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 Alternatively, our unpublished data suggest that Cy3 dye-labeled RNAs can be used in non-denaturing PAGE experiments to measure the tight binding events.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.

<table style="border-collapse:collapse;width:491pt" width="654">
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			<td class="xl25" height="60" style="width:176pt;height:45.0pt" width="235">Molecular sieves, 4A, 1-2 mm diameter pellets&#160;</td>
			<td class="xl26" style="width:116pt" width="155">Alfa Aesar</td>
			<td class="xl74" height="60" style="width:132pt;height:45.0pt" width="176">87956</td>
			<td style="width:66pt" width="88"></td>
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			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">4-Methylbenzhydrylamine hydrochloride (MBHA&#376;HCl)</td>
			<td class="xl30">Sigma-Aldrich</td>
			<td class="xl68" height="60" style="width:132pt;height:45.0pt" width="176">532444</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">N,N-diisopropylethylamine (DIPEA)</td>
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			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">A11801</td>
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			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">(Benzotriazol-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)</td>
			<td class="xl30">Alfa Aesar</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">B25251</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">Acetic anhyride</td>
			<td class="xl30">Sigma-Aldrich</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">320102</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">Kaiser Test Kit&#160;</td>
			<td class="xl30">Sigma-Aldrich</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">60017</td>
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			<td class="xl29" height="60" style="width:176pt;height:45.0pt" width="235">Trifluoroacetic acid (TFA)</td>
			<td class="xl30">Alfa Aesar</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">L06374</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Unmodified PNA monomers</td>
			<td class="xl36" style="width:116pt" width="155">ASM Research Chemicals GmbH</td>
			<td class="xl70" height="60" style="width:132pt;height:45.0pt" width="176">5004007, 5004008, 5004009, 5004010</td>
			<td></td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Boc-Lys(Z)-OH / Fmoc-Lys(Boc)-OH</td>
			<td class="xl36" style="width:116pt" width="155">Sigma-Aldrich</td>
			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">B8389 / 47624</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Thioanisole</td>
			<td class="xl36" style="width:116pt" width="155">Alfa Aesar</td>
			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">A14846</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">1,2-Ethanedithiol</td>
			<td class="xl36" style="width:116pt" width="155">Alfa Aesar</td>
			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">L12865</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Trifluoromethanesulfonic acid</td>
			<td class="xl36" style="width:116pt" width="155">Alfa Aesar</td>
			<td class="xl72" height="60" style="width:132pt;height:45.0pt" width="176">A10173</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">LiChrosper&#174; 100 RP-18 endcapped (5 &#181;m) LiChroCART&#174; 250-4</td>
			<td class="xl41">Merck Millipore</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">150838</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">RNA Oligos&#160;</td>
			<td class="xl41">Sigma-Aldrich</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">Customized&#160;</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">&#945;-cyano-4-hydroxycinnamic acid (CHCA)</td>
			<td class="xl41">Sigma-Aldrich</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">39468</td>
			<td></td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td class="xl41">Alfa Aesar</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">J15694</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">HEPES&#160;</td>
			<td class="xl41">Lonza</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">17-737E</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Acrylamide</td>
			<td class="xl41">Sigma-Aldrich</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">A8887</td>
			<td></td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">N.N&#39;-methylenebisacrylamide</td>
			<td class="xl41">Sigma-Aldrich&#160;</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">146072</td>
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			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Ammonium persulfate (APS)</td>
			<td class="xl41">Bio-rad</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">161-0700</td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">Tetramethylethylenediamine (TEMED)</td>
			<td class="xl41">Bio-rad</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">161-0800</td>
			<td></td>
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		<tr height="60" style="height:45.0pt">
			<td class="xl35" height="60" style="width:176pt;height:45.0pt" width="235">10X Tris-Borate-EDTA (TBE) Buffer, pH 8.3</td>
			<td class="xl41">1st Base</td>
			<td class="xl57" height="60" style="width:132pt;height:45.0pt" width="176">BUF-3010-10X1L</td>
			<td></td>
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			<td class="xl44" height="60" style="width:176pt;height:45.0pt" width="235">Glycerol&#160;</td>
			<td class="xl45">Promega&#160;</td>
			<td class="xl55" height="60" style="width:132pt;height:45.0pt" width="176">H5433</td>
			<td></td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl44" height="60" style="width:176pt;height:45.0pt" width="235">Ethidium bromide (10 mg/mL)</td>
			<td class="xl45">Bio-rad&#160;</td>
			<td class="xl64" height="60" style="width:132pt;height:45.0pt" width="176">161-0433</td>
			<td></td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl48" height="60" style="width:176pt;height:45.0pt" width="235">High Precision Cell (Quartz Suprasil, 200-2500 nm)</td>
			<td class="xl49">Hellma Analytics&#160;</td>
			<td class="xl61" height="60" style="width:132pt;height:45.0pt" width="176">105.250-QS</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

sequence-specific-selective-recognition-double-stranded-rnas-over

Nanyang Technological University

Research

JoVE Journal

Genetics

9.4K Views.  Nanyang Technological University. 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.

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

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...