 (---) and DSC (—).
DNA concentration=2 µM (UV), 25 µM (DSC). All experiments
run in 1X PBS, pH 7.4.
pair between loop (enzyme) and stem (substrate) in a binary
hammerhead system. Despite the sensitivity of the hammerhead
ribozyme to cations,38–42 tetrabasic permethylated t4M (t4MMe
)
did not activate RNA cleavage in any of the U-ribozymes
studied. We first set out to test the extent to which t2M and t4M
could restore catalysis by generating secondary structure alone.
U(2,3) is a minimal ribozyme in which stems II and III are
replaced with oligo-U loops. It therefore lacks secondary
structure in stems II and III, and further, does not have the
loop-stem sequences required for rate-enhancing tertiary
interactions; bond scission is completely deactivated. Consistent
with secondary structural rescue, addition of t2M or t4M
restored cleavage, with t4M exhibiting much greater efficacy
(Figure 4). We then tested the extent to which t4M
Titration of t3M against Fl-dT10C4T10-Dab DNA resulted
in fluorescein (Fl) quenching by dabcyl (Dab), supportive of
formation of a hairpin structure (S.I.) with 5:1 t3M:DNA
stoichiometry. Given the expected 2:1 thymine to melamine
(T:M) ratio,8 we hypothesized that only 2 melamine rings
per t3M unit were needed to bind dT10C4T10. Indeed, t2M,
with 2 melamine rings, exhibited the same 5:1 DNA
stoichiometry, similar heat of binding (-226 kcal/mole) and
has an available primary amine for labeling (Figure 3).
Fluorescein-derivative t2M-Fl* bound to a tetrathymidine
bulge in the DNA structure 12-T2-12, a putative t2M binding
site flanked by two 12mer duplex regions (Figure 3).
Anisotropy measurements indicated micromolar affinity 1:1
binding to the tetrathymidine site for t2M-Fl* (Figure 3),
confirmed by native gel-staining 12-T2-12 with Cy5-labeled
t2M (S.I.). Alternative scaffolds were also prepared, but
failed to improve upon binding or synthetic versatility of the
tren derivatives (S.I.). However, we hypothesized that
increasing the number of bases while maintaining the t2M
pattern would increase binding affinity. Thus, t4M, with
four melamine rings on tren, was prepared by reductive
Figure 4. (Left) U23 deactivated ribozyme, treated with t4M to
alkylation of mono-protected tren (Figure 1). Gratifyingly, restructure stems with oligo-U domains (red) as t4M binding sites.
t4M exhibited enhanced DNA affinity, with an expected
2.5:1 stoichiometry of binding to a Fl-dT10C4T10-Dab probe,
(Right) Cleavage of U23 (blue arrow) upon treatment with 10 µM t4M
(■) or t4MMe (□) and RNA only (○) at 10 mM Mg2+ with 500 nM
ribozyme, 10 µM t4M in 1X Tris-Cl buffer (pH=7.6) at 37 °C.
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could simultaneously rescue secondary and tertiary
interactions using S-U4n, a ribozyme in which stem II is
replaced with 4 UU mismatches, but retains the GUGA
sequence in loop II and U in position 1.7 in stem I, required
for tertiary AU contact.37,43 The partially destructured S-U4n
still cleaves itself at 10 mM magnesium, but was only weakly
active at 0.1 mM Mg2+. Under low magnesium conditions,
addition of t4M significantly enhanced S-U4n cleavage
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Figure 6. (Left) L-U4 ribozyme (boxed) with the HHmin (wt) substrate
(grey) with U in position 1.7 indicated (bold black) and ablated tertiary
interaction indicated (---). L-U4 reacts with the 5U substrate (grey)
with loop-stem binding mediated by t4M. (Right) L-U4 cleavage of 5U
substrate with 10 µM t4M (■), 10 µM t4MMe (□), no additive (○) and
cleavage of wt substrate with t4M (■). Reaction conditions: 4 µM
enzyme, 320 nM Cy3 substrate, 10 µM t4M/t4MMe, 0.1 mM Mg2+ in 1X
Tris-Cl buffer, pH=7.6, 27°C
diminished cleavage activity with all substrates. Addition of t4M
increased cleavage rate of 2U and 5U substrates by ~2 and 4-fold,
respectively, while having no effect on the wt substrate,
consistent with rescue of the loop-substrate tertiary interaction
via t4M bridging of the U-domains (Figure 6). Interestingly, the
reported structure44 of the full-length hammerhead ribozyme
revealed a UAU base triple between the adenine of the enzyme
loop and 2U’s in the substrate; similar triples have been observed
in other functional RNAs.26,45,46 It is intriguing to speculate that
t4M creates a similar type of tertiary interaction via bifacial
Figure 5. Cleavage reactions (0.1 mM Mg2+, 500 nM ribozyme,
with t4M in 1X Tris-Cl, pH=7.6, 37 °C) of the indicated melamine-uridine recognition.
U-ribozymes is illustrated, with U4 domains indicated (red) as t4M
We have described herein design and synthesis of a minimal
binding sites. (A) S-U4n ribozyme, treated with t4M (■) at
indicated concentrations, 10 µM t4MMe (-□-) and alone (○). (B)
S-U4n-2bp ribozyme (extended by 2 bp on stem I) upon treatment
with 10 µM t4M (▲) and alone (△).
tren-based molecular motif for binding oligo T/U sequences in
DNA and RNA, resulting in cooperative folding and
structure-function turn-on.10 While elegant systems are known
in which small molecules trigger folding and chemistry in
aptamers,47–49 to our knowledge, this is the first report of
synthetic small molecules that can trigger global folding of
unstructured oligo T/U nucleic acid. The t4M derivative acts as a
chemical inducer of dimerization50 for two short oligo-U
domains, effectively generating tertiary interactions in RNA by
serving as a synthetic molecular bridge. Binding sites for these
small molecules are readily engineered into partially folded
nucleic acids, potentially generalizing the design of compact,
functional allosteric10,51,52 switches sensitive to t2M derivatives in
a wide range of RNAs. Finally, the t2M motif may be easily
installed on any primary amine, thus enabling versatile
molecular targeting to nucleic acids for packaging, delivery,
labeling and other biotechnology48,49,53,54 applications.
(Figure 5). Further, extension of stem I by two base pairs in
S-U4n-2bp resulted in greatly diminished activity (Figure 5).
The two base extension in stem I is remote from the catalytic
site, and thus the loss of activity likely indicates a disruption
of stem I tertiary interactions essential to catalysis. Together,
these data support the notion that t4M restructures stem II
in such a way that facilitates both secondary and tertiary
interactions within the RNA fold.
We hypothesized that tertiary RNA interactions between
loop II and stem I could be directly bridged by t4M upon
replacement of discrete RNA contacts with U:M:U base
triples. With all secondary structures intact in a binary
hammerhead ribozyme system, tetraloop II (GUGA) was
replaced with either GUUU or UUUU to yield L-GU3 and
L-U4, respectively. Substrates with U at positions 1.7
(native), 1.7-1.8 (UU), and 1.7-1.11 (UUUUU) in the
unpaired region were studied under low magnesium
conditions. While L-GU3 was inactive with native substrate,
Supporting Information
Detailed experimental procedures, chemical structures, sequences,
additional titration calorimetry, fluorescence anisotropy, DSC, thermal
denaturation (UV) data, compound characterization, ribozyme cleavage
considerable cleavage of the 2U substrate was observed, data. This material is available free of charge via the Internet at
http://pubs.acs.org.
despite the absence of AU pairing. This suggests a plastic
tertiary structure in this minimal hammerhead ribozyme,
with other interactions, such as a GU wobble, possibly taking
Acknowledgements
the place of the AU contact between loop II and stem I. Such Research was supported in part by NSF-DMR and NIH.
a possibility is eliminated in L-U4, which exhibited greatly
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Corresponding Author
120, 1.
(42) Mir, A.; Chen, J.; Robinson, K.; Lendy, E.; Goodman, J.; Neau, D.; Golden,
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*bong.6@osu.edu
B. L. Biochemistry 2015, 54, 6369.
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S.; Cedergren, R.; Eckstein, F.; Gerlach, W. L.; Hodgson, R.; Symons, R. H.
Nucleic Acids Res. 1992, 20, 3252.
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