A Macrocyclic Peptide Ligand Binds the Oncogenic MicroRNA-21

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A macrocyclic peptide ligand binds the oncogenic microRNA-21 precursor and suppresses Dicer processing Matthew D Shortridge, Matthew J Walker, Tom Pavelitz, Yu Chen, and Gabriele Varani ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 25, 2017

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ACS Chemical Biology

A macrocyclic peptide ligand binds the oncogenic microRNA-21 precursor and suppresses Dicer processing

Running Title: Disruption of pre-miR21 processing by a β-hairpin peptide

Matthew D. Shortridge¶, Matthew J. Walker¶, Tom Pavelitz¶, Yu Chen¶† and Gabriele Varani¶*

¶

Department of Chemistry, University of Washington, Seattle, Box 351700, Seattle, USA 98195 †Current address Seattle Children’s Research Institute, 1900 9th Ave, Seattle, 98101*

Address correspondence to: GV [email protected] 1 206 543 7113 (Tel) 1 206 685 8665 (Fax)

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MicroRNAs (miRNAs) help orchestrate cellular growth and survival through posttranscriptional mechanisms. The dysregulation of miRNA biogenesis can lead to cellular growth defects, chemotherapeutic resistance and plays a direct role in the development of many chronic diseases. Among these RNAs, miR-21 is consistently overexpressed in most human cancers leading to the down regulation of key tumor suppressing and proapoptotic factors; suggesting that inhibition of miR-21 biogenesis could reverse these negative effects. However, targeted inhibition of miR-21 using small molecules has had limited success. To overcome difficulties in targeting RNA secondary structure with small molecules, we developed a class of cyclic β-hairpin peptidomimetics which binds to RNA stem loop structures, such as miRNA precursors, with potent affinity and specificity. We screened an existing cyclic peptide library and discovered a lead structure which binds to pre-miR21 with a KD=200nM and prefers it over other premiRNAs. The NMR structure of the complex shows the peptide recognizes the Dicer cleavage site and alters processing of the precursor to the mature miRNA in vitro and in cultured cells. The structure provides a rational for the peptide binding activity and clear guidance for further improvements in affinity and targeting.


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The multitude of functionally active non-coding RNAs (ncRNAs) and the presence of functional SNPs within them have revolutionized our understanding of gene expression

1-5

. These RNAs fold into complex secondary and higher order structure that

are responsible for their activity, and their function can be affected in patients by single nucleotide changes, suggesting that they could be targeted for therapeutic intervention 1, 2

. However, there are only limited options to target RNA. Anti-sense oligonucleotides

6

can bind to the mature functional form of microRNAs and engineered proteins7 can selectively target ncRNA sequences and structures in vitro and in cells, but both have well-known pharmacological limitations. Therefore, there is increasing interest in developing RNA-targeting small molecules8-10, encouraged by the successful targeting of riboswitches11 and the development of small molecule sensing aptamers12,

13

.

However, riboswitches and aptamers represent uniquely complex structured RNAs, which were naturally or artificially selected to bind to small molecules12-14. Developing small molecules to specifically target RNA structures typically involved in protein-RNA interactions that subtend ncRNA function (stem loops, internal loops, and bulges) remains extremely challenging, despite significant efforts over the last two decades

8, 9,

15-21

. As in the case of targeting protein-protein interactions, peptide chemistry offers

an attractive alternative to small molecules to targeting RNA and RNA-protein interactions22,

23

because the increased surface area facilitates competition for large

macromolecular interfaces. Our group has successfully used cyclic β-hairpin peptide mimetics to target RNA secondary structures, including the REV response element RNA24 and TAR RNAs from HIV and BIV25-29. The peptides are constrained by a D-



Page 4 of 43

pro/L-Pro template to induce type-2’ β-hairpin folding and improve cyclization during synthesis30. In aqueous solution, the peptides are further stabilized by extensive crossstrand hydrogen bonding30. Head-to-tail backbone cyclization reduces cellular exopeptidase activity while the structural rigidity provides greatly improved binding selectivity, compared to linear or cyclic unstructured peptide mimics (e.g. peptoids15, oligo-carbamates or γ-amino acids31). Once a lead peptide is identified, its activity and selectivity can be increased to reach pM by structure based rational methods. Importantly, the stability of cyclic β-hairpin follows a distinct pattern allowing the molecules to be lengthened or shortened in a predictable manner, and peptide stability mirrors their RNA binding affinity27, 32. Our

previous

work

demonstrated

this

class

of

peptidomemetic

structures

preferentially binds to junctions between A-form helices and single stranded or bulge regions; sites also important for RNA-protein interactions33. These peptides have excellent cellular uptake and are active against many HIV isolates in cells26. We reasoned that this chemistry could also provide a general approach to targeting RNA stem-loops as found in microRNA precursors. Therefore, we screened a library of cyclic, β-hairpin peptides against a shortened hairpin structure mimic of precursor miRNA-21 to identify a lead structure for subsequent optimization. MicroRNAs (miRNAs) are small endogenous non-coding RNAs (21-23 nts long) which recognize mRNA sequences, typically within the 3’-untranslated region to posttranscriptionally regulate gene expression. Interfering with the biogenesis of microRNAs by targeting the stem loop structure common to the primary (pri-miRNA) and precursor (pre-miRNA)

structures

may

provide

an

alternative


strategy

to

antisense

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oligonucleotides to reducing mature miRNA levels in disease conditions where they are over-expressed

34

. Peptoids, aminoglycosides and small RNA binding molecules with

very broad and non-specific activity, have all been identified to bind the terminal stemloop region of several pre-miRNAs, including miR-10b17, miR-2123, 35, 36, miR-9616 and miR-15537. Based on preliminary analysis of the structure of pre-miR21, which contains a large unfolded loop, we reasoned that it would be challenging to identify small molecules that bind to this RNA with high affinity because of the entropic penalty associated with loop rigidification. Therefore, we screened a cyclic peptide library. Herein we report the identification of a cyclic peptide that binds to pre-miR21 with a Kd of 200 nM selectively over related miRNAs and the first high resolution structural analysis of pre-miR21 in both free and bound forms. We use NMR to determine the structure of the peptide-RNA complex and provide a structure rational for its binding and activity in inhibiting pre-miR21 processing by disrupting the Dicer cleavage site.



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RESULTS AND DISCUSSION The pre-miR21 hairpin is capped by an unstructured apical loop – Primary miRNA (pri-miRNA) transcripts are first cleaved in the nucleus by the microprocessor complex including proteins Drosha and DGCR8 generating a 60-70 nts precursor (premiRNA) stem loop (Fig. 1A; in the case of miR-21, the pre-miRNA is 59 nts long). The pre-miRNA is exported to the cytoplasm by exportin 5 and further processed by Dicer to release the functional mature miRNA. The mature pre-miR21 sequence is located on the 5’ strand of the stem loop between residues U8 and A29 (figure 1A, red) as numbered from the primary sequence found in miRBASE v21

38, 39

. The Dicer cleavage

site is located within the apical portion of the stem-loop between A29 on the 5’ strand and C46 on the 3’ strand. We reasoned that the most significant effects of an inhibitor would be observed for ligands that bind to this region of the pre-miRNA and therefore chose to assay new ligands using this portion of the stem loop. Assaying the complete pre-miRNA sequence might have led to the more facile discovery of ligands, at the cost of identifying non-specific ligands whose affinity would be probably boosted by avidity. The model pre-miR21 sequence used in this study spans residues 22-52 (figure 1A, highlighted by the blue box) and encompasses the entire DICER cleavage site. Comparing the imino proton region of the NMR spectra for both the full-length premiRNA (figure 1B, black) to our shortened apical section (figure 1B blue) reveals the two sequences have similar resonances for the common base pairs. This result suggests that our smaller hairpin is a good model to identify ligands which recognize the Dicer cleavage site in pre-miR21.


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To further characterize the target sequence prior to ligand screening, we used NMR to establish the structure of free wild type pre-miR21 stem-loop. To date, high resolution structural information on pre-miRNA hairpins has been limited to computational models or mutated structures

40-43

. Surprisingly, only one wild type pre-

miRNA hairpin loop structure (miR-20b) is deposited in the PDB

43

. The NMR analysis

of both full length and our shortened pre-miR21 demonstrates a relaxed terminal loop structure, where most of the loop imino signals in the 1D 1H and 2D 1H NOESY spectra are missing due to the absence of base pairing. Eight stable base pairs in the short premiR21 apical loop give rise to clear NOE walks typical of nucleic acid helices (figure 1C), and generally have corresponding resonances in the complete pre-miR21 (figure 1B). In

15

N-1H HSQC spectra recorded at 5C, we also observed four weak and broad

peaks with chemical shift values corresponding to tandem U31:G44/G32:U43 pairs that breathe and open frequently, leading to rapid exchange with solvent and peak broadening (figure 1D). Altogether, the NMR data are consistent with the relaxed structure suggested by mutational analysis, NMR, SHAPE, in-line probing and nuclease digestion 7, 23, 35, 40, 41, 44. To establish the structure of wild type free pre-miR21 hairpin apical loop and provide a starting point to study ligand binding, we obtained nearly complete chemical shift

assignments

of

exchangeable

and non-exchangeable resonances

using

established methods45, 46. However, initial attempts to fold the structure using traditional methods (starting from an extended conformation, folding the RNA using NMR-derived distance constrains, followed by refinement) failed to produce a converged structure



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(figure 2A). The absence of long range NOEs from imino signals along with many missing long range and sequential NOEs within the loop region was the reason the apical loop portion would not converge. Thus, we turned to a mixed in silico and experimental method to help converge the apical loop structure. First we used the secondary structure and non-exchangeable proton chemical shift values at 37oC to model the free structure using the FARFAR de novo RNA structure tool

47, 48

. The converged structure from FARFAR modeling was

then subjected to refinement with 349 NOEs, 131 dihedral constraints, and database potential using NIH-XPLOR methods, as previously described

49, 50

(SI table 1). This

method yielded a set of converged structures consistent with the NMR results (figure 2B, and SI table 1), but the overall rmsd values for the apical loop portion remain high for the top 10 structures (SI table 1), consistent with the absence of base pairing. The NMR refined models in figure 2 suggests the pre-miR21 hairpin used in this study is folded with the bulged A29 residue sandwiched between the G28:C46 and C30:G45 base pairs, but the apical loop is unstructured. The good convergence of the stem region and lack of resolution in the terminal loop are not an artifact but a direct result of stark differences in NOE density. We observed an average of 24 NOEs per nucleotide in the helical region but, above A29, the average number of NOEs dropped to 9 per residue; with the majority of these NOEs originating from intrabase connectivities. Interestingly, the amino signals of A29 are clearly visible with interactions to the GC base pairs above and below it, suggesting that A29 is stacked within the helix.


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The relaxed structure of the hairpin suggests that it would be difficult to discover small molecules that would bind to this pre-miRNA with strong activity and especially specificity, because of the entropic penalty associated with rigidification of the RNA structure upon ligand binding. While riboswitches and aptamers, as well as ribosomal RNA, provide examples of specific RNA recognition by small molecules, these interactions usually require the formation of tertiary interactions, sometimes stabilized or induced by binding of the small molecule. Larger ligands with increased surface area, such as peptides, would be more likely to gain sufficient binding energy to conformationally constrain the apical loop and compensate for the entropic loss associated with rearrangements and loss of flexibility of the loop. Discovery of a cyclic peptide that binds to pre-miR21 hairpin with nM affinity: We used EMSA to screen a cyclic peptide library designed to model the RNA binding domain of BIV TAT

29

. Later

generation of these peptides were optimized and elaborated to bind to either the BIV or HIV TAR sequence with high affinity and specificity

27, 28

; to improve the likelihood of

finding hit ligands to pre-miR21, we prioritized screening with non-optimized peptide libraries generated in the early phases of those projects which often bound to those RNAs with µM activity. An initial screen identified 21 peptides (out of 54, 38%), which reported a “hit” response when screened by EMSA at 4µM peptide and 1nM pre-miR21. These screens were conducted in the presence of 250x fold excess of tRNA to reduce nonspecific binding, which has dogged many projects aimed at discovering RNA-binding ligands (SI figure 1). The peptides showing the strongest, fully bound shift by EMSA in SI figure 1 were further screened to measure dissociation constants (eq. 1) and identify the L50



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peptide {cyclo(RVRTRGKRRIRRpP) to bind to the pre-miR21 hairpin with an apparent KD of 200nM, measured in the presence of 250x fold excess of yeast tRNA (figure 3). Thus, we focused our investigation on identifying the binding site and understanding the mechanism of binding and structural basis for the activity of this hit structure. Previous studies on small molecule inhibitors of pre-miR21, suggested that the bulge A29 residue plays an important role in ligand recognition

51

. When we screened a

miR-21:∆A29 mutant, we observed a 5-fold reduction in affinity towards L50 (KD = 1µM) in the presence of excess tRNA (figure 3). However, inspection of the NMR data for the mutant indicate that the decrease in binding may be related to changes in RNA structure, rather than A29 playing a direct role in ligand recognition. The miR-21:∆A29 mutant stabilizes at least one of the U:G tandem wobble pairs in the terminal loop, as shown in the NOESY spectrum for this sample (SI figure 3). Removing this bulge nucleotide could therefore reduce apparent ligand binding by restricting the size of the loop without directly participating in the interaction with the peptide. To further probe the binding site selectivity and pre-miRNA specificity of L50, we measured the binding of L50 towards pre-miR-145, an unrelated pre-miRNA, and a premiRNA chimera composed of the helix of pre-miR21 and the loop sequence of pre-miR145 (miR21-loop145) (figure 3). L50 binding to pre-miR-145 (KD 1-4µM) was reduced 10-fold compared to wild type pre-miR21 and by a lesser extent compared to the miR21:∆A29 mutant. Conversely, the miRNA chimera retained comparable affinity for L50 relative to pre-miR21, suggesting that the lower helix structure and not the apical loop, is responsible for the peptide binding affinity. However, the result on the ∆A29 mutant indicates that a large unpaired loop is required for this interaction to occur at all.


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Lastly, we conduced competition-binding studies with L50-bound pre-miR21 and unlabeled pre-miR-18a or pre-miR21 (in addition to 250x fold excess tRNA) (figure 3B). L50 was first titrated with 1nM labeled pre-miR21 until the concentration equaled the affinity of the complex to form a 50-50 complex. Next, unlabeled pre-miR-18a or premiR21 was titrated into this complex. While the addition of unlabeled pre-miR21 titrated away L50 from labeled pre-miR21, pre-miR18 failed to do so. Altogether, these data suggest that L50 binds to the pre-miR21 hairpin with mid nM affinity and some selectivity against other pre-miRNA sequences. L50 targets a binding site within pre-miR21 common to other ligands. The binding results for L50 suggest the ligand binds to pre-miR21 by straddling the A29 bulge, with specific contacts to the lower helix but also secondary contacts to the loop residues. This would mimic the proposed binding site of the previously reported ligand streptomycin

35

suggesting the two ligands recognize a common binding site in the pre-

miRNA-21 sequence. To explore this possibility, we used both ligand- and targetdetected NMR screening methods to establish if the two ligands (figure 4A) were competitive, non-competitive or exhibited cooperative binding. We first collected ligand detected 1D 1H proton competition assays to establish if L50 can displace streptomycin (figure 4B). A 1D-1H NMR spectrum was collected for a 30µM sample of streptomycin sulfate; to this sample, we added 10µM pre-miR21 (figure 4B bottom). Binding to RNA (or other macromolecules) increases the rotational correlation time of the small molecule ligand, leading to broadened peaks compared to the free molecule (figure 4B middle)

9, 35

. To this ligand-bound sample, we added

increasing amounts of L50 and recorded spectra until the concentration of L50 and



streptomycin were equal. If L50 were competitive with streptomycin, the small molecule ligand will be displaced by the peptide and show an increase in peak intensity comparable to free ligand. However, if the two ligands are non-competitive or exhibit cooperative binding, the streptomycin signals will remain broadened upon addition of L50 peptide. In figure 4B top we observe, at equal ligand concentrations, that the streptomycin signals return to the sharp, intense peaks observed for the free small molecule, suggesting L50 is competitive with the streptomycin binding site. To establish if non-specific contacts or site-specific binding drive the L50-premiR21 interaction, a 700µM sample of pre-miR21 was titrated with 700µM peptide and changes in chemical shift of pyrimidine H5/H6 resonances were recorded by 1H-1H 2D TOCSY experiment (figure 4C black). At 37oC, the 16 cross peaks associated with the pyrimidine H5/H6 protons are clearly identified, with C30 and C46 broadened; these resonances are located near the A29 bulge. Consistent with the binding results above, the terminal loop resonances show negligible changes in chemical shift upon binding of the L50 peptide, but residues near the Dicer cleavage site (U26, U27, C30, U43 and C46) are significantly shifted (figure 4C red). These data support site-specific binding and corroborate the mutational analysis above. By comparison, after running the same experiment with streptomycin, we were only able to detect weak changes in chemical shift; even at 4-fold excess of the molecule (figure 4D). The most notable changes were for C30 and C45 near the Dicer cleavage site. Thus, despite having reportedly 2-fold stronger affinity than L50 (L50 ~200nM vs streptomycin 100nM), streptomycin induced much smaller changes in the TOCSY chemical shifts. We suspect this apparently higher affinity coupled with ease of displacement by L50, might be due to diffuse, non-site


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specific binding of streptomycin to the complete RNA molecule leading to apparent higher affinity through avidity as opposed to specific site-specific contacts with the A29 bulge. Lastly, we were able to show that competition between L50 and streptomycin was due to ligand displacement and not induced by unfolding of the RNA helix. When bound to L50, the pre-miR21 structure maintains the lower stem base pairing observed in the free RNA (figure 5A). Similar to the free pre-miR21 structure (figure 5A bottom), we did not observe imino resonances for the apical loop portion of pre-miR21 above A29 when bound to L50 (figure 5A top). We did notice, however, the amino resonance signals from A29 were significantly broadened and no longer observable in the L50 bound complex. We also observed the largest chemical shift changes for U27 H3 and G28 H1 resonance while the remaining imino peaks of the lower stem showed only minimal changes in chemical shift. Lastly, at 37oC we observed nearly 100 intermolecular NOEs (figure 5B) for the L50:pre-miR21 complex, indicative of a welldefined site specific complex. This is a first for a ligand bound to a pre-miRNA and suggested formal structure determination of the complex was possible. Conversely, we did not observe a significant number of intermolecular NOEs between streptomycin and the pre-miR21 hairpin (SI figure 4); only 6 NOEs were identified and did not mark a consistent binding site, further suggesting this interaction is driven by non-site specific binding. Structure of the L50-pre-miR21 complex: Peptide backbone and side chain assignments of the free L50 peptide were initially made using the pattern of i−i + 1 Hα to NH NOESY cross-peaks characteristic of an antiparallel β hairpin, and assignments of



the arginine residues was aided by recording natural abundance

Page 14 of 43

15

N- and

13

C-HSQC

experiments. After establishing HN and Hα chemical shifts, side-chain resonances were assigned using the 2D 1H-1H TOCSY and 1H-1H NOESY spectra in 99% D2O and 95%H2O/5% D2O. In the RNA complex, the overall pattern was the same for the peptide, however, even at 800MHz, many of the side chain peaks were significantly overlapped, which limited their unambiguous characterization, particularly for Hβ, Hγ and Hδ protons. This is exemplified in figure 5C, where nearly 50% of the peptide Arginine Hα chemical shift values fall within ~0.2ppm and the side chain chemical shifts are nearly uniform. Therefore, despite having nearly 100 intermolecular noes, we were only able to unambiguously assign 14 of them to fold the RNA:peptide complex in XPLOR-NIH 49, 50. The peptide bound RNA spectrum was also significantly broader than the free RNA (figure 5C), but the overall pattern of RNA peaks for the L50-bound pre-miR21 NOESY was similar to the free structure; which helped establish RNA assignments for the complex. Unlike the free structure, we were able to fold the peptide:RNA complex using traditional methods (starting from an extended structure, folding by NOEs, followed by refinement with dihedral, planarity and database potential restraints). However, similar to the free structure, the apical loop region remains largely unstructured as the peptide does not make strong interactions with and constrain apical loop bases (figure 6A&B). The structure identifies critical contacts, the relative orientation of the peptide within the binding site and generates clear hypotheses for ligand improvement. Peptide L50 sits in the major groove at the junction between the single stranded RNA loop and


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the double stranded RNA; as predicted from the binding data (figure 6). The peptide is flipped compared to the TAR structures25,

28, 29

with the Dpr13/Pro14 turn pointing

towards the apical loop and the Gly6/Lys7 turn pointing down towards the helical stem. Two sets of intermolecular NOEs were particularly important to establish the position of the peptide against pre-miR21. Unambiguous intermolecular NOEs between U26 and Ile 10 (figure 5C) are distinctive and this amino acid is necessary for binding. Any change to Ile10, even to other branched aliphatic residues, for example, abolished observable binding to the pre-miR21 sequence. In addition, Val2 has clear NOEs with U31, but in this case, the pre-miR21 binding results suggest Val and Thr are accepted at position 2. The combination of these intermolecular NOEs with the rigid structure of the peptide gives us full confidence in the position of the peptide against the RNA surface. Significantly, the L50 peptide was the only ligand in our library that bound to premiR21 with sufficient affinity to allow us to determine a structure. Its positively charged residues (7 Arg and 1Lys) all appear to interact with phosphate backbone rather than with specific bases, as observed instead in HIV TAR 28. As such, there might be ‘sliding’ in the binding pocket, resulting in overall peak broadness (figure 5C) and relatively high rmsd values for the full complex (figure 6B). L50 is also the only peptide in our library that contains an arginine at position 12, suggesting Arg12 is necessary for binding. However, although Arg12 is clearly resolved in the free peptide spectrum, we were unable to unambitiously assign the side chain chemical shifts in the pre-miR21 bound spectrum; even at 800MHz. From the structure, Arg12 appears to interact with the backbone phosphate of G28. Peptides in our library that contain either Val or Ile in



Page 16 of 43

position 12 do not bind at all, and would not be able to form this strong electrostatic interaction. Peptide L50 disrupts miR21 biogenesis in vitro and in cells: In order to test whether peptide binding would inhibit processing of pre-miR21 to the mature miR-21, we conducted Dicer assays on a chemically synthesized full-length pre-miRNA. At first, we used commercially available enzyme (Genlantis) titrated with increasing amount of L50 peptide (0-10µM). Dicer reaction conditions mimicked single turnover state, with higher levels of enzyme compared to limiting RNA amounts. The reactions were carried out for 90min at 37oC and analyzed on 12% denaturing polyacrylamide gel. Under these conditions, we observe 25% reduction in Dicer processing at 100nM ligand but do not see 50% reduction until nearly 10µM ligand concentration is reached (SI figure 5). These results are surprising, but comparable to those published for Dicer inhibition assays with different chemistries including peptides, small molecules, and even a protein that bind pre-miR21 with low nM affinity

7, 35, 41, 42

. Clearly, inhibition does not

match the affinity of the ligands. To explore this issue, we looked at the time course of the processing reaction for pre-miR21 in the absence and presence of L50 and noticed that processing is very inefficient (figure 7). This feature has been reported by others for this specific premiRNA, but not for other miRNA precursors such as Let7

52-55

. Inefficient processing in

vitro might be due to the absence of ancillary proteins, such as TRBP in the commercial enzyme kit

54-56

. Therefore, we reasoned that addition of TRBP might stimulate pre-

miR21 processing and perhaps even lead us to re-evaluate the inhibitory activity of the


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lead peptide. We expressed and purified in house the DICER-TRBP complex using established protocols

57

, then reaction conditions were optimized to use 35nM enzyme

complex, 200pM RNA in reaction buffer (20mM Tris pH 7.5, 25mM NaCl, 1mM DTT and 1.5mM MgCl2). The in house purified Dicer complex processed pre-miR21 much more efficiently compared to the commercial kit, with clear product formation starting to show after 1.3min for in house purified Dicer-TRBP (figure 7B) compared to 5min for Genlantis dicer (figure 7A). We also found that 40min were sufficient to process pre-miR21 substantially, compared to 80min for the commercial kit (figure 7). By comparison, published inhibition studies using commercial kit assay activity at between 4-16hr reaction times 7, 35, 41, which might be too long to report on genuine enzyme inhibition. Pre-miR21 is processed inefficiently and this is not a characteristic of the source of Dicer enzyme, but activity of in house purified enzyme and/or the presence of TRBP render the enzymatic activity much more robust. Satisfactorily, inhibition of pre-miR21 processing is substantial (though not complete) at 10µM peptide concentration (figure 7B). Strikingly, when bound to L50, the in house purified Dicer-TRBP complex generates new intermediate (red dagger, figure 7B) and product (red double dagger, figure 7B) bands, compared to the commercial enzyme kit (black star figure 7A&B). To test whether this promising activity is sustained in cells, we conducted pre-miRNA processing assays in HEK293 cells where miR-21 is produced from an over-expressing plasmid. We reasoned that expression from a plasmid would be necessary to test an inhibitor of the maturation pathway and resorted to this cell line because it has low natural abundance of the mature miR-21 sequence. It would be very difficult to observe



Page 18 of 43

decreased processing efficiency in cells containing high endogenous levels of miR-21 (e.g. HeLa, MCF7 or PC3) because mature miR-21 has a life-time of 3-5 days; we question whether even an exceptionally efficient inhibitor of maturation would lead to significant decreases in levels of mature miR-21 when the endogenous level is very high and in fact, when we attempted to study changes in miR-21 against the background of high levels of endogenous RNA, changes were not significant. When miR-21 was over-expressed from a plasmid in HEK-293 cells, we are able to induce a 5-fold increase in mature miR-21 (figure 8), but a much greater level of primiR-21, suggesting that in HEK cells as well, pre-miR21 is processed inefficiently. Upon addition of the L50 peptide, we noticed a concentration-dependent decrease in the concentration of processed mature miR-21, except at the highest peptide concentration (100µM), where toxicity might stimulate expression of endogenous miR-21 by an independent mechanism (data not shown). While the activity is observed at relatively high concentration, we have not optimized the peptide for cell penetration (we did not use any transfecting agent to simulate realistic conditions), nor peptide stability against endopeptidases. CONCLUSIONS: Disrupting

the

biogenesis

of

miRNA

expressed

in

cancer

with

non-

oligonucleotide chemistry has many attractive features but is also very challenging10, 16, 35, 41, 58-62

. Furthermore, the absence of structural data has limited progress towards the

optimization of promising hit structures. Here we report the discovery of a peptidic inhibitor of microRNA processing and the structure of a human pre-miRNA bound to the


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cyclic peptide. We focused our assays on peptide mimics because the structure of free pre-miR21 revealed a highly flexible large apical loop that presents a very challenging target for small molecule chemistry. Outside of the larger apical loop, the pre-mIR21 hairpin appears devoid of unique structural features that would provide binding site for small molecules, as observed for example in other RNAs targeted for drug discovery such as TAR or RRE from HIV, or the Hep C IRES

9, 24, 63

. The relaxed structure of the

hairpin suggested to us that ligands with larger interface area, such as peptides, would be required to gain the binding energy required to conformationally constrain the apical loop and compensate the entropic loss associated with loop rigidification. Thus, we identified a cyclic peptide from a small library that binds to pre-miR21 with a KD = 200 nM at the ssRNA/dsRNA junction between the terminal loop and helix. The peptide binds to a site that overlaps the Dicer cleavage site and inhibits pre-miR21 processing by Dicer both in biochemical assays with purified components, and in cellbased assays. The larger, structured peptide easily displaces the small molecule ligand despite streptomycin having an apparent tighter affinity. We suspect the increased affinity for streptomycin is a result of avidity from multiple weak binding sites as evidence in our NMR data and mapped onto free pre-miR21 structure (SI figure 4) and not specific binding. The in vitro Dicer assays showed that the peptide inhibitory activity is lower than the binding activity (200 nM), a gap observed for other pre-miRNA inhibition chemistries, including streptomycin

35, 41, 42

and a designed RNA-binding protein 7. The

NMR structure of peptide L50 bound to pre-miR21 shows that the peptide binds to the RNA major groove, while Dicer chemistry appears to take place on the minor groove



side of the RNA, about ~21 nts away from the 5’ phosphate group recognized by the PAZ domain which acts like a molecular ruler

56

. The lower in vitro activity might be due

to the lack of direct competition between the ligand and the enzyme, or with magnesium ions important for in vitro Dicer cleavage. The discovery of peptide L50, as described here, provides a starting point for the identification of such a ligand by extending the peptide interactions to target the apical loop of pre-miR21. Cyclic peptides of this class represent the most robust ligands to specifically bind RNA secondary structures we have investigated to date and paths for optimization by rational and combinatorial methods have been demonstrated in our previous work 24, 25, 27-29

Methods:

Please see the Supporting Information for details.


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ASSOCIATED CONTENT Accession Codes: PDB and BMRB codes Free pre-miR21: 5UZT, 30257 Bound pre-miR21: 5UZZ, 30258

Acronyms used: TAR: Trans-activation Response element miRNA: mature microRNA pre-miRNA: precursor miRNA, after Drosha cleavage pri-miRNA: Transcribed RNA hairpin prior to Drosha cleavage. NMR: Nuclear Magnetic Resonance NOESY: Nuclear Overhauser Enhancement Spectroscopy TOCSY: Total Correlation Spectroscopy HMQC/HSQC: Homonuclear multiple quantum coherence/ Homonuclear single quantum coherence KD: Dissociation constant AUTHOR CONTRIBUTIONS M.D.S, Y.C. and G. V. conceived the project; M.D.S performed NMR experiments, resonance assignments and structure calculations; Y. C. performed the peptide screening; M.J.W performed Dicer assays and T.P. performed cell based assays. M.D.S and G. V. analyzed the data and wrote the manuscript. All authors have read and edited the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

SUPPORTING INFORMATION AVAILABLE: The Supporting Information is available free of charge on the Internet. Experimental procedures, supporting figures S1-



Acknowledgments: This work was supported by grant NIH-NIGMS 1RO1 GM103834. M. Shortridge was partially supported by American Cancer Society fellowship PF-13056-01-RMC. We would like to thank K. Sarka and W.Yang for help preparing RNA and Dicer-TRBP complex samples.


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Figure for Graphical Abstract: Representative free and bound pre-miR21 structures described in this manuscript



Figure 1: A) Secondary structure prediction of the pre-miR21 hairpin with the mature miR21 sequence in red. The full length sequence is shown and boxed with black outline, G8 and U65*** were swapped compared to the wild type sequence to improve in vitro transcription. The apical loop portion used in this report is boxed with a blue line and A23 was switched to G23* to improve in vitro transcription. Orange dashes are potential weak base pairs observed 1 only at 5C. B) The 1D H imino region for both the apical loop (top, blue) and the full length (bottom, black) show corresponding signals (black line) and that both transcripts lack imino signals from the apical loop region. C) The 2D 1 1 15 1 H- H NOESY walk and D) N- H HSQC for the short pre-miR21 sequence from which NH assignments were derived. Similar data were collected for the full pre-miR21 to obtain the assignments shown in part B.


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Figure 2: The top 20 low energy structures of the wild type pre-miR21 hairpin. A) Using a traditional folding scheme, we were unable to identify a converged set of structures for the apical loop region of pre-miR21. This is largely due to 47, 48 the lack of long range and sequential NOEs. B). To model the structure, we first used FARFAR with 123 nono exchangeable chemical shift values obtained at 37 C to generate an initial fold, followed by refinement using NIH49, 50 XPLOR protocol with 349 noes and 131 dihedral angle constraints.



Figure 3: A) Binding of L50 peptide to pre-miR21 apical loop and variants by EMSA; all RNAs were at 1nM. Both wild type and the miR-21/miR-145 chimera bind to L50 with a KD near 200 nM. Removing the bulge A29 or binding to premiR145 shifts the binding to KD>1µM; non-specific binding is suggested by the smearing observed at 4µM peptide concentration. B) Competition gel between pre-miR21 and pre-miR18 for L50 binding. Secondary structures are found in SI figure 2.


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Figure 4: A) Pre-miR21 ligands L50 and streptomycin B) NMR competition assay; when the free sample of 30µM streptomycin (bottom) is titrated with 10µM pre-miR21 (middle) binding is confirmed by the increased streptomycin linewidth (*). When 30µM of L50 (+) is further titrated into the streptomycin-bound sample (top), the streptomycin resonances (*) approach the RNA-free ligand intensity and linewidth; indicating that L50 is competitive with o streptomycin and probably binds to an overlapping site C) Free pre-miR21 hairpin at 37 C (black) was titrated with L50 peptide (red). The largest chemical shift changes (cyan lines) were observed for residues near the Dicer cleavage site. Terminal loop residues do not shift, suggesting the peptide binds to a specific location near the Dicer cleavage site generating clear changes in the chemical shifts of resonances near the binding site. D) When the same experiment was repeated with streptomycin sulfate, we observed only small diffuse changes in the RNA chemical shifts (cyan lines).



Figure 5: A) The L50 bound pre-miR21 imino spectrum (top) maintains the base pairing of the free RNA spectrum (bottom) indicating that competition between streptomycin and L50 is not due to RNA unfolding. G28 was the most significantly shifted imino resonance and the A29 NH2 peaks broaden further in the bound spectrum supporting the 1 1 idea the peptide straddles the A29 bulge B) The 2D H- H NOESY spectrum of the L50-pre-mIR21 complex in D2O buffer shows nearly 100 intermolecular NOEs between the RNA and peptide (many in the red-boxed region of the spectrum). C) Zoomed-in regions of the NMR spectrum of part B. Unfortunately, many of the intermolecular NOEs were from severely overlapped Arginine side chains and we were not able to unambiguously identify many contacts, even with increased resolution at 800MHz.


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Figure 6: A) Cartoon representation of the lowest energy structure for pre-miR21 bound to L50 peptide. The D-pro/LPro turn is pointed towards the top of the apical loop with the Gly/Lys turn pointing down towards the helical stem. The nucleotides that exhibited significant changes in chemical shift and/or direct intermolecular NOE contacts to the peptide are colored in red. B) The top 10 low energy structures overlaid to show convergence of the stem and flexibility of the loop. C) Surface rendering of the peptide:RNA complex. Unlike HIV TAR, the pre-miR21 hairpin retains a featureless helical structure in the complex, but flexibility in the apical loop accommodates our peptide and could even allow larger ligands to bind productively.



Figure 7: Dicer assay time course measurements highlight the intrinsically inefficient processing of pre-miR21 in vitro. A) Commercial Dicer kit activity begins to show buildup of intermediate (*) at 1.3 min and mature product (lower band) near 5min; significant mature product builds up at >80min. Existing inhibition studies using commercial kit 7, 35, 41 report assay activity at between 4-16hr reaction times . B) Processing efficiency is stimulated in the in house prepared enzyme in complex with TRBP, when the mature product begins to build up at 1.3min while intermediate (*) build-up does not appear until ~5min. Furthermore, with added TRBP, we observe a new intermediate (†) and mature product (‡).


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Figure 8: HEK293 cells, which have low background miR-21 (empty), were transfected with pCMV-miR21 containing vector and we recorded a near 5-fold increase in mature miR-21 with no cyclic peptide added (none). Upon addition of cell penetrating L50 peptide, without adding any transfecting agent to facilitate penetration, we observed a decrease in miR-21 expression, suggesting miR-21 processing is targetable by cyclic peptide chemistry. Error bars represent standard deviation from the average of three biological replicates.



References: [1] Redis, R. S., Vela, L. E., Lu, W., Ferreira de Oliveira, J., Ivan, C., Rodriguez-Aguayo, C., Adamoski, D., Pasculli, B., Taguchi, A., Chen, Y., Fernandez, A. F., Valledor, L., Van Roosbroeck, K., Chang, S., Shah, M., Kinnebrew, G., Han, L., Atlasi, Y., Cheung, L. H., Huang, G. Y., Monroig, P., Ramirez, M. S., Catela Ivkovic, T., Van, L., Ling, H., Gafa, R., Kapitanovic, S., Lanza, G., Bankson, J. A., Huang, P., Lai, S. Y., Bast, R. C., Rosenblum, M. G., Radovich, M., Ivan, M., Bartholomeusz, G., Liang, H., Fraga, M. F., Widger, W. R., Hanash, S., Berindan-Neagoe, I., Lopez-Berestein, G., Ambrosio, A. L., Gomes Dias, S. M., and Calin, G. A. (2016) Allele-Specific Reprogramming of Cancer Metabolism by the Long Noncoding RNA CCAT2, Mol Cell 61, 520-534. [2] Lee, A. S., Kranzusch, P. J., Doudna, J. A., and Cate, J. H. (2016) eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation, Nature 536, 96-99. [3] Di Leva, G., Garofalo, M., and Croce, C. M. (2014) MicroRNAs in cancer, Annu Rev Pathol 9, 287-314. [4] Huarte, M. (2015) The emerging role of lncRNAs in cancer, Nat Med 21, 1253-1261. [5] Wapinski, O., and Chang, H. Y. (2011) Long noncoding RNAs and human disease, Trends Cell Biol 21, 354361. [6] Bennett, C. F., and Swayze, E. E. (2010) RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform, Annu Rev Pharmacol Toxicol 50, 259-293. [7] Chen, Y., Yang, F., Zubovic, L., Pavelitz, T., Yang, W., Godin, K., Walker, M., Zheng, S., Macchi, P., and Varani, G. (2016) Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins, Nat Chem Biol 12, 717-723. [8] Shortridge, M. D., and Varani, G. (2015) Structure based approaches for targeting non-coding RNAs with small molecules, Curr Opin Struct Biol 30, 79-88. [9] Abulwerdi, F. A., Shortridge, M. D., Sztuba-Solinska, J., Wilson, R., Le Grice, S. F., Varani, G., and Schneekloth, J. S., Jr. (2016) Development of Small Molecules with a Noncanonical Binding Mode to HIV-1 Trans Activation Response (TAR) RNA, J Med Chem 59, 11148-11160. [10] Velagapudi, S. P., Vummidi, B. R., and Disney, M. D. (2015) Small molecule chemical probes of microRNA function, Curr Opin Chem Biol 24, 97-103. [11] Howe, J. A., Wang, H., Fischmann, T. O., Balibar, C. J., Xiao, L., Galgoci, A. M., Malinverni, J. C., Mayhood, T., Villafania, A., Nahvi, A., Murgolo, N., Barbieri, C. M., Mann, P. A., Carr, D., Xia, E., Zuck, P., Riley, D., Painter, R. E., Walker, S. S., Sherborne, B., de Jesus, R., Pan, W., Plotkin, M. A., Wu, J., Rindgen, D., Cummings, J., Garlisi, C. G., Zhang, R., Sheth, P. R., Gill, C. J., Tang, H., and Roemer, T. (2015) Selective small-molecule inhibition of an RNA structural element, Nature 526, 672-677. [12] Filonov, G. S., Moon, J. D., Svensen, N., and Jaffrey, S. R. (2014) Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution, J Am Chem Soc 136, 16299-16308. [13] Paige, J. S., Wu, K. Y., and Jaffrey, S. R. (2011) RNA mimics of green fluorescent protein, Science 333, 642646. [14] Roth, A., and Breaker, R. R. (2009) The structural and functional diversity of metabolite-binding riboswitches, Annu Rev Biochem 78, 305-334. [15] Hamy, F., Felder, E. R., Heizmann, G., Lazdins, J., Aboul-ela, F., Varani, G., Karn, J., and Klimkait, T. (1997) An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication, Proc Natl Acad Sci U S A 94, 3548-3553. [16] Velagapudi, S. P., Gallo, S. M., and Disney, M. D. (2014) Sequence-based design of bioactive small molecules that target precursor microRNAs, Nat Chem Biol 10, 291-297. [17] Velagapudi, S. P., and Disney, M. D. (2014) Two-dimensional combinatorial screening enables the bottom-up design of a microRNA-10b inhibitor, Chem Commun (Camb) 50, 3027-3029. [18] Sztuba-Solinska, J., Shenoy, S. R., Gareiss, P., Krumpe, L. R., Le Grice, S. F., O'Keefe, B. R., and Schneekloth, J. S., Jr. (2014) Identification of biologically active, HIV TAR RNA-binding small molecules using small molecule microarrays, J Am Chem Soc 136, 8402-8410. [19] Abulwerdi, F. A., and Schneekloth, J. S., Jr. (2016) Microarray-based technologies for the discovery of selective, RNA-binding molecules, Methods. [20] Disney, M. D., Labuda, L. P., Paul, D. J., Poplawski, S. G., Pushechnikov, A., Tran, T., Velagapudi, S. P., Wu, M., and Childs-Disney, J. L. (2008) Two-dimensional combinatorial screening identifies specific aminoglycoside-RNA internal loop partners, J Am Chem Soc 130, 11185-11194.


Page 32 of 43

Page 33 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[21] Palacino, J., Swalley, S. E., Song, C., Cheung, A. K., Shu, L., Zhang, X., Van Hoosear, M., Shin, Y., Chin, D. N., Keller, C. G., Beibel, M., Renaud, N. A., Smith, T. M., Salcius, M., Shi, X., Hild, M., Servais, R., Jain, M., Deng, L., Bullock, C., McLellan, M., Schuierer, S., Murphy, L., Blommers, M. J., Blaustein, C., Berenshteyn, F., Lacoste, A., Thomas, J. R., Roma, G., Michaud, G. A., Tseng, B. S., Porter, J. A., Myer, V. E., Tallarico, J. A., Hamann, L. G., Curtis, D., Fishman, M. C., Dietrich, W. F., Dales, N. A., and Sivasankaran, R. (2015) SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice, Nat Chem Biol 11, 511-517. [22] Pai, J., Yoon, T., Kim, N. D., Lee, I. S., Yu, J., and Shin, I. (2012) High-throughput profiling of peptide-RNA interactions using peptide microarrays, J Am Chem Soc 134, 19287-19296. [23] Diaz, J. P., Chirayil, R., Chirayil, S., Tom, M., Head, K. J., and Luebke, K. J. (2014) Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha, RNA 20, 528-539. [24] Moehle, K., Athanassiou, Z., Patora, K., Davidson, A., Varani, G., and Robinson, J. A. (2007) Design of betahairpin peptidomimetics that inhibit binding of alpha-helical HIV-1 Rev protein to the rev response element RNA, Angew Chem Int Ed Engl 46, 9101-9104. [25] Leeper, T. C., Athanassiou, Z., Dias, R. L., Robinson, J. A., and Varani, G. (2005) TAR RNA recognition by a cyclic peptidomimetic of Tat protein, Biochemistry 44, 12362-12372. [26] Lalonde, M. S., Lobritz, M. A., Ratcliff, A., Chamanian, M., Athanassiou, Z., Tyagi, M., Wong, J., Robinson, J. A., Karn, J., Varani, G., and Arts, E. J. (2011) Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR) RNA, PLoS Pathog 7, e1002038. [27] Davidson, A., Patora-Komisarska, K., Robinson, J. A., and Varani, G. (2011) Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein, Nucleic Acids Res 39, 248256. [28] Davidson, A., Leeper, T. C., Athanassiou, Z., Patora-Komisarska, K., Karn, J., Robinson, J. A., and Varani, G. (2009) Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein, Proc Natl Acad Sci U S A 106, 11931-11936. [29] Athanassiou, Z., Dias, R. L., Moehle, K., Dobson, N., Varani, G., and Robinson, J. A. (2004) Structural mimicry of retroviral tat proteins by constrained beta-hairpin peptidomimetics: ligands with high affinity and selectivity for viral TAR RNA regulatory elements, J Am Chem Soc 126, 6906-6913. [30] Robinson, J. A., Demarco, S., Gombert, F., Moehle, K., and Obrecht, D. (2008) The design, structures and therapeutic potential of protein epitope mimetics, Drug Discov Today 13, 944-951. [31] Niu, Y., Jones, A. J., Wu, H., Varani, G., and Cai, J. (2011) gamma-AApeptides bind to RNA by mimicking RNA-binding proteins, Org Biomol Chem 9, 6604-6609. [32] Gibbs, A. C., Kondejewski, L. H., Gronwald, W., Nip, A. M., Hodges, R. S., Sykes, B. D., and Wishart, D. S. (1998) Unusual beta-sheet periodicity in small cyclic peptides, Nat Struct Biol 5, 284-288. [33] Lunde, B. M., Moore, C., and Varani, G. (2007) RNA-binding proteins: modular design for efficient function, Nat Rev Mol Cell Biol 8, 479-490. [34] Schoniger, C., and Arenz, C. (2013) Perspectives in targeting miRNA function, Bioorg Med Chem 21, 61156118. [35] Bose, D., Jayaraj, G., Suryawanshi, H., Agarwala, P., Pore, S. K., Banerjee, R., and Maiti, S. (2012) The tuberculosis drug streptomycin as a potential cancer therapeutic: inhibition of miR-21 function by directly targeting its precursor, Angew Chem Int Ed Engl 51, 1019-1023. [36] Chirayil, S., Chirayil, R., and Luebke, K. J. (2009) Discovering ligands for a microRNA precursor with peptoid microarrays, Nucleic Acids Res 37, 5486-5497. [37] Maiti, M., Nauwelaerts, K., and Herdewijn, P. (2012) Pre-microRNA binding aminoglycosides and antitumor drugs as inhibitors of Dicer catalyzed microRNA processing, Bioorg Med Chem Lett 22, 1709-1711. [38] Ambros, V., Bartel, B., Bartel, D. P., Burge, C. B., Carrington, J. C., Chen, X., Dreyfuss, G., Eddy, S. R., Griffiths-Jones, S., Marshall, M., Matzke, M., Ruvkun, G., and Tuschl, T. (2003) A uniform system for microRNA annotation, RNA 9, 277-279. [39] Kozomara, A., and Griffiths-Jones, S. (2014) miRBase: annotating high confidence microRNAs using deep sequencing data, Nucleic Acids Res 42, D68-73. [40] Chirayil, S., Wu, Q., Amezcua, C., and Luebke, K. J. (2014) NMR characterization of an oligonucleotide model of the miR-21 pre-element, PLoS One 9, e108231. [41] Bose, D., Nahar, S., Rai, M. K., Ray, A., Chakraborty, K., and Maiti, S. (2015) Selective inhibition of miR-21 by phage display screened peptide, Nucleic Acids Res 43, 4342-4352.



[42] Pai, J., Hyun, S., Hyun, J. Y., Park, S. H., Kim, W. J., Bae, S. H., Kim, N. K., Yu, J., and Shin, I. (2016) Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays, J Am Chem Soc 138, 857-867. [43] Chen, Y., Zubovic, L., Yang, F., Godin, K., Pavelitz, T., Castellanos, J., Macchi, P., and Varani, G. (2016) Rbfox proteins regulate microRNA biogenesis by sequence-specific binding to their precursors and target downstream Dicer, Nucleic Acids Res 44, 4381-4395. [44] Zeng, Y., Yi, R., and Cullen, B. R. (2005) Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha, EMBO J 24, 138-148. [45] Aboul-ela, F., Karn, J., and Varani, G. (1996) Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge, Nucleic Acids Res 24, 3974-3981. [46] Varani, G., Aboul-ela, F., and Allain, F. (1996) NMR investigation of RNA structure, Progress in Nuclear Magnetc Resonance Spectroscopy 29, 51-127. [47] Lyskov, S., Chou, F. C., Conchuir, S. O., Der, B. S., Drew, K., Kuroda, D., Xu, J., Weitzner, B. D., Renfrew, P. D., Sripakdeevong, P., Borgo, B., Havranek, J. J., Kuhlman, B., Kortemme, T., Bonneau, R., Gray, J. J., and Das, R. (2013) Serverification of molecular modeling applications: the Rosetta Online Server that Includes Everyone (ROSIE), PLoS One 8, e63906. [48] Das, R., Karanicolas, J., and Baker, D. (2010) Atomic accuracy in predicting and designing noncanonical RNA structure, Nat Methods 7, 291-294. [49] Schwieters, C. D., Kuszewski, J. J., Tjandra, N., and Clore, G. M. (2003) The Xplor-NIH NMR molecular structure determination package, J Magn Reson 160, 65-73. [50] Clore, G. M., and Kuszewski, J. (2003) Improving the accuracy of NMR structures of RNA by means of conformational database potentials of mean force as assessed by complete dipolar coupling crossvalidation, J Am Chem Soc 125, 1518-1525. [51] Shi, Z., Zhang, J., Qian, X., Han, L., Zhang, K., Chen, L., Liu, J., Ren, Y., Yang, M., Zhang, A., Pu, P., and Kang, C. (2013) AC1MMYR2, an inhibitor of dicer-mediated biogenesis of Oncomir miR-21, reverses epithelial-mesenchymal transition and suppresses tumor growth and progression, Cancer Res 73, 55195531. [52] Taylor, D. W., Ma, E., Shigematsu, H., Cianfrocco, M. A., Noland, C. L., Nagayama, K., Nogales, E., Doudna, J. A., and Wang, H. W. (2013) Substrate-specific structural rearrangements of human Dicer, Nat Struct Mol Biol 20, 662-670. [53] Lee, H. Y., Zhou, K., Smith, A. M., Noland, C. L., and Doudna, J. A. (2013) Differential roles of human Dicerbinding proteins TRBP and PACT in small RNA processing, Nucleic Acids Res 41, 6568-6576. [54] Lee, H. Y., and Doudna, J. A. (2012) TRBP alters human precursor microRNA processing in vitro, RNA 18, 2012-2019. [55] Chakravarthy, S., Sternberg, S. H., Kellenberger, C. A., and Doudna, J. A. (2010) Substrate-specific kinetics of Dicer-catalyzed RNA processing, J Mol Biol 404, 392-402. [56] Wilson, R. C., Tambe, A., Kidwell, M. A., Noland, C. L., Schneider, C. P., and Doudna, J. A. (2015) DicerTRBP complex formation ensures accurate mammalian microRNA biogenesis, Mol Cell 57, 397-407. [57] De, N., and Macrae, I. J. (2011) Purification and assembly of human Argonaute, Dicer, and TRBP complexes, Methods Mol Biol 725, 107-119. [58] Shah, M. Y., and Calin, G. A. (2014) MicroRNAs as therapeutic targets in human cancers, Wiley Interdiscip Rev RNA 5, 537-548. [59] Li, Z., and Rana, T. M. (2014) Therapeutic targeting of microRNAs: current status and future challenges, Nat Rev Drug Discov 13, 622-638. [60] Connelly, C. M., Boer, R. E., Moon, M. H., Gareiss, P., and Schneekloth, J. S., Jr. (2016) Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays, ACS Chem Biol. [61] Young, D. D., Connelly, C. M., Grohmann, C., and Deiters, A. (2010) Small molecule modifiers of microRNA miR-122 function for the treatment of hepatitis C virus infection and hepatocellular carcinoma, J Am Chem Soc 132, 7976-7981. [62] Gumireddy, K., Young, D. D., Xiong, X., Hogenesch, J. B., Huang, Q., and Deiters, A. (2008) Small-molecule inhibitors of microrna miR-21 function, Angew Chem Int Ed Engl 47, 7482-7484. [63] Charrette, B. P., Boerneke, M. A., and Hermann, T. (2016) Ligand Optimization by Improving Shape Complementarity at a Hepatitis C Virus RNA Target, ACS Chem Biol 11, 3263-3267.


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Figure 1: A) Secondary structure prediction of the pre-miR21 hairpin with the mature miR21 sequence in red. The full length sequence is shown and boxed with black outline, G8 and U65*** were swapped compared to the wild type sequence to improve in vitro transcription. The apical loop portion used in this report is boxed with a blue line and A23 was switched to G23* to improve in vitro transcription. Orange dashes are potential weak base pairs observed only at 5C. B) The 1D 1H imino region for both the apical loop (top, blue) and the full length (bottom, black) show corresponding signals (black line) and that both transcripts lack imino signals from the apical loop region. C) The 2D 1H-1H NOESY walk and D) 15N-1H HSQC for the short pre-miR21 sequence from which NH assignments were derived. Similar data were collected for the full pre-miR21 to obtain the assignments shown in part B. 879x632mm (72 x 72 DPI)



Figure 2: The top 20 low energy structures of the wild type pre-miR21 hairpin. A) Using a traditional folding scheme, we were unable to identify a converged set of structures for the apical loop region of pre-miR21. This is largely due to the lack of long range and sequential NOEs. B). To model the structure, we first used FARFAR 51-52 with 123 non-exchangeable chemical shift values obtained at 37oC to generate an initial fold, followed by refinement using NIH-XPLOR protocol 53-54 with 349 noes and 131 dihedral angle constraints. 726x444mm (72 x 72 DPI)


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Figure 3: A) Binding of L50 peptide to pre-miR21 apical loop and several variants by EMSA; all RNAs were at 1nM. Both wild type and the miR-21/miR145 chimera bind to L50 with a KD near 200 nM. Removing the bulge A29 or binding to pre-miR145 shifts the binding to KD>1µM; non-specific binding is suggested by the smearing observed at 4µM peptide concentration. B) Competition gel between pre-miR21 and pre-miR18 for L50 binding. Secondary structures are found in SI figure 2. 210x186mm (96 x 96 DPI)



Figure 4: A) Pre-miR21 ligands L50 and streptomycin B) NMR competition assay; when the free sample of 30µM streptomycin (bottom) is titrated with 10µM pre-miR21 (middle) binding is confirmed by the increased streptomycin linewidth (*). When 30µM of L50 (+) is further titrated into the streptomycin-bound sample (top), the streptomycin resonances (*) approach the RNA-free ligand intensity and linewidth; indicating that L50 is competitive with streptomycin and probably binds to an overlapping site C) Free pre-miR21 hairpin at 37oC (black) was titrated with L50 peptide (red). The largest chemical shift changes (cyan lines) were observed for residues near the Dicer cleavage site. Terminal loop residues do not shift, suggesting the peptide binds to a specific location near the Dicer cleavage site generating clear changes in the chemical shifts of resonances near the binding site. D) When the same experiment was repeated with streptomycin sulfate, we observed only small diffuse changes in the RNA chemical shifts (cyan lines). 62x46mm (600 x 600 DPI)


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Figure 5: A) The L50 bound pre-miR21 imino spectrum (top) maintains the base pairing of the free RNA spectrum (bottom) indicating the competition between streptomycin and L50 is not due to RNA sample unfolding. G28 was the most significantly shifted imino resonance and the A29 NH2 peaks broaden in the bound spectrum further supporting the idea the peptide straddles the A29 bulge B) The 2D 1H-1H NOESY spectrum of the L50-pre-mIR21 complex in D2O buffer shows nearly 100 intermolecular NOEs between the RNA and peptide (many in the red-boxed region of the spectrum). C) Zoomed-in regions of the NMR spectrum of part B. Unfortunately, many of the intermolecular NOEs were from severely overlapped Arginine side chains and we were not able to unambiguously identify many contacts, even with increased resolution at 800MHz. 56x46mm (600 x 600 DPI)



Figure 6: A) Cartoon representation of the lowest energy structure for pre-miR21 bound to L50 peptide. The D-pro/L-Pro turn is pointed towards the top of the apical loop with the Gly/Lys turn pointing down towards the helical stem. The nucleotides that exhibited significant changes in chemical shift and/or direct intermolecular NOE contacts to the peptide are colored in red. B) The top 10 low energy structures overlaid to show convergence of the stem and flexibility of the loop. C) Surface rendering of the peptide:RNA complex. Unlike HIV TAR, the pre-miR21 hairpin retains a featureless bulge structure in the complex, but flexibility in the apical loop accommodates our peptide and could even allow larger ligands to bind productively. 1538x1028mm (72 x 72 DPI)


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Figure 7: Dicer assay time course measurements highlight the intrinsically inefficient processing of premiR21 in vitro. A) Commercial Dicer kit activity begins to show buildup of intermediate (*) at 1.3 min and mature product (lower band) near 5min; significant mature product builds up at >80min. Existing inhibition studies using commercial kit report assay activity at between 4-16hr reaction times 8, 40, 45. B) Processing efficiency is stimulated in the in house prepared enzyme in complex with TRBP, when the mature product begins to build up at 1.3min while intermediate (*) build-up does not appear until ~5min. Furthermore, with added TRBP, we observe a new intermediate (†) and mature product (‡). 204x263mm (96 x 96 DPI)



Figure 8: HEK293 cells, which have low background miR-21 (empty), were transfected with pCMV-miR21 containing vector and we recorded a near 5-fold increase in mature miR-21 with no cyclic peptide added (none). Upon addition of cell penetrating L50 peptide, without adding any transfecting agent to facilitate penetration, we observed a decrease in miR-21 expression, suggesting miR-21 processing is targetable by cyclic peptide chemistry. Error bars represent standard deviation from the average of three biological replicates. 174x89mm (96 x 96 DPI)


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Figure for Graphical Abstract: Representative free and bound pre-miR21 structures described in this manuscript 1303x925mm (72 x 72 DPI)


A Macrocyclic Peptide Ligand Binds the Oncogenic MicroRNA-21

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