Name: sequence-specific and selective recognition of double-stranded rnas over single-stranded rnas by chemically modified peptide nucleic acids
Uploaded: 2017-09-21T14:00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids at JoVE.com

This work was supported by the Singapore Ministry of Education (MOE) Tier 1 (RGT3/13 and RG42/15 to G.C.) and MOE Tier 2 (MOE2013-T2-2-024 and MOE2015-T2-1-028 to G.C.).

A patent application (PAT/179/14/15/PCT) based on the work reported here has been filed.

RNA duplex-binding PNA oligomers (e.g., 10-mers) are medium-sized molecules and thus can show electrophoretic mobility shift upon binding to RNAs with a comparable or slightly larger size (e.g., 50-mer or smaller). If an RNA is significantly larger than the PNA, titration of PNA into RNA may not work due to a limited gel mobility shift. Thus, the large RNA may be truncated for the non-denaturing PAGE assays. Titration of a large RNA into a fluorophore-labeled PNA allows for the monitoring of the triplex formation by a non-denaturing agarose gel with the sample loaded in the middle of the gel40.
For a titration experiment by non-denaturing PAGE with a constant total concentration of RNA, we typically use an unlabeled RNA concentration of 1 &#181;M for efficient post staining of the free RNA and triplex bands by ethidium bromide. An RNA concentration as low as 0.2 &#181;M may also be sufficient depending on the RNA construct31. The concentration of the unlabeled RNA (0.2 &#181;M) determines that the Kd values that can be accurately measured should be about 0.2 &#181;M or larger. Other staining dyes may be used to enhance the staining efficiency. Alternatively, our unpublished data suggest that Cy3 dye-labeled RNAs can be used in non-denaturing PAGE experiments to measure the tight binding events.
Due to the fact that 2-aminopurine is only moderately fluorescent, 2-aminopurine fluorescence titration is also limited to the measurement of the binding with Kd values close to or above 0.2 &#181;M31. The RNA or the PNA may be labeled with a relatively bright dye for quantifying a relatively tight binding in solution through fluorescence titration, if the binding results in changes in fluorescence signals53,54,55.
The strategy of targeting RNA structures by dsRNA-binding PNAs has been tested for a limited number of RNAs. It is likely that binding properties may vary for dsRNAs with different sequences and base pair compositions. One may always choose the purine-rich strand of a duplex for the design of TFPNAs. It is critical to understand how consecutive Q&#183;C-G triples may affect the stability of a triplex. More extensive sequence-dependent studies are clearly needed to understand the sequence-dependent binding properties of TFPNAs.
The binding affinity of TFPNAs can be further enhanced by increasing the length and/or further modifying the bases and backbones56,57 of TFPNAs. However, a continuous duplex region may not often consist of more than 10 consecutive base pairs without the disruption by non-Watson-Crick structures. One may conjugate TFPNAs with small molecules for the recognition of non-Watson-Crick structures adjacent to dsRNA regions. In principle, a TFPNA-small molecule conjugate is expected to have enhanced binding affinity and specificity compared to a TFPNA or small molecule alone. However, the chemical and physical properties of the linker for the conjugation58,59,60,61,62,63,64 must be optimized.
The fact that TFPNAs can selectively bind to dsRNAs over ssRNAs and dsDNAs suggests that it is possible to develop TFPNAs as very useful chemical probes and potential therapeutic ligands through the regulation of RNA structural dynamics and interactions with proteins and metabolites. Cellular uptake of TFPNAs may be facilitated through the conjugation with cell-penetrating moieties such as small molecules, peptides, and nanoparticles, or complexation with supramolecular structures such as liposomes5,6,12,17,25,31,41,65,66. Further functionalization of TFPNAs with bioimaging tags such as fluorophores and radioisotopes may facilitate the detection, imaging, and targeting of functional RNA structures in living organisms.

RNAs are emerging as important biomarkers and therapeutic targets, due to the recent advances in the discoveries of the RNAs&#39; roles in the regulation and catalysis of diverse biological processes1,2,3. Traditionally, antisense strands have been used to bind to ssRNAs through Watson-Crick duplex formation3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27. Recently, triplex-forming peptide nucleic acids (TFPNAs) have been designed to bind to dsRNAs via Hoogsteen hydrogen bonding (Figure 1)3,28,29. dsRNA regions are present in the majority of the traditional antisense-targeted RNAs, including pre-mRNAs and mRNAs, pre- or pri-miRNAs3, and many other non-coding RNAs1,26,27. Targeting dsRNAs through triple helix formation using TFPNAs may be advantageous due to its structure specificity and is of great potential for use in restoring the normal functions of the RNAs, which are dysregulated in diseases, for example.
The recently published work by Rozners et al., and us3,28,29,30,31,32,33,34,35,36,37,38,39,40,41, reported the efforts&#160;on improving the selective binding of modified TFPNAs towards dsRNAs with enhanced affinity. We have developed synthesis methods for rationally designed PNA monomers (Figure 2) including thio-pseudoisocytosine (L) monomer30 and guanidine-modified 5-methyl cytosine (Q) monomer31. Through various biochemical and biophysical characterization methods, we have demonstrated that relatively short PNAs (6-10 residues) incorporating L and Q residues show improved recognition of Watson-Crick G-C and C-G base pairs, respectively, in dsRNAs. Moreover, compared to unmodified PNAs, PNAs containing L and Q residues show more selective binding towards dsRNA over ssRNA and dsDNA. The guanidine functionality42 in the Q base enables PNAs to enter HeLa cells31.
In our laboratory, we synthesize PNAs by the manual Boc-chemistry (Boc or t-Boc stands for tert- butyloxycarbony (see Figure 2) solid-phase peptide synthesis method4. The synthesis of the PNA monomer with Boc as the amine protecting group is convenient as the Boc group is sterically less bulky in comparison to fluorenylmethyloxycarbonyl (Fmoc) amine protecting group, which may be beneficial during PNA monomer coupling on the solid support. The Boc group is acid-labile and can be easily removed on the solid support by 20-50% trifluoroacetic acid (TFA) in dichloromethane (DCM) during PNA synthesis. An automated peptide synthesizer can be employed to synthesize PNA oligomers; however, 3-5-fold excess of PNA monomer is needed for an automated peptide synthesizer. Manual synthesis requires significantly less PNA monomer (2-3-fold excess), with each coupling easily monitored by the Kaiser test43. Furthermore, many automated synthesizers are not compatible with the Boc strategy synthesis due to the use of corrosive TFA during the Boc removal step.
The PNA oligomers can be purified by reverse-phase high-performance liquid chromatography (RP-HPLC) followed by molecular weight characterization by MALDI-TOF (Figures 3 and 4)30,31. We employ non-denaturing PAGE to monitor triplex formation, due to the fact that free RNA duplex constructs and PNA bound triplexes often show different migration rates (Figure 5)30,31. No labeling is needed if efficient post-staining can be achieved for both of the RNA duplex and PNA&#183;RNA2 triplex bands. A relatively small amount of sample is needed for non-denaturing PAGE experiments. However, the loading (incubation) buffers and the running buffers (pH 8.3) may not be the same, resulting in the measurements being limited to the kinetically stable triplexes, because a relatively high pH of 8.3 may significantly destabilize a triplex.
2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity (with an emission peak at around 370 nm) is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site (Figure 6)31. Unlike many other dyes that show fluorescence emission in the visible range, 2-aminopurine-labeled RNA can be exposed to room light without photo bleaching. Unlike PAGE experiment in which a running buffer of pH 8.3 is often needed, 2-aminopurine based fluorescence titration allows the measurement of binding in one solution at a specified pH, and thus may allow the measurement and detection of relatively weak and kinetically unstable binding at equilibrium.
UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of duplexes (Figure 7)31 and triplexes30,32,44,45. Depending on the length and sequence composition, the melting of triplexes may or may not show a clear transition. Thermodynamic parameters may be obtained if the heating and cooling curves overlap. Accurate thermodynamic parameters can be obtained by isothermal titration calorimetry (ITC)32; however, relatively large amounts of samples are generally required for ITC.

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sequence-specific and selective recognition of double-stranded rnas over single-stranded rnas by chemically modified peptide nucleic acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Reverse-phase HPLC allows the purification of PNA oligomers. We can obtain pure PNA oligomers with two rounds of HPLC purification (Figure 3). The identity of the PNAs can be confirmed by MALDI-TOF analysis (Figure 4).
Non-denaturing PAGE is an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. We typically use multiple RNA hairpins or duplexes with single base pair mutations to characterize binding properties (Figure 5). The non-denaturing PAGE data shown in Figure 5 clearly suggest that the Q- and L-modified PNA can recognize a dsRNA region with a C-G pair (Figure 5B, bottom panel) but not the one without a C-G pair (Figure 5B, top panel). This specific and enhanced recognition is through the T&#183;A-U, L&#183;G-C, and Q&#183;C-G PNA&#183;RNA2 base triple (Figure 1A, C, D) formation. Various PNAs with single or multiple mutations may also be used to demonstrate the improved binding properties of a modified PNA. We have shown that adding 2 mM Mg2+ in the incubation buffer does not affect the binding significantly31.
We have demonstrated by 2-aminopurine fluorescence titration that a Q- and L-modified PNA binds to a targeted dsRNA region (Figure 6A, 6C, 6D) but not ssRNA (Figure 6B, 6E, 6F). PNA P3 binds to the 2-aminopurine-labeled dsRNA with a Kd value of 0.8 &#177; 0.1 &#181;M. The fluorescence intensity at 370 nm for the 2-aminopurine-labeled ssRNA remains relatively constant with varied P3 concentration, indicating the lack of binding of PNA P3 to the ssRNA.
PNAs containing Q residues (P2 and P3) show no thermal melting transitions (Figure 7), suggesting no binding to the ssRNA. This is due to the steric clash present in Watson-Crick like Q-G pair. Compared to unmodified PNA P1, PNAs P4 and P5 containing modified L residues but no Q residues, show decreased melting temperatures for the corresponding RNA-PNA duplexes due to the steric clash present in Watson-Crick like L-G pair. The UV-absorbance-detected thermal melting data are consistent with the 2-aminopurine fluorescence titration data, which also show that a PNA containing Q and L residues does not bind to ssRNA appreciably (Figure 6B, 6E, 6F). Incorporating a Q base is more destabilizing than&#160;an L base, as a Q base has a more significant steric clash in the formation of a Watson-Crick-like Q-G pair (Figure 1F) compared to a Watson-Crick-like L-G pair (Figure 1E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56221/56221fig1v2.jpg" /> 
Figure 1: Chemical structures of stable base triple and unstable base pair structures. (A-D) Major-groove PNA&#183;RNA2 base triples of T&#183;A-U (A), C+&#183;G-C (B), L&#183;G-C (C), and Q&#183;C-G (D). (E, F) Unstable Watson-Crick like PNA-RNA base pairs of L-G (E) and Q-G (F). The letter R represents the sugar-phosphate backbone of RNA. Hydrogen bonds are indicated by black dashed lines. The figure is reproduced from reference31. <a href="https://cloudflare.jove.com/files/ftp_upload/56221/56221fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56221/56221fig2v2.jpg" /> 
Figure 2: Chemical structures of PNA monomers. Four PNA monomers (T, C, L, and Q) are shown. <a href="https://cloudflare.jove.com/files/ftp_upload/56221/56221fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56221/56221fig3.jpg" /> 
Figure 3: Chemical structure of a PNA oligomer and purification by RP-HPLC. (A) Chemical structure of the PNA sequence P3. (B, C) RP-HPLC data of crude PNA P3 (B) and re-purified PNA P3 (C). <a href="https://cloudflare.jove.com/files/ftp_upload/56221/56221fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56221/56221fig4.jpg" /> 
Figure 4:&#160;MALDI-TOF spectrum of purified PNA P3. <a href="https://cloudflare.jove.com/files/ftp_upload/56221/56221fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56221/56221fig5.jpg" /> 
Figure 5:&#160;RNA hairpin and PNA sequences and binding characterization by non-denaturing PAGE. (A) RNA hairpins (rHP1 and rHP2), PNA P3, and a PNA&#183;RNA2 triplex formed between PNA P3 and rHP2. (B) Non-denaturing PAGE (12%) results for rHP1 and rHP2 binding to PNA P3. The incubation buffer is 200 mM NaCl, 0.5 mM EDTA, 20 mM HEPES, pH 7.5. The loaded RNA hairpins (rHP1 and rHP2) are at 1 &#181;M in 20 &#181;L. The PNA concentrations in lanes from left to right are 0, 0.2, 0.4, 1, 1.6, 2, 4, 10, 16, 20, 28, and 50 &#181;M. PNA P3 does not bind to rHP1 (top panel) but binds to rHP2 (bottom panel). (C) Kd determination for P3 binding to rHP2. The fraction of triplex formation (Y) was plotted against the PNA concentration. The figure is adapted from reference31. <a href="https://cloudflare.jove.com/files/ftp_upload/56221/56221fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56221/56221fig6.jpg" /> 
Figure 6: Fluorescence titration study of PNA P3 binding to 2-aminopurine-labeled RNAs. The 2-aminopurine residue is designated as &#39;2&#39; in the RNA sequence. The incubation buffer is 200 mM NaCl, 0.5 mM EDTA, 20 mM HEPES, pH 7.5. (A) A PNA&#183;RNA2 triplex formed between P3 and a 2-aminopurine-labeled dsRNA (dsRNA2-2AP). (B) A hypothetical PNA-RNA duplex formed between P3 and a 2-aminopurine-labeled ssRNA (ssRNA2-2AP). (C, E) Fluorescence emission spectra for the 2-aminopurine-labeled RNA duplex (1 &#181;M) and ssRNA (1 &#181;M), respectively, with varied P3 concentration at pH 7.5. The peak at around 475 nm is due to the weak fluorescence emission of the L base in the PNA. (D, F) Kd determination based on the plots of 2-aminopurine fluorescence intensity (at 370 nm) of the RNA duplex and ssRNA, respectively, versus PNA P3 concentration. The figure is adapted from reference31. <a href="https://cloudflare.jove.com/files/ftp_upload/56221/56221fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56221/56221fig7.jpg" /> 
Figure 7: Thermal melting results for RNA-PNA duplexes. The incubation buffer is 200 mM NaCl, 0.5 mM EDTA, 20 mM NaH2PO4, pH 7.5. All samples contain 5 &#181;M of single-stranded RNA (ssRNA1) and PNA in 130 &#181;L. (A) Single-stranded RNA (ssRNA1), PNAs (P1, P2, P3, P4, and P5) and a hypothetical PNA-RNA duplex formed between PNA P3 and ssRNA1 in a parallel orientation. The steric clash is indicated for the Watson-Crick like Q-G and L-G pairs. (B) Melting curves for different PNAs binding to ssRNA1. The melting temperatures are shown for the curves with melting transitions. The figure is adapted from reference31.

Watch this Scientific Journal Video about Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids at JoVE.com

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

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Genetics

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