Article
these samples. It was absent in the unmodified streptavidin
bead control sample (Figure 5D). No specific nucleobase
peptide variants were identified from the pre-miRNA-155
enrichments. The biohybrid nucleobase peptide binds to RNA-
hairpins with nanomolar affinity. To validate the binding of the
hit sequence to the pre-miRNA hairpins, we synthesized the
compound with a C-terminal biotin handle (39-biotin). 39-
biotin was immobilized on biolayer interferometry (BLI)
streptavidin tips and the association/dissociation to pre-
miRNA21 in solution at different concentrations was
determined: the dissociation constant (K ) was found to be
d
9
nM for pre-miRNA21 (Figure 4E) and 12 nM for pre-
miRNA-155 (Figure 4F). The affinity toward the pre-miRNA-
1 stem, missing the hairpin loop, was reduced by over 50-fold,
2
3
affinity to an unrelated hairpin derived from HIV RNA (SI
Figure 1). We also determined melting temperatures of pre-
miRNA-21 alone and in the presence of 39 using circular
presence of 39, however, a more defined melting transition was
found.
The binding of the nucleobase peptide hit to RNA hairpins
is selective over DNA hairpins, single stranded DNA (ssDNA)
and double stranded DNA (dsDNA). We wanted to determine
whether the binding of 39-biotin to pre-miRNA-hairpins was
based solely on nonspecific electrostatic interactions due to its
Figure 4. Hybrid nucleobase peptides have higher bulk affinity to
oligonucleotides than canonical peptides. (A) Schematic representa-
tion of the ELISA assay: immobilized nucleobase peptide or canonical
peptide library, respectively, is incubated with a biotinylated DNA
library. (B) ELISA readout diagram: nucleobase peptides have higher
affinity toward DNA than canonical peptides.
4
positively charged residues. Other negatively charged
oligonucleotides, such as a DNA sequence with the same
primary sequence as pre-miR-21, ssDNA, or dsDNA, in that
case, would be expected to bind to the biohybrid compound
with similarly high affinity. BLI measurements indicated that
5G).
expression in cells is controlled by miRNAs and miRNAs are
42,43
correlated with disease.
Binders to structured hairpin
precursor miRNA can potentially inhibit the processing to the
44
mature and active miRNA. Two pre-miRNAs involved in
45
46
Single-point nucleobase substitutions in 39 to alanine were
tolerated, as mutation of all four nucleobases caused up to 100-
fold decrease in affinity for RNA. To investigate the role of the
nucleobase monomers and individual residues in the hit
sequence, we synthesized several analogues. First, the 4
nucleobase monomers were mutated to either alanine,
tryptophan, or glutamine. These variants showed reduced
binding affinity for pre-miRNA21 (>50−100-fold decrease;
Figure 5G). The need for a combination of side chains might
provide clues regarding why one sequence was identified in the
affinity selection and no other consensus compounds as
disease are pre-miRNA-21 and pre-miRNA-155.
We envisioned our nucleobase peptide library could be used
to discover high affinity pre-miRNA-hairpin binders. Biotiny-
lated pre-miRNAs (21 and 155) were immobilized on
magnetic streptavidin beads and incubated with the 100-
million membered nucleobase peptide library ([library] = 1
mM; [individual library member] = 10 pM; Figure 5A). Yeast
RNA was added to the selection conditions to scavenge for
nonspecific RNA binders. After washing to remove unbound
material, potential hits were eluted and subjected to nanoLC-
MS/MS. As a negative control, the same workflow was done
with unmodified streptavidin beads. The resultant MS spectra
were visualized with the sequencing software PEAKS 8.5 and
further refined with a Python script that nominated variants
matching the library design. Finally, an extracted ion
chromatogram was created for each variant (based on the
precursor ion used by PEAKS for sequencing): only
compounds resulting in a clear peak in the chromatogram
were considered “hits” (Figure 5D). Cross-analysis of the same
ion in the control chromatograms (beads only) allowed to
exclude potential unspecific bead or streptavidin binders. After
these steps, for enrichments with the pre-miRNA21, only one
nucleobase peptide was identified: Lys-DabG-hAlaA-Arg-Asp-
DabA-Leu-hAlaG-Ala-Lys (Figure 5B,C). The variant ions
were also identified in the LCMS chromatograms resulting
from enrichments with pre-miRNA-155: due to their low
intensity, however, no sequencing data were obtained from
29
observed in other target based AS-MS approaches. Next, we
performed an alanine scan at each position. Mutation of the N-
terminal lysine and the arginine in position 4 depleted the
binding. Single mutations all other positions were tolerated
and did not significantly affect the binding to pre-miR21. A
scrambled variant of 39 showed ∼10-fold decreased binding
affinity (Figure 5G).
DISCUSSION
■
Abiotic sequence defined polymers might be new modalities
for the development of functional materials or therapeu-
47−50
tics.
In this work, we showed the design and synthesis of
SPPS-ready nucleobase monomers. The robust synthesis
procedures allowed for preparation of multimillion membered
combinatorial libraries and this class of materials had enhanced
affinity for nucleic acids, compared to canonical peptides.
F
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Pomplun, Sebastian
