CCR5 RNA Pseudoknots: Residue and Site-Specific Labeling correlate Internal Motions with microRNA Binding

Article abstract of DOI:10.1002/chem.201705948

Conformational dynamics of RNA molecules play a critical role in governing their biological functions. Measurements of RNA dynamic behavior sheds important light on sites that interact with their binding partners or cellular stimulators. However, such measurements using solution-state NMR are difficult for large RNA molecules (>70 nt; nt=nucleotides) owing to severe spectral overlap, homonuclear ¹³C scalar couplings, and line broadening. Herein, a strategic combination of solid-phase synthesis, site-specific isotopic labeled phosphoramidites, and enzymatic ligation is introduced. This approach allowed the position-specific insertion of isotopic probes into a 96 nt CCR5 RNA fragment. Accurate measurements of functional dynamics using the Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion (RD) experiments enabled extraction of the exchange rates and populations of this RNA. NMR chemical shift perturbation analysis of the RNA/microRNA-1224 complex indicated that A90-C1′ of the pseudoknot exhibits similar changes in chemical shift observed in the excited state. This work demonstrates the general applicability of a NMR-labeling strategy to probe functional RNA structural dynamics.

Full text of DOI:10.1002/chem.201705948

A Journal of
Accepted Article
Title: Residue and site-specific labeling of CCR5 RNA pseudoknot
reveals internal motions that correlate with microRNA binding
Authors: Bin Chen, Andrew P Longhini, Felix Nußbaumer, Christoph
Kreutz, Jonathan Dinman, and T Kwaku Dayie
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201705948
Link to VoR: http://dx.doi.org/10.1002/chem.201705948
Supported by

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COMMUNICATION
Residue and site-specific labeling of CCR5 RNA pseudoknot
reveals internal motions that correlate with microRNA binding
Bin Chen[a,b], Andrew P. Longhini[b], Felix Nußbaumer[c], Christoph Kreutz[c], Jonathan Dinman[a]*, & T.
Kwaku Dayie[b]*
stimulate -1 PRF, thus fine-tuning gene expression by controlling
Abstract: Conformational dynamics of RNA molecules play a
mRNA abundance, suggesting that this element may be novel
for controlling HIV-1 viral replication.[4] Unlike other non-coding
functional RNA elements currently being pursued as targets for
various diseases,[5] the pseudoknot motif is within the coding
region of CCR5 mRNA.[4] Therefore, any information about the
CCR5 pseudoknot structure and interaction with miRNAs will
provide insight into gene regulation connected with -1 PRF
mechanism and foundation for RNA/miRNA-based drug design
and development.
critical role in governing their biological functions. Measurements
of RNAs′ dynamic behaviour shed important light on sites that
interact with their binding partners or cellular stimulators.
However, such measurements using solution NMR are difficult
for large RNAs (>70 nt) due to severe spectral overlap,
homonuclear 13C scalar couplings, and linebroadening. In this
work, a strategic combination of solid-phase synthesis, site-
specific isotopic labeled phosphoramidites and enzymatic
ligation is introduced. This approach allowed us to position-
specifically insert isotopic probes into a 96 nt CCR5 RNA.
Accurate measurements of functional dynamics using the Carr-
Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD)
experiments enabled extraction of the exchange rates and
populations of this RNA. NMR chemical shift perturbation
analysis of the RNA/microRNA-1224 complex indicated that
A90-C1′ of the pseudoknot exhibits similar changes in chemical
shift observed in the excited state. This work demonstrates the
While structures are desirable, not all RNA molecules are
amenable to high resolution structural characterization by X-ray
crystallization and nuclear magnetic resonance (NMR)
spectroscopy, mostly because RNAs are highly flexible. Their
inherent plasticity renders them capable of sampling a vast
ensemble of conformations.[6] RNA structures also typically
undergo conformational changes in response to ligand binding,
cellular signals or environmental fluctuations; indeed,
interconversions among different conformational states are
crucial for RNAs to fulfil their diverse biological functions.[7]
Therefore, to better understand how CCR5 functions in -1 PRF
and gene regulation, we need to not only determine its high
resolution structures in free and in complex with miRNA-1224,
but also understand its internal motions and dynamic behavior.
general applicability of
a NMR-labeling strategy to probe
functional RNA structural dynamics.
The C-C chemokine receptor type 5 (CCR-5) is a coreceptor
involved in membrane fusion of human immunodeficiency type 1
virus (HIV-1) particle and plays a critical role in facilitating viral
entry into human cells.[1] Significant efforts have been made to
develop therapeutic agents or strategies either targeting the
receptor itself [2] or manipulating its gene [3] for treatment of HIV-
1 infection. Recently, a highly-conserved pseudoknot motif was
identified in the CCR-5 encoding mRNA that stimulates
programmed -1 ribosomal frameshifting (-1 PRF) and that acts
as an mRNA destabilizing element by directing translating
NMR, as a proven and powerful approach, has been applied
extensively to characterize RNA dynamics over a wide range of
timescales.[8] However, these applications have mainly been
limited to small RNA systems (<50 nucleotides, nt) due to severe
spectral overlap and line broadening, in addition to
conformational heterogeneity in larger RNA species.[8a,9] To
bypass these problems, “divide-and-conquer” strategies have
been employed for structural characterization of sub-domains
derived from larger functional RNA molecules. However, these
approaches have led to questions regarding whether the
isolated sub-domains adopt the same structures, behave with
the same dynamic properties, and carry out the same functions
as those encoded by the full-length RNA molecules.[10] A hybrid
solid-liquid phase transcription method allowing region-specific
isotope labeling for RNAs larger than 50 nt was previously
reported.[11] This, however, was found to be highly sequence
restrictive and generated very low yields for large RNAs.[11]
Recently, coupling of site-specific isotope-labeling techniques
with enzymatic ligation has expanded the size limitation of RNA
NMR.[12] Of particular promise are approaches combining site-
specific isotope-labeling, solid-phase synthesis, and enzymatic
ligation in investigating RNA structure and dynamics. Indeed,
this chemo-enzymatic approach significantly simplifies the NMR
spectra, allows unambiguous resonance assignment and
identification of key residues involved in molecular recognition,
ribosomes to
between this element and a microRNA was proposed to
a
-1 frame termination codon.[4] Interaction
[a]
[b]
Dr. B. Chen, Prof. Dr. J. D. Dinman
Department of Cell Biology and Molecular Genetics
University of Maryland
4062 Campus Dr., College Park, MD 20742 (USA)
E-mail: dinman@umd.edu
Dr. A. P. Longhini, Prof. Dr. T. K. Dayie
Center for Biomolecular Structure & Organization
Department of Chemistry & Biochemistry
University of Maryland
8314 Paint Branch Dr, College Park, MD 20782 (USA)
Email: Dayie@umd.edu
[c]
Mr. F. Nußbaumer, Prof. Dr. C. Kreutz
Institute of Organic Chemistry and Center for Molecular
Biosciences
Innsbruck (CMBI), University of Innsbruck
Innrain 80/82, 6020 Innsbruck (Austria)
E-mail:christoph.kreutz@uibk.ac.at
Supporting information for this article is given via a link at the end
of the document
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10.1002/chem.201705948
Chemistry - A European Journal
COMMUNICATION
and enables accurate measurements of dynamic features at
atomic resolution that are typically difficult to obtain for large
RNA molecules.[8b,e]
deprotected
these
donor
fragments
using
methylamine/ammonium hydroxide followed by treatment with
3M triethylamine trihydrofluoride (TEA, 3HF) in anhydrous
DMSO.[14] This desilylation agent was very efficient even in the
presence of low moisture. After purification to homogeneity by
denaturing polyacrylamide gel electrophoresis (PAGE), we
obtained 0.3 µmol of the donor fragments typically from a 1 µmol
synthesis scale column (Supplementary Table 1).
We report herein the characterization of site-specific motions of
a 96nt CCR5 RNA -1 PRF pseudoknot element that are
correlated to interaction with miR-1224 based on the strategy
described above using NMR spectroscopy. We first prepared a
key precursor (8,1′-13C) modified ATP by coupling (8-13C)-
adenine with (1′-13C)-ribose using chemo-enzymatic synthesis
with an excellent yield above 90%,[13] followed by
Next, we ligated an unlabeled T7 RNA polymerase synthesized
72 nt acceptor fragment to
residue/site-specific labeled 24-nt donor fragment (Figure 1).
The unlabeled acceptor fragment had protective 5′-
triphosphate group and a reactive 3′-OH group by virtue of using
T7 RNA polymerase for the in vitro transcription. The
synthesized residue/site-specific labeled 24-nt donor fragment
was phosphorylated by T4 polynucleotide kinase to obtain the
desired monophosphorylated group at the 5′-terminus. A donor
fragment with different labeling patterns could also be prepared
by in vitro transcription with T7 RNA polymerase and
dephosphorylated using RNA 5′-polyphosphatase prior to
ligation (Figure 1b). In general (Detailed in Supplementary
Materials), excess amounts of the acceptor fragment and the
DNA splint were used in the ligation reaction to achieve
maximum conversion of the donor fragment into the ligated
a solid phase synthesized
dephosphorylation
with
recombinant
shrimp
alkaline
phosphatase to afford (8,1′-13C)-adenosine (Supplementary
Figure 1) (compound 1 in Scheme 1). The exocyclic amino N6
group of (8,1′-13C)-adenosine (1) was then benzoylated to yield 2.
The selective protection of the 2ʹ-hydroxyl group with tert-
butyldimethylsilyl chloride via transient blocking of the 5′- and 3′-
hydroxyl groups using di-tert-butylsilandiylditriflate gave
a
intermediate 3.
Selective removal of the 3′,5′-di-tert-
butylsilanediyl group and subsequent tritylation gave N6-Bz-5ʹ-O-
DMT-(8,1′-13C) adenosine (4). Lastly, the (8,1′-13C)-adenosine
phosphoramidite building block 5, which is not commercially
available, was obtained for the first time by treatment with 2′-
cyanoethyl N,N-diisopropylchlorophosphoramidite (Scheme 1).
This building block is crucial for insertion of isotopic probes into
CCR5 RNA residue-specifically to facilitate subsequent NMR
studies.
residue/site-specific isotope labeled product with
ligation yield (based on the donor fragment) of >60% (Figure
1b).
a typical
Scheme 1. Synthesis of N6-Benzoyl-8,1′-13C-2′-O-TBDMS-adenosine
phosphoramidite 5. Reaction conditions: (a) Benzoylchloride, trimethylsilyl
chloride, pyridine, 2.5h, rt, then water, 28% NH3, 39%. (b) Di-tert-butylsilyl
bis(trifluoromethanesulfonate) in DMF, 0°C, 1h, then tert-butyldimethylsilyl
chloride, 3 h, 60°C, 40%. (c) HF.Py in CH2Cl2, 0°C, 1h, then DMT-Cl in
pyridine, rt, 3h, 91%. (d) CEP-Cl, DiPEA in THF, rt, 3h, 80%. Orange dot = 13C.
Figure
1 Ligation of the CCR5 pseudoknot element. (a) Scheme for
preparation of segmentally and selectively 13C-labeled CCR5 RNA. The
unlabeled 72 nt acceptor fragment was synthesized by in vitro transcription.
The donor was prepared by either in vitro transcription followed by enzymatic
dephosphorylation using RNA 5′ polyphosphatase or solid phase synthesis
followed by phosphorylation with T4 polynucleotide kinase to obtain the
desired monophosphorylated group at the 5′-terminus. A 40mer DNA splint
was used to facilitate RNA ligation. (b) A 12% denaturing PAGE showing the
ligation results. L1: de-phosphorylated donor fragment obtained from in vitro
transcription; L2: phosphorylated donor fragment from solid phase synthesis;
L3: DNA splint; L4: acceptor fragment; L5: ligation reaction at 0 h using the
donor from L1; L6: after 3 h reaction of L5; L7: ligation reaction at 0 h using the
donor from L2; L8: after 3 h reaction of L7; L9: CCR5 mRNA pseudoknot
prepared by in vitro transcription. Similar to prior observation,[8f] addition of the
DNA splint to the reaction system resulted in changes of the migration pattern
of all components in the system. The numbers by the right side of the image (1,
2, 3, and 4) correspond to full length CCR5, 72nt acceptor, DNA splint, and
24nt donor fragment, respectively. Typically, excess amounts of the acceptor
fragment and the DNA splint were applied and the ligation yield was >60%
based on the conversion of the donor fragment which, in most cases, was
prepared with different isotope labels.
Based on previous mutational studies that suggested Stem 2 of
CCR5 pseudoknot motif interacts with miR-1224,[4] the RNA was
split into two fragments such that a 24 nt donor fragment
containing eight adenosine residues spans the proposed
interacting sequence (Figure 1a). The donor fragments bearing
(8,1′-13C)-modified adenosine at each of five positions were
synthesized using RNA solid-phase synthesis (Supplementary
Table 1 & Supplementary Figure 2). Unlabeled 2′-O-TBDMS-
protected nucleoside phosphoramidite building blocks were first
utilized to optimize the conditions for synthesizing the 24 nt
CCR5 donor fragments. A prolonged coupling time (6 min) was
employed to guarantee yields greater than 98%. Next, the
synthesized 2′-O-TBDMS-(8,1′-13C)-adenosine phosphoramidite
was introduced into the donor fragments at different locations of
the RNA in combination with three other unlabeled 2′-O-TBDMS-
protected nucleoside phosphoramidite building blocks. After
successful incorporation of the (8,1′-13C)-adenosine label, we
The residue/site-specific insertion of 13C probes into CCR5 RNA
was confirmed by NMR spectroscopy. 2D H-13C heteronuclear
1
multiple quantum coherence (HMQC) spectra obtained for the
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The last adenosine A88 within the donor fragment region was
exclusively determined (Supplementary Figure 3). The eight
adenosine ribose anomeric atoms in the donor fragment of the
ligated CCR5 RNA were well-resolved in the 2D HMQC
spectrum (Supplementary Figure 3b).
ligated and residue/site-specific labeled CCR5 RNA in the
absence of miR-1224 exhibited
a
single cross peak
corresponding to either ribose C1′ or base C8 of the residue A95
(Figure 2a & 2b). Unambiguous assignments of A95 C1′/H1′
was readily achieved by overlaying the ribose C1′ HMQC
spectrum with that of the CCR5 RNA containing all seventeen
adenosine residues prepared from in vitro transcription with
atom-specifically labeled (8,1′-13C)-ATP and unlabeled
CTP/GTP/UTP (Figure 2c). Continued incorporation of 13C
probes at different positions of the RNA resulted in identification
of ribose C1′/H1′ atoms of A76, A79, A85, and A90 (Figure 2d &
2e) and base C8/H8 atoms of these residues (Supplementary
Fig. 3a & 3b).
In sum, this labeling strategy provides a number of advantages.
First, use of isotopic probes residue/site-specifically helps to
resolve substantial ambiguities in resonance assignments of
RNAs of this size. Second, these labels are effective and reliable
for quickly assigning large (>60 nt) RNAs. This represents an
important advance for RNA structural studies by NMR
spectroscopy, currently stuck at a median size of 25nt in the
PDB. Third, these labels remove complicated scalar coupling
effects or dipolar interactions with adjacent nuclei, thereby
providing an ideal platform for performing, especially, CPMG
RD experiments to investigate CCR5 conformational exchanges
[13b,15]
on micro- to millisecond timescales.
;
other RD
experiments such as R1 and CEST will also benefit from this
ρ
labeling pattern[8b,f,g]
.
Figure 2 Heteronuclear multiple quantum coherence (HMQC) spectra of the
CCR5 pseudoknot element. (a) Ribose C1′ and (b) base C8 HMQC spectra of
ligated CCR5 RNA bearing site-specific (8,1′)-13C-labeled probes at residue
A95. The donor fragment was prepared by solid phase synthesis on a DNA
synthesizer first using unlabeled RNA amidites followed by incorporation of a
selective (8, 1′)-13C-labeled adenosine phosphoramidite at different positions.
Upon ligation to the unlabeled acceptor fragment, the resulting ligated full
length CCR5 pseudoknot element afforded a single peak at either ribose C1′
or base C8 region. The projected 1D 1H spectrum in (a) showed a single peak
across a broader H1′ region, demonstrating the success in isotope labeling of
a specific residue of the full length 96nt RNA. (c) Overlay of ribose HMQC
spectra obtained from two CCR5 RNA pseudoknot samples. The spectrum in
blue was recorded for a full length CCR5 RNA with all 17 adenosine residues
labeled selectively with (8,1′)-13C atoms; while the other in red was from the
ligated CCR5_A95 RNA. (d) Ligation between the unlabeled acceptor and four
different residue-specific labeled donor fragments resulted in resonance
assignments of four additional residues: A90, A85, A79, and A76 at both
ribose C1′ and base C8 regions. The spectrum in blue is the same as that in
(c). This method allowed for straightforward resonance assignments and
provided staring points for assignment of the neighboring residues based on
2D/3D NOESY experiments. (e) Secondary structure of CCR5 pseudoknot
RNA based on previous SHAPE experiments.[4]
Figure
3
Chemical exchange in CCR5 revealed by 13C CPMG RD
experiments. (a) A 2D 1H–13C HMQC spectrum of a ligated CCR5 RNA
sample labeled only with site-specific (8,1′-13C)-adenosines within the donor
fragment. This was recorded in the absence of miR-1224, at 298 K, at 600
MHz proton Larmor frequency, with Trelax = 0 ms. The expected eight anomeric
H-C cross-peaks are well-resolved. (b) 13C CPMG RD profiles of the ligated
CCR5 RNA at 150 MHz carbon Larmor frequency. Red dots represent
experimental data and black ones represent repeated experiments. Solid lines
are best RD curve fittings of the CPMG profiles. Residues A75, A76, A88, A90,
and A95 show non-flat dispersion profiles while A85 and A96 show flat
dispersion profiles. Note that A79′s resonance was too broad to fit. (c) 13C
CPMG RD profiles of A90 ribose anomeric carbon of the ligated CCR5 RNA at
150 (red dots) and 200 MHz (blue dots) carbon Larmor frequency. Black
circles represent repeated experiments and solid lines are the best fits of the
CPMG profiles. (d) NMR titration of the ligated CCR5_A90 RNA with miR-
1224. The 2D HMQC spectrum in magenta recorded in the absence of miR-
1224 exhibits a single cross peak corresponding to ribose C1′ of the residue
A90. Upon addition of miR-1224, the ribose A90-C1′ chemical shift changed
dramatically from 90.3 ppm to 88.3 ppm (in cyan). The large chemical shift
change (up to 2.0 ppm) indicates that the interaction with miR-1224 alters the
local chemical environment of the A90 residues. In the presence of an
equimolar amount of miR-1224, the CCR5 RNA is not completely converted to
its bound form, with ~9% remaining in the free conformation. This is consistent
To distinguish the three remaining adenosine residues in the
donor fragment, we used conventional NMR techniques and
prepared a CCR5 RNA sample by ligating the unlabeled
acceptor fragment and a donor fragment labeled only with eight
site-specific (8,1′-13C)-adenosines. A 2D edited-edited NOESY
spectrum obtained for the RNA sample showed inter-residue
cross-peaks between A75H1′ and A76H8, and between A95H1′
and A96H8, allowing straightforward and unambiguous
assignments of residues A75 and A96 (Supplementary Fig. 3).
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with the EMSA result (Supplementary Figure 6). However, adding 20% extra
amount of miR-1224 converts it to the completely bound form (data not shown).
pairs or is structurally constrained to a fixed conformation. The
presence of miR-1224 triggered a highly specific interaction,
flipping A90 out to participate in an intermolecular interaction,
possibly base stacking with the miR-1224 bases but not base
pairing as indicated by its up field chemical shift value (δC 88.3
ppm). The A90-C1′ chemical shift of the minor-populated
invisible state (|Δω|: 0.8 ± 0.1 ppm) likely samples local base-
flipping motions to facilitate intermolecular recognition. The base
C8 and ribose C1′ of CCR5_G89 also showed large chemical
shift changes (data not shown), suggesting that the predicted
internal bulge involving residues G89 and A90 is vital to the
intermolecular interaction with miR-1224. More detailed analysis
using these and other RD measurements will be needed to
clearly delienate the sign of chemical shift changes.
A pseudo-3D 1H–13C HMQC version of CPMG RD experiment
was used to minimize relaxation loses and the effect of magnetic
field inhomogeneity.[16] The eight well-dispersed 13C probes of
the ligated CCR5 RNA in the 2D 1H–13C correlation map with
Trelax = 0 (Figure 3a) allowed for accurate measurements of the
functional dynamics and subsequent extraction of information
about the exchange processes in which the pseudoknot
participates. As a result, the 1′ anomeric carbons of adenosines
A75, A76, A88, A90, and A95 of the ligated CCR5 RNA at 150
MHz carbon Larmor frequency showed non-flat dispersion
profiles while that of A85 and A96 exhibited flat dispersion
profiles (Figure 3b). Unfortunately the signal intensity of A79-C1′
rapidly decreases during the CPMG relaxation delay which
prevents further characterization and analysis. Additionally A76
and A96 appear to have shoulder peaks suggestive of multiple
conformations. More detailed analyses are needed to elucidate
the precise nature of these conformations.
Our NMR data indicate that the secondary structure of the CCR5
RNA pseudoknot is slightly different from that derived from
SHAPE experiments.[4] We are continuing with resonance
assignment using this strategy to obtain a more accurate
secondary structure of the CCR5 RNA pseudoknot using NMR
spectroscopy.
CPMG RD experiments were also conducted at 200 MHz carbon
Larmor frequency (magnet field strength of 18.8 T) (Figure 3c &
Supplementary Figure 4). Subsequent global fitting resulted in
extracting kinetic and thermodynamic parameters of the
conformational transition.[13b,17] Initially, we expected to obtain
the same dynamic parameters because the seven probes report
on the same biological exchange process. Surprisingly, fits to a
two-state exchange model resulted in residue-specific variations
in dynamic behaviors: two probes (A85 and A96) showed no
conformational exchange, whereas the other five displayed
different thermodynamic parameters (Table 1). A76 samples a
minor-populated state of PB ≈ 30%, while residues A75, A88,
A90 and A95 sample an invisible-populated state of PB <10%
(Table 1). The exchange rate constants (kex) of A75, A88, and
A90 were very close to one another within experimental error,
whereas the rate parameters of A76 and A95 were roughly one
magnitude higher (Table 1). Longitudinal relaxation rates were
also recorded for these sites. Larger longitudinal relaxation rate
of residues A85 and A96 indicated the presence of rapid local
motion and high flexibility, indicating complicated motions
occuring on various time scales (Supplementary Table 2 and
Supplementary Figure 5). Thus these probes experience local
independent complex motions or conformational exchanges.
In summary, in this study we have expanded the labeling
protocol from previously reported (6,1′-13C) cytidine labels to
(8,1′-13C) base and ribose building blocks for preparing
residue/site-specifically isotopic labeled 96nt CCR5 RNA to
study conformational dynamics.[8f,15a] The strategic combination
of solid-phase synthesis of RNA with labeled phosphoramidites,
enzymatic ligation, and NMR spectroscopy allows investigations
into the structures and dynamics of large RNA systems, than
have been possible until now, with biologically important
functions that are otherwise impeded by severe spectral overlap.
Experimental Section
See details in Supplementary materials.
Acknowledgements
The authors would like to thank Dr. Paul Paukstelis for access to
the DNA synthesizer and help with setup of DNA/RNA synthesis.
This work was funded by grants to JDD from the NIH (R01
GM117177), and to TKD from NSF for NMR instrumentation
(DBI1040158) and NIH (U54GM103297).
A 2D 1H-13C HMQC spectrum of the ligated and residue/site-
specific labeled CCR5_A90 RNA in the absence of miR-1224
exhibited a cross peak corresponding to the ribose C1′/H1′
(Figure 3d). Upon addition of miR-1224, the ribose H1′ chemical
shift was barely perturbed from 5.86 ppm to 5.83 ppm. In
contrast, the ribose C1′ chemical shift was strongly perturbed
from 90.3 ppm to 88.3 ppm (Figure 3d). A similar result was
obtained for the A90-C8/H8 chemical shift changes (data not
shown here). The large chemical shift adjustment (up to 2.0
ppm) unambiguously indicated that miR-1224 binding alters the
local chemical environment of the A90-C1′ atom. It is notable
that the chemical shift (δC 90.3 ppm) of A90-C1′ in the free form
CCR5 pseudoknot suggests that the A90 residue either base-
Keywords: CCR5 mRNA pseudoknot • MicroRNA • NMR
spectroscopy • RNA solid-phase synthesis • Dynamics
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10.1002/chem.201705948
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Table 1 Kinetic and thermodynamic parameters extracted from CPMG RD experiments[a]
Residues (ribose C1')
kex [S-1]
PB [%]
χ2
Δω [ppm]
A75
A76
A88
A90
A95
179 ± 138
1014 ± 188
356 ± 97
8 ± 5
30 ± 12
3 ± 1
0.8 ± 0.1
0.3 ± 0.2
0.6 ± 0.1
0.8 ± 0.1
0.5 ± 0.2
1.08
1.12
0.86
1.46
0.96
165 ± 121
943 ± 197
9 ± 5
6 ± 2
[a]: The CPMG RD experiments were carried out on the ligated CCR5 RNA sample labeled with site-specific (8,1′-13C)-adenosines within the donor fragment in
the absence of miR-1224 at 298 K. The CPMG data of the ribose C1' of all five adenosine residues were fitted together to slow exchange two-state models and
the results were derived from 1000 Monte Carlo runs (Detailed in Supplementary Materials). Note that strictly PB and Δw are not separable in the absence of other
data.
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A highly conserved pseudoknot
element within the mRNA encoding
Author(s), Corresponding Author(s)*
human C-C chemokine receptor type
Page No. – Page No.
5 regulates its own gene’s expression
through a translational recoding event.
A residue/site-specific labeling
Title
strategy was applied to insert isotopic
probes into the pseudoknot. This
approach enables unambiguous
assignments of key residues involved
in intermolecular interaction with
miRNA-1224 and results in accurate
measurements of their dynamics at
atomic resolution.
((Insert TOC Graphic here))
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Bin Chen[a], Andrew P. Longhini[b], Felix
Nußbaumer[c], Christoph Kreutz[c],
Jonathan Dinman[a]*, & T. Kwaku
Dayie[b]*
Residue and site-specific labeling of
CCR5 RNA pseudoknot reveals
internal motions that correlate with
microRNA binding
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