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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Structural Features of a 3′ Splice Site in
Influenza A</title><author><name sortKey="Chen, Jonathan X0a L" sort="Chen, Jonathan X0a L" uniqKey="Chen J" first="Jonathan X0a L." last="Chen">Jonathan X0a L. Chen</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Kennedy, Scott D" sort="Kennedy, Scott D" uniqKey="Kennedy S" first="Scott D." last="Kennedy">Scott D. Kennedy</name><affiliation><nlm:aff id="aff2">Department of Biochemistry and Biophysics,<institution>University of Rochester School of Medicine and Dentistry</institution>, Rochester, New York 14642,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Turner, Douglas H" sort="Turner, Douglas H" uniqKey="Turner D" first="Douglas H." last="Turner">Douglas H. Turner</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Center for RNA Biology,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25909229</idno><idno type="pmc">4455060</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4455060</idno><idno type="RBID">PMC:4455060</idno><idno type="doi">10.1021/acs.biochem.5b00012</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000087</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000087</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Structural Features of a 3′ Splice Site in
Influenza A</title><author><name sortKey="Chen, Jonathan X0a L" sort="Chen, Jonathan X0a L" uniqKey="Chen J" first="Jonathan X0a L." last="Chen">Jonathan X0a L. Chen</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Kennedy, Scott D" sort="Kennedy, Scott D" uniqKey="Kennedy S" first="Scott D." last="Kennedy">Scott D. Kennedy</name><affiliation><nlm:aff id="aff2">Department of Biochemistry and Biophysics,<institution>University of Rochester School of Medicine and Dentistry</institution>, Rochester, New York 14642,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Turner, Douglas H" sort="Turner, Douglas H" uniqKey="Turner D" first="Douglas H." last="Turner">Douglas H. Turner</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Center for RNA Biology,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></nlm:aff></affiliation></author></analytic><series><title level="j">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="bi-2015-000127_0014" id="ab-d31e164"></graphic></p><p>Influenza A is an RNA virus with
a genome of eight negative sense
segments. Segment 7 mRNA contains a 3′ splice site for alternative
splicing to encode the essential M2 protein. On the basis of sequence
alignment and chemical mapping experiments, the secondary structure
surrounding the 3′ splice site has an internal loop, adenine
bulge, and hairpin loop when it is in the hairpin conformation that
exposes the 3′ splice site. We report structural features of
a three-dimensional model of the hairpin derived from nuclear magnetic
resonance spectra and simulated annealing with restrained molecular
dynamics. Additional insight was provided by modeling based on <sup>1</sup>H chemical shifts. The internal loop containing the 3′
splice site has a dynamic guanosine and a stable imino (<italic>cis</italic> Watson–Crick/Watson–Crick) GA pair. The adenine bulge
also appears to be dynamic with the A either stacked in the stem or
forming a base triple with a Watson–Crick GC pair. The hairpin
loop is a GAAA tetraloop closed by an AC pair.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Thompson, W X0a W" uniqKey="Thompson W">W.
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<front><journal-meta><journal-id journal-id-type="nlm-ta">Biochemistry</journal-id><journal-id journal-id-type="iso-abbrev">Biochemistry</journal-id><journal-id journal-id-type="publisher-id">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American
Chemical Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25909229</article-id><article-id pub-id-type="pmc">4455060</article-id><article-id pub-id-type="doi">10.1021/acs.biochem.5b00012</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>Structural Features of a 3′ Splice Site in
Influenza A</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Chen</surname><given-names>Jonathan
L.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Kennedy</surname><given-names>Scott D.</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath3"><name><surname>Turner</surname><given-names>Douglas H.</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff3" ref-type="aff">§</xref></contrib><aff id="aff1"><label>†</label>Department of Chemistry,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></aff><aff id="aff2"><label>‡</label>Department of Biochemistry and Biophysics,<institution>University of Rochester School of Medicine and Dentistry</institution>, Rochester, New York 14642,<country>United States</country></aff><aff id="aff3"><label>§</label>Center for RNA Biology,<institution>University of Rochester</institution>, Rochester, New York 14627,<country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>turner@chem.rochester.edu</email>. Phone: <phone>(585)
275-3207</phone>. Fax: <fax>(585) 276-0205</fax>.</corresp></author-notes><pub-date pub-type="epub"><day>24</day><month>04</month><year>2015</year></pub-date><pub-date pub-type="ppub"><day>02</day><month>06</month><year>2015</year></pub-date><volume>54</volume><issue>21</issue><fpage>3269</fpage><lpage>3285</lpage><history><date date-type="received"><day>05</day><month>01</month><year>2015</year></date><date date-type="rev-recd"><day>21</day><month>04</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015 American Chemical
Society</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>American Chemical
Society</copyright-holder><license><license-p>This is an open access article published under an ACS AuthorChoice <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_termsofuse.html">License</ext-link>, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="bi-2015-000127_0014" id="ab-d31e164"></graphic></p><p>Influenza A is an RNA virus with
a genome of eight negative sense
segments. Segment 7 mRNA contains a 3′ splice site for alternative
splicing to encode the essential M2 protein. On the basis of sequence
alignment and chemical mapping experiments, the secondary structure
surrounding the 3′ splice site has an internal loop, adenine
bulge, and hairpin loop when it is in the hairpin conformation that
exposes the 3′ splice site. We report structural features of
a three-dimensional model of the hairpin derived from nuclear magnetic
resonance spectra and simulated annealing with restrained molecular
dynamics. Additional insight was provided by modeling based on <sup>1</sup>H chemical shifts. The internal loop containing the 3′
splice site has a dynamic guanosine and a stable imino (<italic>cis</italic> Watson–Crick/Watson–Crick) GA pair. The adenine bulge
also appears to be dynamic with the A either stacked in the stem or
forming a base triple with a Watson–Crick GC pair. The hairpin
loop is a GAAA tetraloop closed by an AC pair.</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi5b00012</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>bi-2015-000127</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id="sec1">Influenza
virus infections annually
contribute to 3300–49000 deaths<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> and more than 200000 hospitalizations in the United States.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> The largest influenza pandemic, known as the
Spanish flu (H1N1, 1918–1919), killed as many as 40–50
million people worldwide.<sup><xref ref-type="bibr" rid="ref3">3</xref></sup> Lesser pandemics
consist of the Asian (H2N2, 1957), Hong Kong (H3N2, 1968), and Russian
(H1N1, 1977) flus.<sup><xref ref-type="bibr" rid="ref3">3</xref></sup> Available drugs are
neuraminidase inhibitors and M2 ion channel blockers (adamantanes).<sup><xref ref-type="bibr" rid="ref4">4</xref></sup> However, the emergence of influenza strains with
resistance to both classes of drugs, especially neuraminidase inhibitors,<sup><xref ref-type="bibr" rid="ref5">5</xref>,<xref ref-type="bibr" rid="ref6">6</xref></sup> has led to interest in identifying new antiviral therapeutics.<sup><xref ref-type="bibr" rid="ref7">7</xref></sup> Antiviral agents may selectively target viral
RNA structure with small molecules,<sup><xref ref-type="bibr" rid="ref8">8</xref>−<xref ref-type="bibr" rid="ref10">10</xref></sup> oligonucleotides,<sup><xref ref-type="bibr" rid="ref11">11</xref></sup> or synthetic peptides.<sup><xref ref-type="bibr" rid="ref12">12</xref></sup></p><p>The influenza A genome consists of eight segments of negative
sense
vRNA, which encode at least 11 proteins.<sup><xref ref-type="bibr" rid="ref13">13</xref></sup> A pandemic of influenza occurs when RNA segments of human and animal
viruses reassort to give rise to new strains to which humans have
no immunity.<sup><xref ref-type="bibr" rid="ref13">13</xref></sup> The extreme ends of each
segment are highly conserved and base pair to form a promoter for
RNA synthesis.<sup><xref ref-type="bibr" rid="ref13">13</xref></sup></p><p>With bioinformatics
approaches, Moss et al.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> identified conserved
and stably folded secondary structures
of influenza A mRNAs. Three of the secondary structures have been
confirmed by chemical mapping,<sup><xref ref-type="bibr" rid="ref15">15</xref>,<xref ref-type="bibr" rid="ref16">16</xref></sup> and a fourth was found
to fold into a hairpin rather than a predicted multibranch loop.<sup><xref ref-type="bibr" rid="ref17">17</xref></sup> Two of the conserved secondary structures contain
the 3′ splice site of segment 7 mRNA. Segment 7 encodes the
essential M1 and M2 and/or M42 proteins.<sup><xref ref-type="bibr" rid="ref18">18</xref>,<xref ref-type="bibr" rid="ref19">19</xref></sup> An equilibrium between a two-hairpin folding and a pseudoknot folding
may regulate expression of M1 and M2 and/or M42<sup><xref ref-type="bibr" rid="ref15">15</xref>,<xref ref-type="bibr" rid="ref19">19</xref></sup> (Figure <xref rid="fig1" ref-type="fig">1</xref>). For example, the equilibrium
populations of these conformations may depend on factors in the cellular
environment such as pH, protein binding, or the presence of metabolites.
In chemical mapping experiments on the two-hairpin model of the 3′
splice site of segment 7 mRNA (Figure <xref rid="fig1" ref-type="fig">1</xref>), the
smaller hairpin, 14 nucleotides (nt), is dynamic on the basis of high
reactivity to enzymes and small molecules.<sup><xref ref-type="bibr" rid="ref15">15</xref></sup> Herein, we report the NMR structure of the consensus sequence of
the larger hairpin, 37 nt, containing the 3′ splice site (Figure <xref rid="fig1" ref-type="fig">1</xref>).</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Secondary structures of constructs of the 3′ splice
site
region of segment 7 mRNA. A red arrowhead denotes the splice site.
(a) Pseudoknot and hairpin conformations from ref (<xref ref-type="bibr" rid="ref15">15</xref>). The SF2/ASF exonic splicing
enhancer binding site is colored green and a polypyrimidine tract
blue.<sup><xref ref-type="bibr" rid="ref15">15</xref></sup> Numbers in brackets correspond
to numbering of residues of the 39 nt hairpin studied with NMR. (b)
The 39 nt hairpin studied with NMR. (c) The 11 nt hairpin mimic. (d)
The 19 nt duplex model containing the 2 nt × 2 nt internal loop.
The U14 residue in the 39-mer was substituted with a cytidine to stabilize
formation of the target heterodimer over a homodimer.</p></caption><graphic xlink:href="bi-2015-000127_0002" id="gr1" position="float"></graphic></fig><sec id="sec2"><title>Experimental Methods</title><sec id="sec2.1"><title>Preparation of 39 nt Hairpin
Samples</title><p>Milligram quantities
of a 39 nt construct containing the segment 7 hairpin (Figure <xref rid="fig1" ref-type="fig">1</xref>) were prepared with <italic>in vitro</italic> transcription
by T7 RNA polymerase.<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> T7 RNA polymerase
was synthesized from a plasmid supplied by B. S. Tolbert (Case Western
Reserve University, Cleveland, OH) and purified via nickel column
affinity chromatography.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> A pUC18 plasmid
containing an insert for the RNA sequence was constructed and purified
from <italic>Escherichia coli</italic> competent cells with standard
plasmid preparation protocols (see the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref> for the plasmid construct). The 5′ tail
of the hairpin was replaced with a 5′ GG dinucleotide to enhance
the efficiency of transcription initiation.<sup><xref ref-type="bibr" rid="ref22">22</xref></sup> The plasmid was linearized with EcoRV-HF restriction endonuclease
(New England BioLabs) at 37 °C prior to <italic>in vitro</italic> transcription. Transcription mixtures typically consisted of 25
mM Mg<sup>2+</sup>, 1 mg/mL DNA template, rNTPs (12–13 mM each),
40 mM DTT, and 0.65–0.70 mg/mL T7 RNA polymerase at pH 7.5–8.0.
After transcription mixtures had been incubated for 2 h at 37 °C,
2.5 μL of 0.5 M EDTA and 6 μL of 50% glycerol were added
for every 30 μL of transcription mixture to stop reactions.
Transcription mixtures were purified via FPLC using three 5 mL HiTrap
DEAE Sepharose FF columns (GE Healthcare) connected in series.<sup><xref ref-type="bibr" rid="ref23">23</xref></sup> FPLC fractions with purified RNA were concentrated
and then exchanged with an Amicon Ultra-15 Centrifugal Filter Unit
(EMD Millipore) into NMR buffer [80 mM KCl, 20 mM KH<sub>2</sub>PO<sub>4</sub>/K<sub>2</sub>HPO<sub>4</sub>, and 0.02 mM Na<sub>2</sub>EDTA
(pH 6.0)] to yield 3.8 mg of RNA. The final NMR sample had 1.1 mM
RNA in 300 μL, including 15 μL of D<sub>2</sub>O to provide
a lock signal.</p><p>A second sample of the 39 nt hairpin was synthesized
from the linearized plasmid template with T7 High Yield RNA Synthesis
Kits (New England BioLabs). This sample was initially purified via
FPLC and concentrated, as described above. The second sample was further
purified via denaturing polyacrylamide gel electrophoresis, extracted
from gels via electroelution, concentrated, and exchanged into NMR
buffer.<sup><xref ref-type="bibr" rid="ref24">24</xref></sup> The final NMR sample had 1.4
mM RNA in 400 μL of NMR buffer, including 15 μL of D<sub>2</sub>O. NMR spectra for the two samples were essentially identical.</p></sec><sec id="sec2.2"><title>Design of 11 nt Hairpin and 19 nt Duplex Model Mimics</title><p>The
relatively large size of the 39 nt hairpin resulted in spectral
overlap of resonances that made resonance assignments difficult. To
aid resonance assignments, two smaller model constructs were assembled:
(1) a 19 nt duplex RNA corresponding to the 2 nt × 2 nt internal
loop region and (2) an 11 nt RNA corresponding to the hairpin loop
region (Figure <xref rid="fig1" ref-type="fig">1</xref>). In the 19-mer duplex, the
residue that corresponds to U14 of the 39-mer was substituted with
cytidine to stabilize formation of the intended heterodimer over a
homodimer (Figure S1 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). The 11 nt hairpin and the bottom strand of the 19 nt duplex each
have a 3′ dangling nucleotide to stabilize their helices.<sup><xref ref-type="bibr" rid="ref25">25</xref>,<xref ref-type="bibr" rid="ref26">26</xref></sup></p></sec><sec id="sec2.3"><title>Preparation of Model Mimic Samples</title><p>Oligoribonucleotides
were purchased from Integrated DNA Technologies, Inc. (IDT), and dissolved
in 315 μL of NMR buffer, including 15 μL of D<sub>2</sub>O. The highest concentration of r(5′CCAGAAACGGA)
was 4.0 mM. The final concentration of the duplex, r(5′GCAGGCCCA)
+ r(5′UGGGAGUGCA), was
1.0 mM. MgCl<sub>2</sub> was added to each sample to a final concentration
of 5 mM.</p></sec><sec id="sec2.4"><title>NMR Spectroscopy</title><p>NMR spectra of samples in Shigemi
NMR tubes (Shigemi, Inc.) were acquired on Varian Inova 500 and 600
MHz spectrometers. For samples in H<sub>2</sub>O, one-dimensional
spectra were recorded for all constructs at a series of temperatures,
with a 1–1–echo pulse to suppress the water signal.<sup><xref ref-type="bibr" rid="ref27">27</xref></sup> For 2D spectra, a WATERGATE pulse with flipback<sup><xref ref-type="bibr" rid="ref28">28</xref>,<xref ref-type="bibr" rid="ref29">29</xref></sup> or an S-pulse<sup><xref ref-type="bibr" rid="ref30">30</xref></sup> was applied during acquisition
to suppress the water signal. 2D NOESY spectra with different mixing
times were acquired at −2 and 20 °C for all constructs
and at additional temperatures for the two smaller constructs. 2D
TOCSY spectra were acquired with mixing times between 30 and 50 ms
for all constructs. Imino chemical exchange peaks of the 39-mer were
detected with a 2D ROESY experiment, where their sign is the opposite
of that of peaks arising from direct cross-relaxation.<sup><xref ref-type="bibr" rid="ref31">31</xref></sup> 2D NOESY spectra<sup><xref ref-type="bibr" rid="ref32">32</xref></sup> were acquired on the smaller constructs in D<sub>2</sub>O at a series
of temperatures to overcome ambiguities due to overlaps.</p><p>Proton
chemical shifts were referenced internally to the frequency of water,
with 2,2-dimethylsilapentane-5-sulfonic acid (DSS) as the external
reference standard, and carbon chemical shifts were referenced indirectly
to DSS on the basis of the absolute proton frequency according to
Biological Magnetic Resonance Data Bank (BMRB) standards.<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref33">33</xref></sup> 2D NMR spectra were processed with NMRPipe.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup></p></sec></sec><sec id="sec3"><title>Modeling Methods</title><sec id="sec3.1"><title>NMR Spectra for Obtaining Restraints</title><p>Resonances were
assigned with standard procedures using <sup>1</sup>H–<sup>1</sup>H NOESY, <sup>1</sup>H–<sup>1</sup>H TOCSY, <sup>1</sup>H–<sup>13</sup>C HSQC, and <sup>1</sup>H–<sup>31</sup>P HETCOR spectra and SPARKY.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> Distance
restraints were generated from spectra with mixing times between 50
and 150 ms, to minimize contributions from spin diffusion.<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref36">36</xref></sup></p></sec><sec id="sec3.2"><title>Methods for Obtaining NOE Restraints</title><p>Most distance
restraints for pairs of hydrogen atoms were obtained by integrating
NOE volumes with SPARKY.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> Some that were
difficult to integrate were manually assigned to a range of distances
based on the relative size of their NOEs. NOE volumes were converted
to distance restraints by referencing to volumes from fixed distances:
H2′–H1′ (2.75 Å), H4′–H1′
(3.35 Å), pyrimidine H5–H6 (2.45 Å), cytosine H42–H41
(1.75 Å), cytosine H41–guanine H1 in a CG pair (2.70 Å),
and adenine H2–uracil H3 in an AU pair (2.85 Å). Hydrogen
bonds between bases were restrained to 2.1 ± 0.3 Å for all
canonical base pairs. H1′–H2′ scalar coupling
information was used to restrain all canonically base paired residues
to the C3′-endo conformation. The χ dihedral angle was
held between 170° and 340° (<italic>anti</italic>) for all
residues except G10, where NMR evidence indicates that it is flexible.</p><p>In general, if an NOE in equivalent chemical environments is present
in spectra of the 39 nt hairpin and either smaller construct, a distance
restraint was obtained from the smaller construct to reduce complications
of peak overlap. Because of different chemical environments, NOEs
from the 39 nt hairpin were used for distance restraints rather than
terminal residues G7, A15, U27, and A36 in the 19 nt duplex and C16
and A26 in the 11 nt hairpin. Intraresidue NOE volumes from those
residues, however, were used to calculate reference distances for
the 19 nt duplex and 11 nt hairpin. Restraints were also not obtained
from C14 in the 19-mer duplex because the C14-G28 pair is structurally
different from the U14-G28 pair in the 39-mer. Restraints for the
11-mer and 19-mer duplex constructs were obtained from spectra acquired
with Mg<sup>2+</sup> present because they generally had narrower and
better-resolved cross-peaks that could be more accurately integrated.</p></sec><sec id="sec3.3"><title>Structure Calculation</title><p>Structures were refined with
a simulated annealing<sup><xref ref-type="bibr" rid="ref37">37</xref>,<xref ref-type="bibr" rid="ref38">38</xref></sup> protocol on a starting structure
built with NUCGEN.<sup><xref ref-type="bibr" rid="ref39">39</xref></sup> Solvent was simulated
with the generalized Born implicit solvent model and 0.1 M NaCl.<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> The system was heated from 0 to 3000 K in 5000
steps for 5 ps and cooled to 100 K in 93000 steps for 93 ps and then
to 0 K in 2000 steps for 2 ps. Force constants were 12 kcal mol<sup>–1</sup> Å<sup>–2</sup> for NOE restraints and
12 kcal mol<sup>–1</sup> rad<sup>–2</sup> for dihedral
angle restraints. The weight of the restraints was increased from
0.1 to 1 during the first 3000 steps, i.e., during heating, and held
at 1 for the remainder of the simulation. These restrained molecular
dynamics calculations were conducted with AMBER 14<sup><xref ref-type="bibr" rid="ref41">41</xref></sup> using the parm99χ_YIL force field.<sup><xref ref-type="bibr" rid="ref42">42</xref></sup> The simulated annealing procedure was repeated with different
initial velocities to generate an ensemble of 200 structures. The
22 structures without violations along with the eight structures with
the lowest distance restraint violation energies and violations between
0.1 and 0.2 Å were refined with the same simulated annealing
protocol except that they were heated to 600 K. Force constants for
refinement were 30 kcal mol<sup>–1</sup> Å<sup>–2</sup> for NOE restraints and 30 kcal mol<sup>–1</sup> rad<sup>–2</sup> for dihedral angle restraints. The 20 structures with the lowest
distance restraint violation energies that also agreed with NMR experimental
restraints were selected as a final ensemble of structures. Similar
structure minimization and refinement protocols were followed for
the 19 nt duplex and 11 nt hairpin. Rmsds of the ensemble of structures
were calculated with VMD.<sup><xref ref-type="bibr" rid="ref43">43</xref></sup> Images of 3D
models of the RNA were generated with PyMOL.</p></sec><sec id="sec3.4"><title>Relationship between Chemical
Shifts and Structure</title><p>Chemical shifts can provide structural
information about RNA.<sup><xref ref-type="bibr" rid="ref44">44</xref></sup> Programs have
been developed to predict 3D structure
from chemical shifts<sup><xref ref-type="bibr" rid="ref45">45</xref>,<xref ref-type="bibr" rid="ref46">46</xref></sup> or chemical shifts from a model
structure.<sup><xref ref-type="bibr" rid="ref47">47</xref>,<xref ref-type="bibr" rid="ref48">48</xref></sup> For RNA, nonexchangeable <sup>1</sup>H chemical shifts calculated with programs such as NUCHEMICS<sup><xref ref-type="bibr" rid="ref47">47</xref></sup> and RNAShifts<sup><xref ref-type="bibr" rid="ref49">49</xref></sup> agreed
well with experiments.<sup><xref ref-type="bibr" rid="ref49">49</xref>,<xref ref-type="bibr" rid="ref50">50</xref></sup></p><p>The ROSETTA software suite<sup><xref ref-type="bibr" rid="ref46">46</xref>,<xref ref-type="bibr" rid="ref51">51</xref></sup> on the ROSIE server<sup><xref ref-type="bibr" rid="ref52">52</xref></sup> was used to model
separately two fragments of the 39 nt hairpin: a 19 nt hairpin containing
residues 12–30 with the hairpin loop and A26 bulge and an 18
nt duplex containing residues 7–15 and 27–35 with the
internal loop. Input files for each computation consisted of the RNA
sequence, a predicted secondary structure, and a set of assigned chemical
shifts (Table S1 and Figure S2 of the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref>). Chemical shifts of nonexchangeable protons of
the 39 and 11 nt hairpins and the 19 nt duplex were also calculated
with NUCHEMICS for ensembles of 20 minimized 3D structures restrained
by NOEs. For comparison with experiment, the chemical shift of each
hydrogen was averaged among the 20 minimized structures for each construct.</p></sec></sec><sec sec-type="results" id="sec4"><title>Results</title><sec id="sec4.1"><title>The Secondary Structure from NMR Is That Predicted from Bioinformatics</title><p>NMR spectra of the 39 nt hairpin confirm the helices predicted
by bioinformatics and supported by chemical mapping (Figures <xref rid="fig2" ref-type="fig">2</xref> and <xref rid="fig3" ref-type="fig">3</xref>).<sup><xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref15">15</xref></sup> Resonance assignments of exchangeable protons in the helices of
the 39-mer commenced with identifying signature GH1–UH3 cross-peaks
of GU pairs in the 10–12 ppm region of NOESY spectra at −2
°C (Figure <xref rid="fig2" ref-type="fig">2</xref>).<sup><xref ref-type="bibr" rid="ref53">53</xref></sup> Imino resonances corresponding to helices U4-A38 to C8-G34 and C12-G30
to U14-G28 were assigned from these cross-peaks according to the imino
proton chemical shifts of AU and GC pairs (Figure <xref rid="fig2" ref-type="fig">2</xref> and Table S2 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>).<sup><xref ref-type="bibr" rid="ref53">53</xref></sup> A table of all assigned chemical
shifts is given in the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>.</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Imino proton region of 1D and 2D proton NMR spectra of the 39 nt
construct showing sequential proton walks with blue and green lines.
The water signal was suppressed with a 1–1–echo pulse
in the 1D spectrum and an S-pulse in the 2D NOESY spectrum. The daggers
in the 1D spectrum mark chemical exchange peaks. The spectra were
acquired at −2 °C with a mixing time of 125 ms for the
2D spectrum. Addition of 5 and 10 mM Mg<sup>2+</sup> caused minor
shifts and sharpening of the imino resonances, including U4, U5, U6,
G11, G19, G24, and U33 (Figure S3 of the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref>).</p></caption><graphic xlink:href="bi-2015-000127_0003" id="gr2" position="float"></graphic></fig><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Schematic of the secondary structure of the 39 nt hairpin with
assigned interresidue NOEs. Blue lines denote NOEs identified in the
39-mer and the 19 nt duplex, green lines NOEs identified in the 39-mer
and the 11 nt hairpin, and red lines NOEs identified only in the 39-mer.</p></caption><graphic xlink:href="bi-2015-000127_0004" id="gr3" position="float"></graphic></fig><p>A cross-peak between resonances
at 12.41 and 12.69 ppm in a NOESY
spectrum at −2 °C was determined from a ROESY spectrum
to be a chemical exchange peak for G34H1. The primary G34H1 peak lies
at 12.69 ppm. The secondary peak overlaps with G7H1 (Figure <xref rid="fig2" ref-type="fig">2</xref>). The A15H2–U27H3 cross-peak is weak because
of solvent exchange (Figure S4 of the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref>). The G24H1 peak was assigned to 11.92 ppm in the
−2 °C NOESY spectrum based on cross-peaks to C17 amino
protons (Figure S4 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>) but is missing from the 20 °C NOESY spectrum. The G25H1 peak
was assigned to 12.41 ppm in the 20 °C NOESY spectrum on the
basis of cross-peaks to C16 amino protons but could not be identified
in the −2 °C NOESY spectrum. These assignments indicate
that C16 and G25, in addition to C17 and G24, form Watson–Crick
base pairs, though an NOE between G24H1 and G25H1 could not be identified.</p><p>The U5H3 resonance is overlapped with the G24H1 peak. U27H3 appears
as a shoulder to the G7/G34 peak. Resonances corresponding to G11H1
and G19H1 were identified at 13.0–13.1 and 10.4–10.5
ppm, respectively (<italic>vide infra</italic>). The resonance at
10.8 ppm was assigned to G10H1 and/or G32H1, and their presence indicates
that they are at least partially protected from solvent exchange.<sup><xref ref-type="bibr" rid="ref54">54</xref></sup> Only the solvent-exposed G1 and G2 could not
be located in any imino proton spectrum.</p><p>The imino proton NOEs
(Figure <xref rid="fig2" ref-type="fig">2</xref>) are consistent
with the predicted secondary structure (Figure <xref rid="fig1" ref-type="fig">1</xref>). Thus, hydrogen bonding restraints for each type of canonical base
pair predicted in the secondary structure were applied during structure
modeling.</p><p>Assignments of nonexchangeable protons in the 39-mer
(Table S2
of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>) were facilitated
by spectra of the smaller constructs (Figure <xref rid="fig3" ref-type="fig">3</xref>), and there was a high degree of correlation between the final chemical
shifts (Figure <xref rid="fig4" ref-type="fig">4</xref> and Tables S3 and S4 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). Assignments were initiated
by identifying pyrimidine H5–H6 cross-peaks from TOCSY spectra.
Cytosine H5 and H6 peaks were assigned by intraresidue NOE cross-peaks
to amino protons and from those to associated guanosine imino protons.
Intraresidue and interresidue H1′–H6/H8 NOEs from G2
to A9 and from U33 to U39 confirm the A-form geometry of the stem
below the internal loop in the constructs (Figure S5 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>).<sup><xref ref-type="bibr" rid="ref53">53</xref></sup> The same types of H1′–H6/H8 NOEs from C12 to C17,
from G24 to G25, and from U27 to G30 confirm the A-form geometry of
the stem above the internal loop. Cross-peaks from an adenine H2 to
the 3′ H1′ on the same strand and the 3′ H1′
on the opposite strand also identify A-form helical regions of the
constructs.<sup><xref ref-type="bibr" rid="ref55">55</xref></sup> Specifically, NOEs are present
from A3H2 to U4H1′, A9H2 to G34H1′, A15H2 to C16H1′
and U27H1′, A36H2 to G7H1′ and G37H1′, and A38H2
to U5H1′ and U39H1′. In RNA helices, a similar pattern
of NOEs from guanine H1 to the 3′ H1′ on the same strand
and the 3′ H1′ on the opposite strand is present (Figure
S4 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>)<sup><xref ref-type="bibr" rid="ref56">56</xref></sup> and was used to confirm H1′ assignments
of residues such as U6, A38, U14, and G29, near GC and GU pairs in
the 39-mer.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>Differences in chemical shifts between the 39 nt hairpin and 19
nt duplex (residues 7–15 and 27–36) and between the
39 nt hairpin and 11 nt hairpin (residues 16–26) for select
nonexchangeable aromatic and sugar protons. Chemical shift data were
obtained from spectra at 20 °C for the 39-mer and 11-mer and
at 25 °C for the 19-mer. Spectra for the 11-mer and 19-mer were
acquired with 5 mM Mg<sup>2+</sup>. Bars colored with light shades
belong to terminal helix residues of the 11-mer (residues 16 and 26)
and 19-mer (residues 7, 15, 27, and 36) that are not at the termini
of any helices of the 39-mer and thus are in structurally inequivalent
regions among the constructs. Residue numbers on the <italic>x</italic>-axis align with the middle of each set of two bars in each plot.
Residue 14 is not included because of a U to C substitution.</p></caption><graphic xlink:href="bi-2015-000127_0005" id="gr4" position="float"></graphic></fig></sec><sec id="sec4.2"><title>The Hairpin Loop Is a GAAA
Tetraloop Closed with an AC Pair</title><p>Thermodynamic calculations
with the nearest neighbor model<sup><xref ref-type="bibr" rid="ref57">57</xref>,<xref ref-type="bibr" rid="ref58">58</xref></sup> predicted that the
11 nt construct would form a hairpin rather than
a duplex (Figure S6 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). To check the prediction, 1D imino proton NMR spectra were recorded
at 0.2 and 4 mM at 2 °C (Figure S7 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>).<sup><xref ref-type="bibr" rid="ref59">59</xref></sup> The
number of chemical shifts and resonances and their relative intensities
were similar. Therefore, the sequence forms a hairpin at a strand
concentration of 4 mM used to measure NOEs for modeling.</p><p>In
the 39 and 11 nt hairpins, G19H1 was assigned to 10.46 and 10.43 ppm,
respectively, consistent with a sheared GA (<italic>trans</italic> Hoogsteen/sugar edge) pair.<sup><xref ref-type="bibr" rid="ref60">60</xref>−<xref ref-type="bibr" rid="ref62">62</xref></sup> Moreover, NOEs from G19 imino
and amino protons to A22H8 in the 39-mer and 11-mer are consistent
with G19N2 and A22N7 being close in a sheared GA-like conformation
in a GNRA tetraloop.<sup><xref ref-type="bibr" rid="ref63">63</xref></sup></p><p>A sequential
walk consisting of H1′–H6/H8 correlations
was completed from C17 to C23 in the 39-mer (Figures <xref rid="fig3" ref-type="fig">3</xref> and <xref rid="fig5" ref-type="fig">5</xref>) and 11-mer (Figure <xref rid="fig6" ref-type="fig">6</xref>). Intraresidue H1′–H6/H8 NOEs for
C17 to A22 were of typical intensity for bases in the <italic>anti</italic> orientation. An NOE between G19H2′ and A21H8 indicates that
A21 lies inside the loop. Cross-peaks from A21H1′ to A20H2,
from A22H1′ to A21H2, and from C23H1′ to A22H2 in the
11-mer agree with the expected H2<sub><italic>i</italic></sub>–H1′<sub><italic>i</italic>+1</sub> pattern of NOE interactions for stacked
bases.<sup><xref ref-type="bibr" rid="ref55">55</xref></sup> In spectra of the 11-mer, H1′
of C23 (4.85 ppm at 20 °C) is relatively upfield compared to
typical A-form values, and the large line width indicates that it
is even further upfield some of the time.<sup><xref ref-type="bibr" rid="ref64">64</xref>,<xref ref-type="bibr" rid="ref65">65</xref></sup> However, the chemical shifts of H1′ of a 3′ uridine
in a loop-closing AU pair and of a 3′ cytidine in a loop-closing
GC pair of two different GAAA tetraloops are 3.82 and 3.28 ppm, respectively.<sup><xref ref-type="bibr" rid="ref66">66</xref>,<xref ref-type="bibr" rid="ref67">67</xref></sup> The different structure of an A-C pair relative to a Watson–Crick
pair may result in a weaker effect of ring current on C23H1′
from the A22 base. On the basis of its large line width, however,
the H1′ chemical shift of C23 in some conformations may be
near the expected upfield range of H1′ chemical shifts of a
3′ tetraloop-closing residue. H3′ of A22 could not be
identified in NMR spectra of the 39-mer because of overlap but was
assigned to 4.62 ppm in a 20 °C spectrum of the 11-mer (4.58
ppm at −2 °C) in D<sub>2</sub>O without Mg<sup>2+</sup>, relatively upfield of the chemical shift (∼5.0 ppm) expected
for the last adenosine of a GAAA tetraloop closed by a canonical base
pair.<sup><xref ref-type="bibr" rid="ref65">65</xref>,<xref ref-type="bibr" rid="ref67">67</xref>−<xref ref-type="bibr" rid="ref70">70</xref></sup></p><fig id="fig5" position="float"><label>Figure 5</label><caption><p>H1′–H6/H8 region of a 2D
proton NOESY spectrum of
the 39 nt hairpin showing sequential proton walks for residues C12–C23
and G24–A31. The C23H1′–H6 and C23H1′–G24H8
NOEs are missing from the walk because the C23H1′ and H<sub>2</sub>O resonances are close to each other. H1′–H6/H8
walk NOEs are labeled in blue. Adenine H2 signals are labeled with
red dashed lines. H1′–adenine H2 NOEs are labeled in
red with only the label of the residue for H1′. A G19H1′
(5.45 ppm)–A21H8 (7.96 ppm) NOE is labeled in orange. The spectrum
was acquired at 20 °C and a mixing time of 350 ms with a WATERGATE
pulse to suppress the water signal. In the secondary structure of
the 39 nt hairpin, residues whose intraresidue H1′–H6/H8
NOEs were identified in the NOESY walks are labeled in blue. Spectrum
and walks for residues G2−G10 and U33−U39 are in Figure
S5 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>.</p></caption><graphic xlink:href="bi-2015-000127_0006" id="gr5" position="float"></graphic></fig><fig id="fig6" position="float"><label>Figure 6</label><caption><p>(a) H1′–H6/H8 region of a 2D proton
NOESY spectrum
of the 11 nt hairpin showing a sequential proton walk with blue lines.
H1′–H6/H8 walk NOEs are labeled in blue. Adenine H2
signals are labeled with red dashed lines. H1′–adenine
H2 NOEs are labeled in red with only the label of the residue for
H1′. The G19H1′ (5.56 ppm)–A21H8 (7.89 ppm) NOE
is labeled in orange and is consistent with the formation of a GNRA-like
U-turn. The spectrum was recorded at −2 °C in D<sub>2</sub>O and 5 mM Mg<sup>2+</sup> with a mixing time of 400 ms. Cross-peaks
from C23H1′ to A22H2 and C23H6 were not observed in this spectrum,
but in a spectrum acquired at 20 °C with a mixing time of 400
ms. (b) Secondary structure of the 11 nt hairpin with assigned interresidue
NOEs. Green lines denote NOEs identified in the 11-mer and the 39
nt hairpin and red lines NOEs identified only in the 11-mer. (c) Geometry
of the G19-A22 sheared GA pair observed in the AMBER-refined structures.
(d) Geometry of the A18-C23 pair observed in most of the AMBER-refined
structures.</p></caption><graphic xlink:href="bi-2015-000127_0007" id="gr6" position="float"></graphic></fig><p>The possible occurrence
of a protonated A<sup>+</sup>C pair at
the base of the hairpin loop was investigated with homonuclear NOESY
and <sup>13</sup>C–<sup>1</sup>H HSQC experiments. A<sup>+</sup>C pairs can have a p<italic>K</italic><sub>a</sub> as high as 6.5
for protonation of adenine N1.<sup><xref ref-type="bibr" rid="ref71">71</xref>−<xref ref-type="bibr" rid="ref73">73</xref></sup> Formation of A<sup>+</sup>C pairs
is accompanied by an upfield shift of adenine C2 by ∼7 ppm
(to ∼145 ppm) relative to other adenine C2 resonances, a downfield
shift of adenine H2 to above 8 ppm, and an adenine H1 shift of ∼14.5
ppm.<sup><xref ref-type="bibr" rid="ref71">71</xref>,<xref ref-type="bibr" rid="ref73">73</xref></sup> The A18H2 chemical shifts of 7.43 and 7.67
ppm in the 39-mer and 11-mer (20 °C), respectively, the absence
of imino proton signals at ≥14.5 ppm, and the absence of adenine
C2 resonances below 150 ppm are inconsistent with an A<sup>+</sup>C pair. Furthermore, the presence of an NOE from A18H2 to C23 amino
proton(s) in a NOESY spectrum<sup><xref ref-type="bibr" rid="ref74">74</xref></sup> of the
11-mer is structurally inconsistent with the orientation of the adenine
H2 and cytosine amino protons in opposite grooves of an A<sup>+</sup>C pair (signal overlap prevented this NOE from being identified in
NOESY spectra of the 39-mer). This NOE, however, is consistent with
a <italic>cis</italic> Watson–Crick bifuricated AC pair (Figure <xref rid="fig6" ref-type="fig">6</xref>).<sup><xref ref-type="bibr" rid="ref62">62</xref></sup> In summary, NMR
spectra are consistent with a GAAA tetraloop closed by an AC pair.</p></sec><sec id="sec4.3"><title>The Internal Loop Has an Imino GA Pair and Is Dynamic</title><p>Fewer
cross-peaks were observed among residues within the internal
loop than within the hairpin loop (Figure <xref rid="fig7" ref-type="fig">7</xref>). The G11H1 peak of the 39 nt hairpin and 19 nt duplex at 13.08
ppm has a cross-peak to A31H2 at 7.96 and 7.99 ppm, respectively,
consistent with an imino (<italic>cis</italic> Watson–Crick/Watson–Crick)
GA pair (Figure <xref rid="fig7" ref-type="fig">7</xref>).<sup><xref ref-type="bibr" rid="ref61">61</xref>,<xref ref-type="bibr" rid="ref62">62</xref>,<xref ref-type="bibr" rid="ref65">65</xref>,<xref ref-type="bibr" rid="ref75">75</xref></sup> Cross-peaks
from H1′ and H2′ of G30 to A31H8 in spectra of the 39-mer,
in addition to a cross-peak from C12H1′ to A31H2 in spectra
of the 19-mer duplex, suggest that A31 is stacked below G30. In both
the 39-mer hairpin and 19-mer duplex, there are weak NOEs from G11H1
and U33H3 to G10H1 and/or G32H1, in addition to an NOE from G11H1
to C12H1′.</p><fig id="fig7" position="float"><label>Figure 7</label><caption><p>(a) H1′–H6/H8 region of a 2D proton NOESY
spectrum
of the 19 nt duplex showing a sequential proton walk with blue lines
for residues 7–15 and green lines for residues 27–36.
H1′–H6/H8 walk NOEs are labeled with the same respective
colors. The G32H1′–H8 cross-peak is small because G32
is dynamic. Adenine H2 signals are labeled with red dashed lines.
H1′–adenine H2 NOEs are labeled in red with only the
label of the residue for H1′. The U27H1′–H6 NOE
overlaps with the U27H5–H6 NOE. The G10H1′ (6.13 ppm)–H8
(7.68 ppm) NOE is labeled in orange. Additional G10 NOEs are absent
because G10 is dynamic. The spectrum was acquired at −2 °C
in D<sub>2</sub>O and 5 mM Mg<sup>2+</sup> with a mixing time of 400
ms. (b) Secondary structure of the 19 nt duplex with assigned interresidue
NOEs. Blue lines denote NOEs identified in the 19-mer and the 39 nt
hairpin and red lines NOEs identified in only the 19-mer duplex. (c)
Geometry of the G11-A31 imino GA pair.</p></caption><graphic xlink:href="bi-2015-000127_0008" id="gr7" position="float"></graphic></fig><p>A strong G10H1′–H8 NOE and weak A9H1′–
and A9H2′–G10H8 NOEs of the 39-mer and 19-mer duplex
indicate that G10 has a <italic>syn</italic> conformation,<sup><xref ref-type="bibr" rid="ref36">36</xref>,<xref ref-type="bibr" rid="ref55">55</xref>,<xref ref-type="bibr" rid="ref76">76</xref>,<xref ref-type="bibr" rid="ref77">77</xref></sup> or an equilibrium of <italic>syn</italic> and <italic>anti</italic> conformations (Figure <xref rid="fig7" ref-type="fig">7</xref> and Figure S4 of
the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). An A9H2–G10H1′
NOE of the 39-mer is weaker than other H2<sub><italic>i</italic></sub>–H1′<sub><italic>i</italic>+1</sub> cross-peaks in
A-form helices. The G10H8 chemical shift (7.60 ppm in the 39-mer at
20 °C) is upfield of its unshielded reference value (8.10 ppm),<sup><xref ref-type="bibr" rid="ref47">47</xref></sup> consistent with populations in which it is stacked
in the helix. On the other hand, few NOEs were detected for G10, consistent
with an extrahelical conformation. Apparently, G10 is in an equilibrium
of conformations in which G10 is stacked in or extruded from the helix
(Figure <xref rid="fig7" ref-type="fig">7</xref> and Figure S4 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). Exchange cross-peaks
from 13.4 to 13.0 ppm and from 10.8 to 11.0 ppm in a ROESY spectrum
correspond to U33 and G10 and/or G32, respectively, consistent with
dynamics at the start of the loop.</p><p>Spectra of the 19-mer duplex
acquired in D<sub>2</sub>O have a
broad G32H8 resonance. The expected intraresidue H1′–H8
NOEs of G11 and G32 are present in a 400 ms spectrum of the 19-mer
duplex in D<sub>2</sub>O, but absent in H<sub>2</sub>O and D<sub>2</sub>O spectra of the 39-mer and 19-mer duplex taken with shorter mixing
times. In 1D spectra of the 19-mer duplex acquired between 0 and 40
°C in D<sub>2</sub>O, a broad peak for G32H8 is observed at 0
°C, which further broadened initially, almost disappearing as
the temperature was increased to 20 °C, and then sharpened above
30 °C (Figure <xref rid="fig8" ref-type="fig">8</xref>). This observation suggests
interconversion of G32 or an adjacent residue between two conformations
in an intermediate time range at low temperatures, changing to fast
exchange at 30 °C, resulting in a single peak.<sup><xref ref-type="bibr" rid="ref78">78</xref></sup> Evidently, G32 is dynamic. Taken together, NMR spectral
properties of the internal loop region demonstrate that the GA pair
is relatively fixed but the GG pair is dynamic.</p><fig id="fig8" position="float"><label>Figure 8</label><caption><p>Aromatic region of 1D
proton NMR spectra of the 19 nt duplex acquired
from 0 to 45 °C in D<sub>2</sub>O and 5 mM Mg<sup>2+</sup>.</p></caption><graphic xlink:href="bi-2015-000127_0009" id="gr8" position="float"></graphic></fig></sec><sec id="sec4.4"><title>Bulge Loop</title><p>NOEs
from A15H1′ and A15H2′
to C16H6 and from A15H2 to C16H1′ indicate that A15 and C16
are close, so the A26 bulge does not prevent their stacking (Figure <xref rid="fig5" ref-type="fig">5</xref>). In contrast, there is no evidence of stacking
between G25 and U27 as there are no NOEs between those residues. NOE
cross-peaks from G25H1′ and G25H3′ to A26H8 and from
A26H1′ to U27H6 have normal A-form intensities. Cross-peaks
from G25H2′ to A26H8 and from A26H2′ to U27H6 could
be present but are overlapped by intraresidue H2′–H6/H8
cross-peaks of A26 and U27, respectively. The seven steps of a sequential
H1′–H6/H8 NOE walk were observed from nucleotides G24
to A31, inclusive, and the A26H8 chemical shift (8.04 ppm) is within
the range of the H8 chemical shifts observed for stacked adenines.
These data indicate that A26 is not completely bulged out of the helix.<sup><xref ref-type="bibr" rid="ref79">79</xref></sup></p><p>RNA FRABASE<sup><xref ref-type="bibr" rid="ref80">80</xref></sup> was searched for 3D structures with an adenine bulge flanked by
canonical base pairs as for A26. One such structure has the bulged
A excluded from the helix<sup><xref ref-type="bibr" rid="ref81">81</xref></sup> on the basis
of evidence for sequential NOE connectivity between residues on each
side of the A and the AH8 chemical shift of 8.48 ppm, close to the
reference value of 8.64 ppm for AH8 when the chemical shift is not
affected by neighboring ring currents.<sup><xref ref-type="bibr" rid="ref47">47</xref>,<xref ref-type="bibr" rid="ref81">81</xref></sup> In contrast,
another structure has a bulged A stacked in the helix on the basis
of cross-peaks of nearly equal intensity from AH2 to H1′ of
consecutive cross-strand 3′ C’s and an AH8 chemical
shift of 8.18 ppm.<sup><xref ref-type="bibr" rid="ref82">82</xref></sup> Similarly, a bulged
A stacked in the helix of a duplex was revealed by interresidue, intrastrand,
and interstrand H2–H1′ cross-peaks, sequential H1′–H6/H8
cross-peaks through the bulged A, and an AH8 chemical shift of 7.88
ppm.<sup><xref ref-type="bibr" rid="ref79">79</xref></sup> The 60 ms NOESY spectrum of the
39-mer has a cross-peak from A26H2 to C17H1′ that is stronger
than that from A26H2 to C16H1′ even though A26 is closer to
C16 than C17 in the secondary structure. This spectral feature can
be explained, however, by formation of a (C16-G25)A26 base triple
of the type seen in crystal structures.<sup><xref ref-type="bibr" rid="ref83">83</xref>,<xref ref-type="bibr" rid="ref84">84</xref></sup></p></sec><sec id="sec4.5"><title>Effect of Magnesium
on the Structure of the GAAA Tetraloop and
2 nt × 2 nt Internal Loop</title><p>Solvated Mg<sup>2+</sup> ions
bind to RNA by diffuse, nonspecific interactions with the backbone
or by specific interactions with the RNA.<sup><xref ref-type="bibr" rid="ref85">85</xref></sup> The former predominate due to the energetic cost of dehydrating
Mg<sup>2+</sup> for site-specific interactions.<sup><xref ref-type="bibr" rid="ref86">86</xref></sup> A sheared GA pair can directly coordinate Mg<sup>2+</sup>, while an imino GA pair binds fully hydrated Mg<sup>2+</sup>.<sup><xref ref-type="bibr" rid="ref61">61</xref>,<xref ref-type="bibr" rid="ref87">87</xref>,<xref ref-type="bibr" rid="ref88">88</xref></sup> To study the effect of Mg<sup>2+</sup> on the structures of the GAAA tetraloop and 2 nt ×
2 nt internal loop, the short RNA constructs were studied in the presence
of 5 mM MgCl<sub>2</sub>.</p><p>The 5 mM Mg<sup>2+</sup> caused some
minor shifts and sharpening of imino resonances of the 11 nt hairpin
(Figure S8 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>) and 19 nt duplex (Figure S9 of the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref>). Nevertheless, exchange cross-peaks of imino resonances
for G10/G32 and U33 remained. The chemical shifts of G11H1 and A31H2
in the 19-mer duplex changed little, but resonances for G11H1 in the
39-mer were easier to observe in the presence of Mg<sup>2+</sup> (Figure
S3 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). These
observations indicate that addition of Mg<sup>2+</sup> did not significantly
stabilize the helical or internal loop regions or induce a conformational
change in the flexible G10 and/or G32. No new imino proton resonances
were observed in the presence of Mg<sup>2+</sup>. Thus, Mg<sup>2+</sup> did not introduce new elements of secondary or tertiary structure.<sup><xref ref-type="bibr" rid="ref65">65</xref>,<xref ref-type="bibr" rid="ref66">66</xref>,<xref ref-type="bibr" rid="ref89">89</xref></sup></p><p>Mg<sup>2+</sup> sharpened
some of the nonexchangeable resonances
of the 11-mer hairpin and 19-mer duplex. Minor shifts (typically <0.1
ppm) of nonexchangeable resonances occurred in these constructs (Figures
S10 and S11 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). On the basis of its H1′–H8 cross-peak, G10 of the
19-mer duplex was in a <italic>syn</italic>–<italic>anti</italic> equilibrium in the presence and absence of Mg<sup>2+</sup>.</p><p>The similarities of chemical shifts obtained on the model mimics
with and without 5 mM Mg<sup>2+</sup> indicate that Mg<sup>2+</sup> did not significantly impact the structures of the 11-mer hairpin
or 19-mer duplex.<sup><xref ref-type="bibr" rid="ref90">90</xref></sup> The data are also consistent
with the expected small effect of counterion charge on <sup>1</sup>H chemical shift.<sup><xref ref-type="bibr" rid="ref45">45</xref>,<xref ref-type="bibr" rid="ref91">91</xref></sup></p></sec><sec id="sec4.6"><title>Modeling Helices and the
Hairpin Loop</title><p>The simulated
annealing protocol provided structures for the 39 nt hairpin that
are consistent with the NMR details described above (Table <xref rid="tbl1" ref-type="other">1</xref> and Figures S12 and S13 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). The helices formed as expected,<sup><xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref15">15</xref></sup> including two wobble GU and Watson–Crick A15-U27 and C16-G25
pairs.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><title>Structural Refinement Statistics for
the 39 nt Hairpin, 19 nt Duplex, and 11 nt Hairpin for the Average
of 20 Structures of Each RNA Construct</title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col></colgroup><thead><tr><th style="border:none;" align="center"> </th><th style="border:none;" align="center">39 nt hairpin</th><th style="border:none;" align="center">19 nt duplex</th><th style="border:none;" align="center">11 nt hairpin</th></tr></thead><tbody><tr><td style="border:none;" align="left">no. of restraints</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left"> </td><td style="border:none;" align="left"> </td></tr><tr><td style="padding-left: 2em;" align="left">all distance restraints,
including hydrogen bonds</td><td style="border:none;" align="left">224</td><td style="border:none;" align="left">106</td><td style="border:none;" align="left">68</td></tr><tr><td style="padding-left: 2em;" align="left">all NOE restraints</td><td style="border:none;" align="left">192</td><td style="border:none;" align="left">87</td><td style="border:none;" align="left">59</td></tr><tr><td style="padding-left: 2em;" align="left">intraresidue</td><td style="border:none;" align="left">89</td><td style="border:none;" align="left">50</td><td style="border:none;" align="left">33</td></tr><tr><td style="padding-left: 2em;" align="left">sequential residues</td><td style="border:none;" align="left">65</td><td style="border:none;" align="left">26</td><td style="border:none;" align="left">20</td></tr><tr><td style="padding-left: 2em;" align="left">long range</td><td style="border:none;" align="left">38</td><td style="border:none;" align="left">11</td><td style="border:none;" align="left">6</td></tr><tr><td style="padding-left: 2em;" align="left">hydrogen bond</td><td style="border:none;" align="left">32</td><td style="border:none;" align="left">19</td><td style="border:none;" align="left">9</td></tr><tr><td style="padding-left: 2em;" align="left">dihedral restraints</td><td style="border:none;" align="left">176</td><td style="border:none;" align="left">93</td><td style="border:none;" align="left">24</td></tr><tr><td style="border:none;" align="left">rmsd of experimental
restraints</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left"> </td><td style="border:none;" align="left"> </td></tr><tr><td style="padding-left: 2em;" align="left">distances (Å)</td><td style="border:none;" align="left">6.4 × 10<sup>–4</sup></td><td style="border:none;" align="left">6.0 × 10<sup>–4</sup></td><td style="border:none;" align="left">7.7 × 10<sup>–4</sup></td></tr><tr><td style="padding-left: 2em;" align="left">dihedral angles (deg)</td><td style="border:none;" align="left">1.5</td><td style="border:none;" align="left">1.1</td><td style="border:none;" align="left">0.0</td></tr><tr><td style="border:none;" align="left">rmsd of structures for heavy atoms
(Å)</td><td style="border:none;" align="left"> </td><td style="border:none;" align="left"> </td><td style="border:none;" align="left"> </td></tr><tr><td style="padding-left: 2em;" align="left">all residues (except
1 and 2 in the 39-mer)</td><td style="border:none;" align="left">2.90 ± 0.57</td><td style="border:none;" align="left">1.23 ± 0.43</td><td style="border:none;" align="left">0.75 ± 0.21</td></tr><tr><td style="padding-left: 2em;" align="left">internal loop (residues
10, 11, 31, and 32)</td><td style="border:none;" align="left">1.65 ± 0.28</td><td style="border:none;" align="left">0.62 ± 0.23</td><td style="border:none;" align="left">–</td></tr><tr><td style="padding-left: 2em;" align="left">base triple (residues
16, 25, and 26)</td><td style="border:none;" align="left">1.14 ± 0.18</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">–</td></tr><tr><td style="padding-left: 2em;" align="left">hairpin loop and AC
pair (residues 18–23)</td><td style="border:none;" align="left">0.83 ± 0.16</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">0.67 ± 0.24</td></tr><tr><td style="padding-left: 2em;" align="left">helix 12/30–15/27
(excluding C16-G25 and C17-G24 base pairs)</td><td style="border:none;" align="left">0.47 ± 0.13</td><td style="border:none;" align="left">0.39 ± 0.20</td><td style="border:none;" align="left">–</td></tr></tbody></table></table-wrap><p>In the 39 and 11 nt hairpins, the
modeled AGAAAC loop has properties
of GNRA tetraloops (Figure <xref rid="fig9" ref-type="fig">9</xref> and Figure S13
of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). A sharp
U-turn exists between G19 and A20 with the following atoms within
hydrogen bonding distance as expected (Figure <xref rid="fig9" ref-type="fig">9</xref>c):<sup><xref ref-type="bibr" rid="ref64">64</xref>,<xref ref-type="bibr" rid="ref92">92</xref></sup> (1) G19H2 and A22OP, (2) G19 2′OH
and A21N7, (3) G19H2 and A22N7, and (4) G19H1 and A22OP. Stacking
of G19 on the 5′ side of the loop and A20–A22 on the
3′ side of the loop is also consistent with models of GNRA
tetraloops.<sup><xref ref-type="bibr" rid="ref64">64</xref></sup> The distance between G19N3
and A22 amino protons, however, is too long (>3.4 Å) for a
hydrogen
bond.<sup><xref ref-type="bibr" rid="ref93">93</xref></sup> This is consistent with observations
for some GNRA tetraloops,<sup><xref ref-type="bibr" rid="ref94">94</xref></sup> including several
in crystal structures of rRNAs.<sup><xref ref-type="bibr" rid="ref83">83</xref>,<xref ref-type="bibr" rid="ref84">84</xref>,<xref ref-type="bibr" rid="ref95">95</xref></sup> Jucker et al.<sup><xref ref-type="bibr" rid="ref64">64</xref></sup> reported that the GA
pair in a GAAA loop has a GN3–AN6 hydrogen bonding distance
ranging from 3.4 to 5.1 Å with an average of 4.28 Å; the
long length may indicate a water-mediated interaction. The unrestrained
sugar puckers of A20–A22 are primarily C3′-endo (δ
near 84°),<sup><xref ref-type="bibr" rid="ref96">96</xref></sup> consistent with relatively
weak or unobservable H2′–H1′ cross-peaks in a <sup>1</sup>H–<sup>1</sup>H TOCSY spectrum acquired at 20 °C
(Figure S15 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>), even though the second to fourth residues of a GNRA loop may experience
C2′-endo states (δ near 147°).<sup><xref ref-type="bibr" rid="ref64">64</xref>,<xref ref-type="bibr" rid="ref96">96</xref></sup> In the modeled structures, a C23 amino proton forms a hydrogen bond
with A18N1 or A18N3, consistent with the previously mentioned cross-peak
between a C23 amino proton and A18H2 in a water spectrum of the 11-mer<sup><xref ref-type="bibr" rid="ref74">74</xref>,<xref ref-type="bibr" rid="ref97">97</xref>,<xref ref-type="bibr" rid="ref98">98</xref></sup> and the absence of a protonated
A<sup>+</sup>C pair (Figure <xref rid="fig9" ref-type="fig">9</xref> and Figure S13
of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>).</p><fig id="fig9" position="float"><label>Figure 9</label><caption><p>(a) Model of
the GAAA loop of the 39 nt hairpin construct calculated
with AMBER, showing the 3′ A<sub>3</sub> stack and an AC pair
with a hydrogen bond from the C23 amino group to A18N1. (b) Space-filling
model of the A18-C23 pair. (c) Space-filling model of the G19-A22
pair.</p></caption><graphic xlink:href="bi-2015-000127_0010" id="gr9" position="float"></graphic></fig></sec><sec id="sec4.7"><title>Modeling the Internal Loop</title><p>Except for terminal residues
in the 19 nt duplex model, all of the residues primarily have a C3′-endo
sugar pucker (Figure S16 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). The modeled 2 nt × 2 nt internal loop contains a G11-A31
imino (<italic>cis</italic> Watson–Crick/Watson–Crick)
pair in the 39 nt hairpin and 19 nt duplex (Figure <xref rid="fig10" ref-type="fig">10</xref> and Figure S16 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). If the force field allowed the exocyclic amine of G11 to be out
of plane, then it could have a favorable interaction with the carbonyl
of C12.<sup><xref ref-type="bibr" rid="ref99">99</xref>−<xref ref-type="bibr" rid="ref102">102</xref></sup> While the G11 and U33 imino peaks are broadened because of solvent
exchange, local conformational dynamics, or both (Figure <xref rid="fig2" ref-type="fig">2</xref>), cross-peaks from these resonances to A31H2 and
A9H2 (Figure S4 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>), respectively, and chemical shifts typical of hydrogen-bonded imino
protons indicate that formation of G11-A31 and A9-U33 (<italic>cis</italic> Watson–Crick/Watson–Crick) pairs is dominant.</p><fig id="fig10" position="float"><label>Figure 10</label><caption><p>Calculated
model of the internal loop of the 39 nt hairpin construct.
(a) G10 stacked in the helix with a <italic>syn</italic> conformation.
(b) G10 flipped out of the helix with an <italic>anti</italic> conformation.
G10 was also observed in <italic>syn</italic> and <italic>anti</italic> conformations flipped out of and stacked in the helix, respectively.
The averages of G10 chemical shifts calculated with NUCHEMICS<sup><xref ref-type="bibr" rid="ref47">47</xref></sup> for the 20 structures with the lowest distance
restraint violation energies generated by simulated annealing are
consistent with the structural ensemble. (c) Space-filling model of
the G11-A31 pair.</p></caption><graphic xlink:href="bi-2015-000127_0011" id="gr10" position="float"></graphic></fig><p>Consistent with the
NMR characteristics, some structural models
have G10 extruded from the helix while others have G10 positioned
within the helix, in a <italic>syn</italic> conformation or an <italic>anti</italic> conformation (Figure <xref rid="fig10" ref-type="fig">10</xref> and
Figure S16 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). On the basis of the G10H1′–H8 distance of 3.02 Å
derived from NOE volumes, G10 is estimated to be in the <italic>syn</italic> and <italic>anti</italic> conformations in 26 and 74% of the populations
of the 39 nt hairpin, respectively. G10 forms no hydrogen bonds with
G32 in two of the 20 lowest-violation energy structures. G32 is also
dynamic but is always within the helix in the ensemble of generated
structures.</p></sec><sec id="sec4.8"><title>The A26 Bulge Can Form a Base Triple</title><p>The A26 bulge
can form a (C16-G25)A26 cWW/cSH base triple (Figure <xref rid="fig11" ref-type="fig">11</xref>),<sup><xref ref-type="bibr" rid="ref103">103</xref></sup> as observed in the 30S and 70S ribosomal subunits of <italic>Thermus thermophilus</italic> [PDB entries 2UXC for (C1260-G1274)A1275
and 2B9N for
(C965-G952)A2267].<sup><xref ref-type="bibr" rid="ref83">83</xref>,<xref ref-type="bibr" rid="ref84">84</xref>,<xref ref-type="bibr" rid="ref104">104</xref></sup> A characteristic feature of this type of base triple is that the
adenine is in the minor groove of a canonical CG pair.<sup><xref ref-type="bibr" rid="ref103">103</xref>,<xref ref-type="bibr" rid="ref105">105</xref></sup> The structures of the base triple in the models of the segment 7
hairpin generally agree with expected structures of the cWW/cSH CGA
base triple.<sup><xref ref-type="bibr" rid="ref103">103</xref>,<xref ref-type="bibr" rid="ref106">106</xref></sup> In most of the modeled structures,
A26N7 and at least one, if not both, of the A26 amino hydrogens are
within hydrogen bonding distance of one of the G25 amino hydrogens
and C16O2, respectively. One of the A26 amino hydrogens is also within
hydrogen bonding distance of C16O2′. The location of A26 in
the minor groove of the C16-G25 pair is consistent with cross-peaks,
including those from A26H2 to C16H1′, C17H1′, and U27H1′,
and A26H8 to G25H3′.<sup><xref ref-type="bibr" rid="ref106">106</xref></sup> The distance
between A26H2 and C16H1′ is greater than that between A26H2
and C17H1′, which agrees with the slightly weaker NOE from
A26H2 to C16H1′ compared to that of A26H2 to C17H1′.
A similar arrangement of a C, a G, and an A was observed in a hairpin
from <italic>Caenorhabditis elegans</italic>, but the adenine appears
to be stabilized by a hydrogen bond between its amino group and the
ribose of a 3′ cross-strand residue rather than the ribose
of the C.<sup><xref ref-type="bibr" rid="ref107">107</xref></sup> In chemical modification experiments,<sup><xref ref-type="bibr" rid="ref15">15</xref></sup> A26 was modified by DEPC, which carbethoxylates
an exposed adenine N7, such as one not buried in the major groove
of an RNA helix.<sup><xref ref-type="bibr" rid="ref108">108</xref></sup> This is inconsistent
with the formation of a hydrogen bond between A26N7 and the amino
group of G25, suggesting that the base triple is dynamic. In short,
the NMR-guided models suitably explain the observed NOE data for residues
around A26, but not the DEPC mapping data. Both types of data can
be rationalized, however, by a dynamic model for A26 (<italic>vide
infra</italic>).</p></sec><sec id="sec4.9"><title>Prediction of Structure from Chemical Shifts</title><p>Predicted
structures for the loops were also generated with CS-ROSETTA-RNA<sup><xref ref-type="bibr" rid="ref46">46</xref></sup> by applying chemical shift restraints from the
NMR spectra to secondary structures containing the same loops. Canonical
base pairs in helical regions of the secondary structure for each
RNA fragment were present in 3D models of the 20 lowest-energy structures.
All 20 lowest-energy structures of a 19 nt hairpin mimic, r(5′CCUACC<underline>AGAAAC</underline>GG<underline>A</underline>UGGG3′) (Figure S2 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>), containing the hairpin
loop and A26 bulge, have a 3′ stack of bases A20–A22
and a sheared-like G19-A22 base pair. A cWC/WC A18-C23 pair forms
in all 20 structures. A26 stacks below G25 in all 20 structures without
forming a (C16-G25)A26 base triple, and the distance from A26H2 to
C17H1′ is much longer than that to C16H1′, contrary
to the larger C17H1′–A26H2 NOE compared to the C16H1′–A26H2
NOE. This again suggests that A26 is dynamic so that one structure
does not satisfy all the data.</p><p>The G11-A31 imino pair was present
in all 20 lowest-energy CS-ROSETTA-RNA structures of an 18 nt duplex
containing the 2 nt × 2 nt internal loop (Figure S2 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). Sixteen structures have
G10-G32 in a base pair that resembles a <italic>trans</italic> Hoogsteen/sugar
edge pair stabilized by a G10O6 to G32H1 hydrogen bond in 10 structures
and a G10O6 to G32H2 hydrogen bond in six structures. The four remaining
structures have a G10-G32 base pair resembling a <italic>cis</italic> Watson–Crick/Hoogsteen pair stabilized by a G10H1 and/or
G10H2 to G32N7 hydrogen bond. In all 20 structures, G10 is in an <italic>anti</italic> conformation, contrary to the <italic>syn</italic> character
revealed by its relatively large H1′–H8 NOE. Taken together,
ROSETTA provides reasonable 3D models of the RNA from sequence, secondary
structure, and assigned chemical shifts. Comparisons between structures
based on chemical shifts and distance restraints, however, reveal
dynamics.</p><fig id="fig11" position="float"><label>Figure 11</label><caption><p>(a) Schematic of a CGA base triple of the cWW/cSH family<sup><xref ref-type="bibr" rid="ref103">103</xref></sup> with expected hydrogen bonds from the adenine
to cytosine and guanine: A-N7 to G-H22 and A-H61 to C-O2. Not shown
is a hydrogen bond from A-H62 to C-O2′. (b) Model of the (C16-G25)A26
base triple of the 39 nt hairpin construct refined by AMBER. (c) Space-filling
model of the (C16-G25)A26 base triple.</p></caption><graphic xlink:href="bi-2015-000127_0012" id="gr11" position="float"></graphic></fig></sec><sec id="sec4.10"><title>Prediction of Chemical Shifts from Structure</title><p>The program
NUCHEMICS<sup><xref ref-type="bibr" rid="ref47">47</xref></sup> was used to predict chemical
shifts for the ensembles of 20 structures of the RNA constructs generated
with distance restraints. Average calculated chemical shifts of H1′,
H2′, H2, H5, and H6/H8 of the 39 nt hairpin agree for most
residues within 0.4 ppm of those assigned in NMR spectra at 20 °C
(Figure <xref rid="fig12" ref-type="fig">12</xref>). Chemical shifts of sugar resonances
of the internal loop are within ∼0.2 ppm of experiment despite
the flexibility of the loop (Figure <xref rid="fig12" ref-type="fig">12</xref> and
Figure S18 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). For the AC closed GAAA loop, larger differences between predicted
and experimental chemical shifts are observed. The A18H2 proton was
predicted to be at 7.03 ppm in the 39-mer and 7.11 ppm in the 11 nt
hairpin, which are ∼0.4 and ∼0.6 ppm smaller than experimental
chemical shifts for their respective structures (Figure <xref rid="fig12" ref-type="fig">12</xref> and Figure S19 of the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref>). The C23H1′ chemical shift for the 11-mer
was 4.85 ppm but was predicted to be 3.77 ppm. To explore whether
the discrepancy is seen with other RNAs, differences were analyzed
between chemical shifts predicted with NUCHEMICS for 3D structures
of three GAAA hairpins<sup><xref ref-type="bibr" rid="ref65">65</xref>,<xref ref-type="bibr" rid="ref67">67</xref>,<xref ref-type="bibr" rid="ref70">70</xref></sup> from the PDB<sup><xref ref-type="bibr" rid="ref104">104</xref></sup> and their assigned chemical
shifts from the BMRB.<sup><xref ref-type="bibr" rid="ref33">33</xref></sup> Each of the three
types of canonical base pairs is represented among these structures
as a closing pair of the GAAA hairpin loop. Indeed, for a given RNA,
experimental and predicted chemical shifts for each of these hairpins
differ most (up to 2.3 ppm) within the GAAA hairpin loop and closing
base pair (Figures S20–S22 of the <xref rid="notes-2" ref-type="notes">Supporting
Information</xref>), confirming that either NUCHEMICS inconsistently
predicts true chemical shifts of these residues or the structures
are inaccurate or dynamic.</p><fig id="fig12" position="float"><label>Figure 12</label><caption><p>Chemical shift differences of the 39 nt hairpin
between experiment,
assigned at 20 °C, and those predicted by NUCHEMICS for H2/H5,
H6/H8, H1′, and H2′ in an ensemble of 20 structures
generated with NMR restraints. Residue numbers on the <italic>x</italic>-axis align with the middle of each set of two bars in each plot.</p></caption><graphic xlink:href="bi-2015-000127_0013" id="gr12" position="float"></graphic></fig><p>Chemical shifts were predicted
and averaged for the 20 lowest-energy
structures generated with CS-ROSETTA for the 19 nt hairpin and 18
nt duplex (Figure S2 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). Except for those of terminal residues in each construct, most
H1′, H2′, H2, H5, and H6/H8 chemical shifts of the CS-ROSETTA
structures, including those of GAAA loop residues, were predicted
to be within 0.3 ppm of those assigned for NMR spectra of the 39 nt
hairpin at 20 °C (Figures S23 and S24 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>). The average predicted chemical shift
of A26H2 in the CS-ROSETTA structures of the 19 nt hairpin is within
0.1 ppm of the experimental chemical shift. Thus, the stacking of
A26 below G25 in the CS-ROSETTA structures may occur in the 39 nt
hairpin, consistent with a dynamic base triple.</p></sec></sec><sec sec-type="discussion" id="sec5"><title>Discussion</title><p>Influenza is a public health problem that is incompletely controlled
by yearly vaccination.<sup><xref ref-type="bibr" rid="ref109">109</xref></sup> Available therapeutics
target neuraminidase and M2 proteins but are not particularly efficacious.
While most therapeutics used clinically target proteins, RNA research
is revealing a wealth of potential RNA targets, including splice sites.
For example, beta thalassemia and Duchenne muscular dystrophy have
been treated or reversed with oligonucleotides that block a splice
site.<sup><xref ref-type="bibr" rid="ref110">110</xref>−<xref ref-type="bibr" rid="ref112">112</xref></sup></p><p>If a splice site has a stable structure,
it should also be possible
to affect splicing with small molecules that bind specifically to
a loop.<sup><xref ref-type="bibr" rid="ref8">8</xref>,<xref ref-type="bibr" rid="ref113">113</xref>,<xref ref-type="bibr" rid="ref114">114</xref></sup> Most splice
sites, though, are thought not to have stable structures.<sup><xref ref-type="bibr" rid="ref115">115</xref>−<xref ref-type="bibr" rid="ref118">118</xref></sup> One exception is the 3′ splice site of segment 7 mRNA from
influenza A.<sup><xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref15">15</xref></sup> Splicing at this site determines
the relative abundance of two essential proteins, M1 and M2.<sup><xref ref-type="bibr" rid="ref119">119</xref></sup> The amount of splicing may be determined by
an equilibrium between a pseudoknot and two-hairpin structure around
the splice site (Figure <xref rid="fig1" ref-type="fig">1</xref>). The results reported
here reveal several interesting characteristics of the hairpin containing
the 3′ splice site.</p><p>The splice site is between G10 and
G11 in a conformationally flexible
internal loop where G10 appears to be in equilibria between intrahelical
and extrahelical conformations as well as <italic>syn</italic>–<italic>anti</italic> conformations for the base relative to the ribose (Figure <xref rid="fig10" ref-type="fig">10</xref>). Local flexibility may be an important characteristic
for a 3′ splice site.<sup><xref ref-type="bibr" rid="ref115">115</xref></sup> In contrast
to G10 and G32, the adjacent G11-A31 and A9-U33 pairs are relatively
stable. Alternative conformations of the splice site region are also
consistent with exchange peaks for imino protons of residues G10 and/or
G32, U33, and G34 and two-site exchange observed for G32H8. The flexibility
of the internal loop and its functional importance to the viral life
cycle as a splice site make it an attractive target for binding and
inhibition by therapeutic agents.<sup><xref ref-type="bibr" rid="ref120">120</xref>,<xref ref-type="bibr" rid="ref121">121</xref></sup></p><p>Specificity
of binding to RNA can be improved by targeting two
loops by coupling together two small molecules.<sup><xref ref-type="bibr" rid="ref8">8</xref>,<xref ref-type="bibr" rid="ref122">122</xref></sup> Presumably, the bulge A loop, the GAAA tetraloop capping the hairpin,
or both could be targeted along with the internal loop. The GAAA tetraloop
is closed by an AC pair, which is an unusual combination. The (C16-G25)A26
base triple formed by the bulged A is known to occur in other RNAs<sup><xref ref-type="bibr" rid="ref83">83</xref>,<xref ref-type="bibr" rid="ref84">84</xref></sup> but is rare.</p><p>Internal loops with adjacent GG and imino GA
pairs were observed
in loop E of <italic>E. coli</italic> 5S rRNA, r(5′C<underline>GAUGGUA</underline>G<sub>79</sub>/3′G<underline>AUGAGAG</underline>C<sub>97</sub>),<sup><xref ref-type="bibr" rid="ref123">123</xref></sup> the HIV Rev responsive element (RRE), r(5′G<underline>GG</underline>C<sub>49</sub>/3′G<underline>GUA</underline>C<sub>74</sub>),<sup><xref ref-type="bibr" rid="ref120">120</xref>,<xref ref-type="bibr" rid="ref121">121</xref></sup> and the 2 nt × 2 nt internal
loop, (5′A<underline>GG</underline>U<sub>271</sub>/3′U<underline>GA</underline>A<sub>282</sub>), from a hairpin of a group II intron
of <italic>Oceanobacillus iheyensis</italic> (Figure S25 of the <xref rid="notes-2" ref-type="notes">Supporting Information</xref>).<sup><xref ref-type="bibr" rid="ref88">88</xref>,<xref ref-type="bibr" rid="ref124">124</xref>,<xref ref-type="bibr" rid="ref125">125</xref></sup> The first two loops participate
in protein recognition but are larger than the 2 nt × 2 nt loop
containing the influenza 3′ splice site.</p><p>The sequence
of the r(5′A<underline>GG</underline>U<sub>271</sub>/3′U<underline>GA</underline>A<sub>282</sub>) loop
is similar to that of the r(5′A<underline>GG</underline>C/3′U<underline>GA</underline>G) influenza splice site loop. To structurally compare
the loops, hydrogen atoms were added to Protein Data Bank (PDB) X-ray
structures of the group II intron (3EOH, 4E8M, 4E8Q, 4FAR, 4FAW, and 4FAX) using Reduce with the NOFLIP option.<sup><xref ref-type="bibr" rid="ref126">126</xref></sup> In each of the crystal structures, G270 and
A283 form an imino GA pair, the same as G11-A31 in the segment 7 hairpin.
G269 and G284, however, form a <italic>trans</italic> Hoogsteen/sugar
edge pair, perhaps as a result of the noncanonical A268-U285 pair
below it. In contrast to the Watson Crick A9-U33 pair closing the
splice site loop, the A268-U285 pair in the group II intron is a <italic>trans</italic> Watson–Crick/Watson–Crick pair in the
2008 structure<sup><xref ref-type="bibr" rid="ref124">124</xref></sup> and a <italic>trans</italic> Watson–Crick/Hoogsteen pair in the 2012 structures.<sup><xref ref-type="bibr" rid="ref125">125</xref></sup> In all six structures, the A268 amino proton
that does not contact U285 points away from the major groove of its
helix and lies within hydrogen bonding proximity of O4′ of
an extrahelical residue, G321. There is also a hydrogen bond from
the amino of G269 to O2′ of G321. Similar extrahelical hydrogen
bonds were found in X-ray crystal structures of ribosomes.<sup><xref ref-type="bibr" rid="ref127">127</xref>−<xref ref-type="bibr" rid="ref129">129</xref></sup> The terminal base pair of the group II intron motif consists of
a canonical GC pair with G267 forming an extrahelical <italic>trans</italic> sugar edge/sugar edge pair with G320 to form a cWW/tSS (C286-G267)G320
base triple.<sup><xref ref-type="bibr" rid="ref62">62</xref>,<xref ref-type="bibr" rid="ref103">103</xref></sup></p><p>Nucleotide details of
the X-ray structures of the r(5′A<underline>GG</underline>U<sub>271</sub>/3′U<underline>GA</underline>A<sub>282</sub>) internal
loop region of the group II intron hairpin
differ from those of the NMR solution structure of the segment 7 hairpin.
G269 of the group II intron has an <italic>anti</italic> conformation,
in contrast to the mix of <italic>syn</italic> and <italic>anti</italic> conformations of G10 of the segment 7 hairpin. A268 of the 2008
X-ray structure<sup><xref ref-type="bibr" rid="ref124">124</xref></sup> has a <italic>syn</italic> conformation, compared to the <italic>anti</italic> conformation
of A9 in the segment 7 hairpin. In the A268-U285 pair of the 2012
X-ray structures, the orientation of A268H2 away from U285H3 is inconsistent
with the presence of an NOE from A9H2 to U33H3 in NMR spectra of the
segment 7 hairpin. Evidently, tertiary interactions from the amino
protons of A268 and G269 to G321 stabilize G269-G284 and noncanonical
A268-U285 base pairs in the group II intron. Similar large differences
have been observed between an NMR structure of an isolated internal
loop and the same sequence loop in crystals of ribosomes.<sup><xref ref-type="bibr" rid="ref130">130</xref></sup> The results suggest certain internal loops
may be poised for molecular recognition by induced fit, structure
capture,<sup><xref ref-type="bibr" rid="ref131">131</xref></sup> adaptive recognition,<sup><xref ref-type="bibr" rid="ref132">132</xref>,<xref ref-type="bibr" rid="ref133">133</xref></sup> or all of them. Independent of mechanism, the results also suggest
that a variety of small molecules could bind tightly to the influenza
internal loop and serve as therapeutics.</p><p>Ultimately, it should
be possible to predict the structure and
dynamics of RNAs and of molecules to bind them. The results presented
here provide a useful benchmark for testing such predictions. The
minimal effect of Mg<sup>2+</sup> on the 2 nt × 2 nt internal
loop and A<underline>GAAA</underline>C tetraloop implies that it will
not be necessary to include Mg<sup>2+</sup> in such calculations.</p></sec></body><back><notes id="notes-2" notes-type="si"><title>Supporting Information Available</title><p>(I) Equilibria of 19
nt duplex
mimics of a portion of the 39 nt hairpin with homodimers of the top
strands of their duplexes. (II) Fragments of the 39 nt hairpin used
for modeling with CS-ROSETTA. (III) 1D and 2D proton spectra of the
constructs studied. (IV) Thermodynamic calculation for formation of
the 11 nt hairpin. (V) Differences in chemical shifts of select nonexchangeable
hydrogens of the 11 nt hairpin and 19 nt duplex with and without 5
mM Mg<sup>2+</sup> at −2 °C. (VI) Ensembles of the 10
structures of the 11 nt hairpin and 19 nt duplex modeled in AMBER
with NMR restraints. (VII) Lowest distance restraint violation energy
models of the GAAA loop and closing AC pair of the 11 nt hairpin and
of the internal loop of the 19 nt duplex calculated in AMBER with
NMR restraints. (VIII) Chemical shift differences of the 19 nt duplex
and 11 nt hairpin between experiment, assigned at 25 and 20 °C,
respectively, in the presence of 5 mM Mg<sup>2+</sup>, and those predicted
by NUCHEMICS for H2/H5, H6/H8, H1′, and H2′ in an ensemble
of 20 structures generated with NMR restraints. (IX) Differences between
experimental and NUCHEMICS-predicted chemical shifts of GAAA hairpins
closed by a GU, GC, or AU pair. (X) Differences between experimental
chemical shifts of the 39 nt hairpin, assigned at 20 °C, and
those predicted by NUCHEMICS for H2/H5, H6/H8, H1′, and H2′
for an ensemble of 20 structures each of the 19 nt hairpin and 18
nt duplex as generated by CS-ROSETTA with chemical shift constraints.
(XI) Secondary structure of a hairpin with a 2 nt × 2 nt internal
loop from a group II intron of <italic>O. iheyensis</italic>. (XII)
Chemical shifts used to model fragments of the segment 7 hairpin with
CS-ROSETTA. (XIII) Assigned chemical shifts of the 39 and 11 nt hairpins
and 19 nt duplex. (XIV) Plasmid insert design for <italic>in vitro</italic> transcription of the 39 nt 3′ segment 7 mRNA hairpin by T7
RNA polymerase. (XV) Distance and dihedral restraints for AMBER modeling
of the 39 and 11 nt hairpins and the 19 nt duplex. The Supporting
Information is available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link> at
DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acs.biochem.5b00012">10.1021/acs.biochem.5b00012</ext-link>.</p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="bi5b00012_si_001.pdf"><caption><p>bi5b00012_si_001.pdf</p></caption></media></supplementary-material></sec><notes id="notes-1" notes-type="accession-codes"><title>Accession Codes</title><p>NMR chemical shifts
for the 11 nt hairpin, 19 nt duplex, and 39 nt hairpin were deposited
in the Biological Magnetic Resonance Data Bank as entries 25414, 25415,
and 25416, respectively. Structural coordinates for these constructs
were deposited in the RCSB Protein Data Bank as entries 2MXJ, 2MXK, and 2MXL, respectively.</p></notes><notes id="notes-3" notes-type="funding-statement"><p>This work was
supported by National Institutes of Health Grant GM22939.</p></notes><notes id="NOTES-d2110e1828-autogenerated" notes-type="conflict-of-interest"><p>The authors declare
no
competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>We thank the following individuals for their contributions
to this work: Dr. Blanton Tolbert for providing a plasmid that expresses
T7 RNA polymerase and a protocol for <italic>in vitro</italic> transcription
of RNA by T7 RNA polymerase; Dr. Jesse Kleingardner, Dr. Kara Bren,
and Hiram Lyon for assistance with purifying RNA transcripts; Dr.
Brendan Mort for assistance with computations; and Dr. Anton Petrov
for providing information about base triples from literature. Minimizations
by simulated annealing were performed with computational resources
provided by the Center for Integrated Research Computing (CIRC).</p></ack><glossary id="dl1"><def-list><title>Abbreviations</title><def-item><term>1D</term><def><p>one-dimensional</p></def></def-item><def-item><term>2D</term><def><p>two-dimensional</p></def></def-item><def-item><term>3D</term><def><p>three-dimensional</p></def></def-item><def-item><term>FPLC</term><def><p>fast protein liquid chromatography</p></def></def-item><def-item><term>HETCOR</term><def><p>heteronuclear
correlation</p></def></def-item><def-item><term>HIV-1</term><def><p>human immunodeficiency virus-1</p></def></def-item><def-item><term>HSQC</term><def><p>heteronuclear single-quantum coherence</p></def></def-item><def-item><term>NMR</term><def><p>nuclear magnetic resonance</p></def></def-item><def-item><term>NOESY</term><def><p>nuclear Overhauser
effect spectroscopy</p></def></def-item><def-item><term>rmsd</term><def><p>root-mean-square deviation</p></def></def-item><def-item><term>ROESY</term><def><p>rotating frame Overhauser spectroscopy</p></def></def-item><def-item><term>TOCSY</term><def><p>total correlation spectroscopy.</p></def></def-item></def-list></glossary><ref-list><title>References</title><ref id="ref1"><mixed-citation publication-type="book" id="cit1"><collab>Centers
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