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Enhanced Triplex Hybridization of DNA and RNA via Syndiotactic

Side Chain Presentation in Minimal bPNAs
Sarah Rundell, Oliver Munyaradzi, and Dennis Bong*
Cite This: Biochemistry 2022, 61, 85−91 Read Online

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ABSTRACT: General design principles for recognition at noncanonical

interfaces of DNA and RNA remain elusive. Triplex hybridization of bifacial
Downloaded via UNIV ESTADUAL PAULISTA on January 22, 2022 at 02:44:31 (UTC).

peptide nucleic acids (bPNAs) with oligo-T/U DNAs and RNAs is a robust
recognition platform that can be used to define structure−function
relationships in synthetic triplex formation. To this end, a set of minimal
(mw < 1 kD) bPNA variants was synthesized to probe the impact of amino
acid secondary structural propensity, stereochemistry, and backbone
cyclization on hybridization with short, unstructured T-rich DNA and U-
rich RNAs. Thermodynamic parameters extracted from optical melting
analyses of bPNA variant hybrids indicated that there are two bPNA
backbone modifications that significantly improve hybridization: alternating
(D, L) configuration in open-chain dipeptides and homochiral dipeptide
cyclization to diketopiperazine. Further, binding to DNA is preferred over
RNA for all bPNA variants. Thymine−uracil substitutions in DNA substrates revealed that the methyl group of thymine accounts for
71% of ΔΔGDNA‑RNA for open-chain bPNAs but only 40% of ΔΔGDNA‑RNA for diketopiperazine bPNA, suggesting a greater
sensitivity to RNA conformation and more optimized stacking in the cyclic bPNA. Together, these data reveal pressure points for
tuning triplex hybridization at the chiral centers of bPNA, backbone conformation, stacking effects at the base triple, and the nucleic
acid substrate itself. A structural blueprint for enhancing bPNA targeting of both DNA and RNA substrates includes syndiotactic
base presentation (as found in homochiral diketopiperazines and D, L peptides), expansion of base stacking, and further investigation
of bPNA backbone preorganization.

■ INTRODUCTION
Triplex formation necessarily includes binding of a non-
Scheme 1. This Work: bPNA Structure−Function
Relationships from Thermodynamic Characterization of
canonical interface in nucleic acids:1,2 a purine-rich strand is Triplex Hybridization of 4 M (Melamine) bPNA Variants
targeted with two pyrimidine strands, forming both Watson− (Blue) with 12nt DNAs (Y = T) and RNAs (Y = U)a
Crick and Hoogsteen pairs. Native triplexes of DNA and RNA
with PNAs3,4 similarly form via the addition of a third strand to
the Hoogsteen face of a pre-existing duplex. Determination of
structure−activity relationships for triplex recognition could
reveal general design principles for targeting noncanonical
structural motifs5 that are abundant in functional noncoding6,7
nucleic acids. Herein, we report such an SAR study on bifacial
peptide nucleic acids (bPNAs), which uniquely bind two
oligopyrimidine (T/U) strands simultaneously by docking two a
Base-triple formation with melamine is shown as expansion.
pyrimidines onto melamine (M) to form a base triple (Scheme
1).8,9 While nesting the oligo-T/U bPNA binding site between can greatly enhance nucleic acid hybridization. Herein, we set
duplex domains can greatly enhance bPNA binding relative to out to test the sensitivity of bPNA hybridization to scaffold
unfolded substrates,10−12 completely unstructured oligopyr-
imidines can be induced to fold into a triplex hybrid structure
with bPNA.13−17 Despite fundamental differences in the Received: October 18, 2021
recognition interface, bPNA hybridization is expected to be Revised: December 9, 2021
tunable via structural manipulation, much like XNAs bearing Published: December 27, 2021
native bases. Structural variants of the aminoethylglycine
(AEG) PNA prototype18 have demonstrated that back-
bone,19−21 side chain,22−25 and electrostatic24,26 modifications

© 2021 American Chemical Society https://doi.org/10.1021/acs.biochem.1c00693

85 Biochemistry 2022, 61, 85−91
Biochemistry pubs.acs.org/biochemistry
modification to develop design principles for improving bPNA
binding to DNA and RNA. Prior studies established that
bPNA binding to oligo-T/U domains is largely side chain-
driven and tolerant of backbone chemistry ranging from α-
peptide, peptoid,27 small molecules,28 to polyacrylate29−31
backbones.
However, there are significant penalties for side chain
shortening, which motivated our recent focus on side chain
optimization.30 We found that DNA/RNA binding, bPNA
solubility, and cell permeability11,12 are greatly enabled by the
ionizable tertiary amino side chain of bivalent base-tripling
residue K2M.10,32 (Figure 1). The use of K2M, which arises from
Figure 1. Structures of 4 M bPNA scaffolds studied: (a) tripeptide,
(b) dipeptide stereoisomers, and (c) diketopiperazine stereoisomers.
The structure of base-tripling residue K2M is shown in the inset with
the second ethyl melamine substituent in back grayed out.
double reductive-alkylation10,32 of the ε-amine of lysine with
melamine (M) acetaldehyde, enables the preparation of
bPNAs without additional solubilizing components. Recent
studies indicating that short bPNAs are sufficient for
intracellular targeting of U-rich internal loop (URIL)-
containing lncRNAs11 and telomeric DNA12 prompted us to
focus our structure−function studies herein on tripeptide and
dipeptide 4 M bPNAs with just two K2M residues. Though
successful PNA targeting of paired bases6,33−36 and inser-
tion37,38 in between paired bases has been reported, it can be
challenging to quantify binding to preformed duplexes.
86
Article
Similarly, while bPNA binding to prestructured T/U internal
bulges is highly favored over unstructured oligos,10,11 only
nuanced changes are observed in the optical melting profile of
structured targets. We thus limited our studies on bPNA
binding to short oligos without significant folding propensity
(d(T4C4T4), r(U4C4U4), r(U4GUGAU4)) such that any
detected thermal transition can be attributed to bPNA
hybridization, with the expectation that our findings would
apply to structured URILs as well. Though the stabilizing
GNRA RNA loop39,40 modestly increases hybrid Tm relative to
the C4 loop, none of the oligos exhibit melting transitions.
A series of 4 M bPNAs were prepared: open-chain
tripeptides (K2MXK2M), dipeptide (K2MK2M, K2Mk2M, k2MK2M,
k2Mk2M), and diketopiperazine (c[K2M]2, c[k2MK2M], c[k2M]2)
diastereomers containing (L)-K2M and (D)-k2M, derived from
(L) and (D)-lysine, respectively. To our surprise, the central X-
residue in the K2MXK2M scaffold could be widely varied or
removed altogether with a minor impact on hybridization. The
dipeptide scaffold was markedly more sensitive to structural
variation, with significant gains in hybrid stability upon
cyclization to diketopiperazine. Hybrids with diastereomeric
(L, D) and (D, L) dipeptide bPNAs were superior to those with
( L , L ) and ( D , D ) bPNAs, while conversely, ( L , L )
diketopiperazine was a much better binder than the (D, L).
These structure−function trends within the bPNA library
largely hold true for both DNA and RNA binding, with all
variants binding more tightly to DNA than RNA. Together,
these data identify sites in bPNA that are most sensitive to
structure−function tuning and point the way forward to
improve targeting of both DNA and RNA.
Tripeptide bPNAs. A library of K2MXK2M bPNAs was
synthesized wherein the central residue X was varied to
examine the impact of secondary structure propensity on
hybridization with DNA and RNA. The study was limited to
nonionizable X residues to maintain bPNA charge at +2 in all
cases, arising from protonation of two K2M tertiary amines.
Melting temperatures (Tm) and thermodynamic parameters
extracted from van’t Hoff analyses reveal that hybridization is
insensitive to changes in the central residue regardless of the
secondary structure propensities41,42 of each X-residue. For
instance, residues that favor φ and ψ angles with high β-sheet
(Val, Thr), α-helix (Ala, Leu), or even PPI/PPII helix (Pro)
propensity led to similar hybrid Tm with DNA (∼40 °C) and
RNA (∼23 °C) at 2 μM concentration (Figure 2, Table S1).
Furthermore, hybrid Tm was not significantly perturbed by
sterics, with Ala, Leu, and t-butylglycine at the X position
yielding similar properties. A small penalty in DNA binding
was observed for structure-breaking unsubstituted residues
(Gly, β-Ala), though this distinction was not seen in RNA
binding. A modest 1.5 °C advantage in Tm was seen with X =
Trp in RNA binding, while the remaining variants exhibited
closely clustered hybrid properties (Figure 2).
Free energy of hybridization remained essentially constant
while a plot of ΔH vs TΔS exhibited high linearity, suggestive
of enthalpy−entropy compensation in both DNA and RNA
hybrids (Figure 3). Interestingly, the ordering of variants was
different along the two lines, consistent with distinct binding
environments. Though confounding with regard to our efforts
to optimize binding, we conclude that triplex hybridization of
tripeptide bPNAs remains largely base-triple driven, with high
tolerance to substitution at the central position. This
observation augurs well for substitution of the central position
https://doi.org/10.1021/acs.biochem.1c00693
Biochemistry 2022, 61, 85−91
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Figure 2. (A) Overlaid van’t Hoff plots of K2MXK2M bPNAs (X = Ala, β-Ala, Gly, Ile, Leu, Ser, Phe, Pro, Thr, Trp, Tyr, Val, and t-butylglycine)
with DNA (d(T4C4T4)) and RNA (r(U4GUGAU4)) substrates, grouped together as indicated. For reference, X = Ala is shown in bold for both
data sets, with only outlying data sets for DNA (X = Gly, β-Ala) and RNA (X = Pro, Trp) indicated. The remaining overlapping data sets are shown
in light gray for both DNA and RNA. (B) Overlaid van’t Hoff plots of dipeptide K2MK2M and diketopiperazine c[K2M]2 bPNAs with (lower) DNA
d(T4C4T4) and (upper) RNA r(U4GUGAU4) substrates with the K2MAK2M bPNA plot (Ala) included for reference. Tm calculated from (A) first-
derivative analysis or (B) direct fit. Error bars (s.d.) from triplicate experiments are shown.

Figure 3. Enthalpy (ΔH) vs entropy (−TΔS) plots at 25 °C for DNA (●) and RNA (■) hybrids with (A) K2MXK2M, X-residue as labeled, and (B)
the indicated dipeptide bPNAs. The data for K2MAK2M is shown in (B) for reference. Tables 1 and S1 show full thermodynamic parameters and
associated standard deviation error.

Table 1. Thermodynamic Parameters for bPNA Hybrid Dissociation from Optical Meltinga
d(T4C4T4) r(U4GUGAU4)
bPNA Tm (°C) ΔG25 (kcal) ΔH (kcal) ΔS (cal) Tm (°C) ΔG25 (kcal) ΔH (kcal) ΔS (cal)
c[K2M]2 47.2 12.5 (0.1) 61.5 (1.5) 164.4 (4.7) 28.1 8.7 (0.1) 55.0 (2.6) 155.3 (8.4)
k2MK2M 46.0 12.9 (0.3) 72.7 (3.9) 201.6 (12) 24.8 8.1 (0.1) 50.9 (3.1) 143 (10)
K2Mk2M 43.0 11.7 (0.2) 61.8 (2.9) 167 (16) 26.2 8.4 (0.1) 52.3 (0.6) 147.3 (2.1)
k2Mk2M 40.2 11.1 (0.1) 59.1 (2.5) 160.8 (7.8) 24.0 8.0 (0.1) 52.9 (0.4) 150.5 (1.4)
K2MK2M 39.7 10.9 (0.1) 57.8 (1.7) 157.4 (5.4) 25.3 8.3 (0.1) 55.8 (3.8) 159 (12)
K2MAK2M 39.4 11.0 (0.1) 62.2 (2.3) 171 (7.3) 23.8 8.1 (0.1) 53.4 (1.4) 151.9 (4.7)
a
Conditions: 2 μM DNA, 2 μM bPNA, 100 mM NaCl, 10 mM phosphate, and pH 7.5. Tm was obtained by direct fit (error <1%) and comparable
to first-derivative analysis. Free energy, enthalpy and entropy parameters are shown are per mole and per mol K, respectively. The changing order of
D, L and L, D dipeptides is indicated in bold.

with large prosthetic groups such as fluorophores or affinity
tags.
Dipeptide bPNAs and the Impact of Cyclization to
Diketopiperazine bPNA. Deletion of the central X-residue
yielded a dipeptide bPNA scaffold (K2MK2M) with hybrid
properties similar to tripeptides, again with DNA hybrids more
stable than RNA hybrids (Figure 2B, Table 1). Remarkably,
87
cyclization of K2MK2M bPNA to diketopiperazine c[K2M]2
resulted in a nearly 8 and 3 °C increase in DNA and RNA
hybrid thermostability over the open-chain dipeptide, as well as
a 5 °C advantage over the RNA hybrid with K2MAK2M. Across
the tripeptide bPNA series, similar entropic costs were
observed for both DNA and RNA binding, while enthalpy of
RNA binding was less favorable (Figure 3). In contrast, the
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Figure 4. (A) Illustration of side chain presentation in (left) dipeptide bPNA stereoisomers when a β-helix backbone is adopted and (right)
diketopiperazine boat conformers for (L, L) and (D, L) diastereomers. (B) Overlaid van’t Hoff plots of dipeptide bPNA stereoisomers K2MK2M (L, L),
k2MK2M (D, L), K2Mk2M (L, D), and k2Mk2M (D, D) and diketopiperazine c[K2M]2 with DNA d(T4C4T4) and (C) RNA r(U4GUGAU4).

enthalpy−entropy relationship for the dipeptide series
exhibited an offset for both enthalpy and entropy, with
diminished binding enthalpy and decreased entropic cost for
RNA vs DNA binding. Despite expectations, van’t Hoff
analysis did not clearly indicate an entropic advantage to the
diketopiperazine relative to open-chain dipeptides (Figure 3,
Table 1). Rather, consistent with the narrow range of ΔΔG for
variants, subtle deviations from the linearity of enthalpy−
entropy compensation enable the diketopiperazine backbone
to emerge as the most efficient binder. Indeed, gains in binding
appear to be enthalpy-driven with nearly compensatory
entropic cost, suggesting that optimization of base-triple
stacking via backbone cyclization tightens binding at the
expense of nucleic acid backbone mobility. We speculated that
the diketopiperazine scaffold improves hybridization by
presentation of the synthetic bases on one face of the molecule
when homochiral dipeptides are cyclized (Figure 4).
Diketopiperazines are known to preferentially adopt boat
conformations in aqueous solution,43 which provides two
possible hybridization interfaces (Figure 4A), di-pseudoequa-
torial or di-pseudoaxial lysine side chains. Though di-
pseudoaxial side chains may have unfavorable intramolecular
steric interactions, these may be offset by more favorable base-
triple stacking.
Dipeptide bPNA Stereochemistry and Hybridization.
The possible advantage of syndiotactic side chains in triplex
hybridization prompted the synthesis of open chain bPNAs
with alternating (D, L) configuration; such peptides are known
to adopt β-helical conformations with side chains projecting
away from a curving backbone (Figure 4A).44−46 Syndiotactic
88
side chain arrangement of homochiral dipeptide backbones
potentially generates backbone steric interactions, though
alternative conformations for hybridization are possible. The
enantiomer of K2M was synthesized from Fmoc-D-Lysine10,12,32
and the resulting derivative (Fmoc-k2M) used to prepare
diastereomeric bPNAs (K2Mk2M, k2MK2M, c[k2MK2M]). As
expected, DNA hybrids with K2Mk2M and k2MK2M were
significantly more stable than those with the homochiral
K2MK2M, k2Mk2M and tripeptide K2MAK2M (Table 1). Notably,
the DNA complex with (D, L) bPNA (k2MK2M) was 3 °C more
stable than the diastereomeric complex with (L, D) bPNA
(K2Mk2M). In RNA, this preference switches to favor the (L, D)
bPNA-RNA hybrid by ∼2 °C more than the (D, L) bPNA-
RNA hybrid. These small but significant differences again
reflect distinctions between DNA and RNA binding. In
contrast to c[K2M]2, the (D, L) diketopiperazine c[k2MK2M]
was the least effective at DNA hybridization. Though a van’t
Hoff analysis of the DNA hybrid was performed, c[k2MK2M]
was unique among bPNAs in that thermal denaturation
showed broad, multitransition melting (Table S3.3), limiting
the validity of data interpretation and indicative of poorly
defined binding. The loss of function upon switching a single
chiral center in c[K2M]2 from L to D illustrates the advantage of
syndiotactic presentation: side chains are on the same side of
the ring in the homochiral c[K2M]2 and on opposite sides in
c[k2MK2M] (Figure 4A). The same general trend was observed
with RNA: c[K2M]2 and K2Mk2M formed the two most stable
hybrids while c[k2MK2M] did not even exhibit a melting
transition.
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Figure 5. (A) Illustration of expected base triples formed with melamine (blue) and thymine or uracil, with the CH3 → H replacement circled with
dashed lines. (B) Overlaid van’t Hoff plots from optical melting data of bPNAs (left) K2MAK2M and (right) c[K2M]2 with RNA rU4C4U4 and DNAs
dT4C4T4, dU4C4T4, dU2T2C4T2U2, and dU4C4U4 as indicated, with a schematic of T → U DNA oligos and RNA in hairpin conformation shown at
center. All melts were triplicated with standard deviation error bars shown.

Origins of Thermodynamic Differences between DNA
and RNA Hybrids with bPNAs. The most obvious global
trend was that bPNA binds more weakly to RNA (r(U4C4U4),
r(U4GUGAU4)) than to DNA (d(T4C4T4)), with ΔTm ∼20
°C and ΔΔG ∼ 4−5 kcal/mole in favor of the DNA hybrid
(Table 1). With structured RNA substrates that bear a U-rich
internal loop (URIL), bPNA affinity can be in the nanomolar
range with hybrid thermal stability ∼70 °C under similar
conditions; this enables the use of bPNA for ligand/label
installation10,29 as well as selective pulldown enrichment from
cellular total RNA.11 However, with short, unstructured
substrates, hybridization is required to drive folding; this
limits the overall binding efficiency and amplifies the
differences resulting from pyrimidine methylation and the
ribose 2′-OH group. We probed the role of the methyl group
by full and half T → U substitution in d(U4C4U4), d(U4C4T4),
and d(U2T2C4T2U2) DNA backbone substrates. For consis-
tency, we used the identical tetracytidine (C4) turn in all
sequences instead of the stabilized GUGA RNA loop; this
resulted in an across the board 8−9 °C drop in hybrid thermal
stability. In an echo of the prior report from Wang and Kool
for DNA and RNA triplexes,47 we found that the thymine
methyl group was always stabilizing to triplex formation.
With K2MAK2M, loss of all eight methyls resulted in a ΔTm of
−17.5 °C and ΔΔG25 = +3.2 kcal/mole, or +0.40 kcal/mole/
methyl (Figure 5, Table 2). This value is somewhat higher than
Kool’s reported methyl effect in DNA triplexes (4.0 kcal/18
methyls = 0.22 kcal/mole/methyl). The T → U replacement
of one side of the triplex (d(U4C4T4)) resulted in a ΔTm of −9
°C and ΔΔG25 + 1.8 kcal/mole, while replacement at the
terminus of the triplex (d(U2T2C4T2U2)) resulted in ΔTm of
−8.2 °C and ΔΔG25 + 1.5 kcal/mole (Table 2). The methyl
impact in half-site T → U substitution (0.38−0.45 kcal/mole/
89
Table 2. bPNA Hybridization with RNA, DNA, and (T →
U) DNAa
a
Conditions: 2 μM DNA/RNA, 2 μM bPNA, 100 mM NaCl, 10 mM
phosphate, and pH 7.5. Tm was obtained by first-derivative analysis
and found comparable to direct fit. ΔG25 calculated at 25 °C (kcal/
mole) for hybrid dissociation. Standard deviation errors from
triplicate measurements are shown in brackets.
methyl) therefore mirrors full-site substitution (0.4 kcal/mole/
methyl). Thus, in K2MAK2M hybrids, methyls account for a
majority of the ΔΔG25 (71%) and ΔTm (71%) observed
between DNA and RNA substrates.
This contribution is diminished with diketopiperazine
c[K2M]2 hybrids: methylation only accounts for 40% of
ΔΔG25 and 47% of ΔTm (Figure 5, Table 2). The majority
of ΔΔG25 (+5.2 kcal/mole) can be attributed to the 2′-OH
group, with ∼60% loss in binding free energy (ΔΔG25 = +3.1
kcal/mole) upon substitution of a DNA backbone (d-
(U 4 C 4 U 4 )) for RNA (r(U 4 C 4 U 4 )). Overall, T → U
replacement at all eight positions accounts for 40% loss
binding free energy (2.1 kcal/mole) with c[K2M]2, while it
accounts for a 71% loss (3.2 kcal/mole) with K2MAK2M.
Accordingly, in the diketopiperazine hybrid, the contribution
per methyl group for full substitution is decreased (0.26 kcal/
methyl/mole), as is half-site substitution (0.23−0.3 kcal/
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Biochemistry 2022, 61, 85−91
Biochemistry pubs.acs.org/biochemistry
methyl/mole), relative to the K2MAK2M hybrid. As the C5
methyl is thought to primarily improve base stacking in DNA
and RNA triplex formation,47 we infer that base-triple stacking
with c[K2M]2 is more optimized in diketopiperazine hybrid-
ization relative to tripeptide, thus minimizing the impact of
methylation. Given the strong influence of the 2′-OH on
backbone conformation,48−50 we conclude that distinct steric
environments, akin to differences between A-form dsRNA and
B-form dsDNA, are another core reason that bPNA binding to
RNA is weaker than to DNA.
■ CONCLUSIONS
Taken together, these data identify tunable structure−function
relationships for bPNA triplex hybridization to unstructured
oligo-T/U DNAs and RNAs. Synthesis and evaluation of
bPNA backbone variants have revealed strategies for bPNA
modification that are well tolerated by triplex hybridization, as
well as modifications that significantly improve or ablate
nucleic acid recognition. In particular, X residues in the
K2MXK2M scaffold may be altered freely or deleted; this finding
identifies this position as a reasonable site for label installation
alternative to the N-terminus, which has been previously used.
Deletion of the central position yields a compact dipeptide
scaffold that is useful in triplex hybridization targeting and the
shortest bPNA to date with molecular weight <1 kD. In
addition, we found that syndiotactic side chain presentation,
accomplished through the alternation of D, L configuration in
the open-chain k2MK2M bPNA and diketopiperazine c[K2M]2,
can significantly improve hybrid stability. This stability derives
in large part from stacking considerations: our studies on T →
U-substituted DNAs revealed that base-stacking accounts for
∼40−71% of the energetic advantage exhibited in DNA vs
RNA hybrids. Thus, bPNA triplex hybridization exhibits clear
structure−function tunability that resonates with prior studies
in triplexes formed from native nucleic acid backbones.47
While the studies herein were carried out at the limit of RNA
binding to unfolded U-rich 12-mers, we anticipate that the
simplified dipeptide scaffolds will be effective in intracellular
targeting of structured URIL RNA substrates as previously
reported for the Ala tripeptide, K2MAK2M.11 Importantly, the
studies herein provide rough general guidelines for targeting
noncoding interfaces in DNA and RNA through iterative
structural modification of bPNAs.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biochem.1c00693.
Detailed protocols, additional optical melting raw and
processed data, and compound characterization (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Dennis Bong − Department of Chemistry & Biochemistry,
The Ohio State University, Columbus, Ohio 43210, United
States; orcid.org/0000-0003-3778-9183;
Email: bong.6@osu.edu
Authors
Sarah Rundell − Department of Chemistry & Biochemistry,
The Ohio State University, Columbus, Ohio 43210, United
States
90
Article
Oliver Munyaradzi − Department of Chemistry &
Biochemistry, The Ohio State University, Columbus, Ohio
43210, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.1c00693
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by NIH (GM111995,
1R01GM143543-01), NSF (DMR 1802432) grants (to
D.B.), and the Center for RNA Biology at OSU.
■
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