Name: Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids (Video) | JoVE Journal
Uploaded: 2017-09-21T14:00:00.000+00:00
Duration: 9 min 4 s
Description: Learn protocols for synthesizing and characterizing modified Peptide Nucleic Acids that selectively recognize double-stranded RNA structures. This method enables precise RNA targeting for chemical biology research.

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5 minutes

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 Alternatively, our unpublished data suggest that Cy3 dye-labeled RNAs can be used in non-denaturing PAGE experiments to measure the tight binding events.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><tbody><tr><td>Molecular sieves, 4A, 1-2 mm diameter pellets&amp;nbsp;</td><td>Alfa Aesar</td><td>87956</td><td></td></tr><tr><td>4-Methylbenzhydrylamine hydrochloride (MBHA&lt;span class=&quot;font6&quot;&gt;&amp;Yuml;&lt;/span&gt;&lt;span class=&quot;font0&quot;&gt;HCl)&lt;/span&gt;</td><td>Sigma-Aldrich</td><td>532444</td><td></td></tr><tr><td>N,N-diisopropylethylamine (DIPEA)</td><td>Alfa Aesar</td><td>A11801</td><td></td></tr><tr><td>(Benzotriazol-1-yl-oxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)</td><td>Alfa Aesar</td><td>B25251</td><td></td></tr><tr><td>Acetic anhyride</td><td>Sigma-Aldrich</td><td>320102</td><td></td></tr><tr><td>Kaiser Test Kit&amp;nbsp;</td><td>Sigma-Aldrich</td><td>60017</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Alfa Aesar</td><td>L06374</td><td></td></tr><tr><td>Unmodified PNA monomers</td><td>ASM Research Chemicals GmbH</td><td>5004007, 5004008, 5004009, 5004010</td><td></td></tr><tr><td>Boc-Lys(Z)-OH / Fmoc-Lys(Boc)-OH</td><td>Sigma-Aldrich</td><td>B8389 / 47624</td><td></td></tr><tr><td>Thioanisole</td><td>Alfa Aesar</td><td>A14846</td><td></td></tr><tr><td>1,2-Ethanedithiol</td><td>Alfa Aesar</td><td>L12865</td><td></td></tr><tr><td>Trifluoromethanesulfonic acid</td><td>Alfa Aesar</td><td>A10173</td><td></td></tr><tr><td>LiChrosper&amp;reg; 100 RP-18 endcapped (5 &amp;micro;m) LiChroCART&amp;reg; 250-4</td><td>Merck Millipore</td><td>150838</td><td></td></tr><tr><td>RNA Oligos&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Customized&amp;nbsp;</td><td></td></tr><tr><td>&amp;alpha;-cyano-4-hydroxycinnamic acid (CHCA)</td><td>Sigma-Aldrich</td><td>39468</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Alfa Aesar</td><td>J15694</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Lonza</td><td>17-737E</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma-Aldrich</td><td>A8887</td><td></td></tr><tr><td>N.N&#039;-methylenebisacrylamide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>146072</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Bio-rad</td><td>161-0700</td><td></td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>Bio-rad</td><td>161-0800</td><td></td></tr><tr><td>10X Tris-Borate-EDTA (TBE) Buffer, pH 8.3</td><td>1st Base</td><td>BUF-3010-10X1L</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>H5433</td><td></td></tr><tr><td>Ethidium bromide (10 mg/mL)</td><td>Bio-rad&amp;nbsp;</td><td>161-0433</td><td></td></tr><tr><td>High Precision Cell (Quartz Suprasil, 200-2500 nm)</td><td>Hellma Analytics&amp;nbsp;</td><td>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

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·A-U, L·G-C, and Q·C-G PNA·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 ± 0.1 µM. 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 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="RNA-DNA base pairing diagram; Hoogsteen vs. Watson-Crick pairs; chemical structure analysis." class="xfigimg" src="/files/ftp_upload/56221/56221fig1v2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Chemical structures of stable base triple and unstable base pair structures. (A-D) Major-groove PNA·RNA2 base triples of T·A-U (A), C+·G-C (B), L·G-C (C), and Q·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="Chemical structures diagram of Boc-PNA variants, illustrating peptide nucleic acid synthesis." class="xfigimg" src="/files/ftp_upload/56221/56221fig2v2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 2"> 
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="Peptide synthesis diagram with crude and repurified chromatography results, retention time analysis." class="xfigimg" src="/files/ftp_upload/56221/56221fig3.jpg" data-altupdatedat="1788250015000" data-alt="Figure 3"> 
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="Mass spectrometry result; m/z vs. intensity graph; analyzing compound P3; calculated vs. observed mass." class="xfigimg" src="/files/ftp_upload/56221/56221fig4.jpg" data-altupdatedat="1788250015000" data-alt="Figure 4"> 
Figure 4: 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="RNA hairpin interactions; diagram, gel electrophoresis, binding curve; triplex formation analysis." class="xfigimg" src="/files/ftp_upload/56221/56221fig5.jpg" data-altupdatedat="1788250015000" data-alt="Figure 5"> 
Figure 5: RNA hairpin and PNA sequences and binding characterization by non-denaturing PAGE. (A) RNA hairpins (rHP1 and rHP2), PNA P3, and a PNA·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 µM in 20 µL. 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 µM. 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="DNA-RNA triplex, duplex stability; fluorescence spectra; binding affinity curves; spectroscopy data." class="xfigimg" src="/files/ftp_upload/56221/56221fig6.jpg" data-altupdatedat="1788250015000" data-alt="Figure 6"> 
Figure 6: Fluorescence titration study of PNA P3 binding to 2-aminopurine-labeled RNAs. The 2-aminopurine residue is designated as '2' in the RNA sequence. The incubation buffer is 200 mM NaCl, 0.5 mM EDTA, 20 mM HEPES, pH 7.5. (A) A PNA·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 µM) and ssRNA (1 µM), 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="RNA duplex stability; melting curves; graph; ssRNA1, P1-P5 interaction; thermal denaturation analysis." class="xfigimg" src="/files/ftp_upload/56221/56221fig7.jpg" data-altupdatedat="1788250015000" data-alt="Figure 7"> 
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 µM of single-stranded RNA (ssRNA1) and PNA in 130 µL. (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.

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 ...

The overall goal of this methods article is to introduce the detailed protocols utilized by our lab for studying the molecular recognition of double-stranded RNAs by chemically modified peptide nucleic acids. This method can help answer key questions in the RNA chemical biology field such as detection and targeting of RNA structures. The main advantage of this technique is that the design principles can be applicable to the targeting of any RNA duplex structures.Demonstrating the procedure will be Desiree Toh, a research associate from my laboratory. To begin this procedure, add four microliters of a 35%glycerol solution to previously prepared RNA PNA samples and mix gently. Using a micropipette and gel loading tips, carefully 20 microliters of each sample to a previously prepared polyacrylamide gel making sure not to introduce any air bubbles.Run the gel at a constant voltage of 250 volts for five hours. After five hours, turn off the power supply and remove the glass plate from the gel stand. Then remove the gel sealing tape and disassemble the glass plate.Gently remove the gel from the glass plate and place it in a container filled with 350 milliliters of deionized water. Carefully add 35 microliters of ethidium bromide to the container and place it on the platform shaker on low speed for 30 minutes. After disposing of the ethidium bromide solution, rinse the gel with 1.5 to two liters of distilled water.Then scan the gel using an imager with a green laser of 532 nanometers and the emission filter set at 610 nanometers. Quantify the gel band intensities using free software. Transfer the appropriate quantity of 2-aminopurine labeled double-stranded RNA into a clean 1.5 milliliter tube.Then concentrate the RNA solutions using a vacuum concentrator. Next, transfer the appropriate volumes of the targeted PNA for various concentrations from the previously prepared main stock to separate 1.5 milliliter tubes. Concentrate the PNA solutions using the vacuum concentrator.Add 975 microliters of incubation buffer to the tube containing the dried RNA and mix thoroughly to ensure all the RNA is dissolved. After briefly centrifuging the RNA solution, subject it to annealing by placing the tube into a preheated heat block for 10 minutes. When finished, turn off the heat block and allow the sample to cool to room temperature.Add 75 microliters of RNA to each of the tubes containing the dried PNA and mix thoroughly. After allowing the samples to sit at room temperature for at least one hour, incubate them at four degrees Celsius overnight. Now, transfer the appropriate quantities of 2-aminopurine labeled single-stranded RNA to separate 1.5 milliliter tubes.Transfer the appropriate volumes of targeted PNA for various concentrations from the main stock to the respective tubes containing the single-stranded RNA. After drying the samples, add 75 microliters of incubation buffer to each of the tubes and mix thoroughly. Subject each sample to annealing by placing each tube into a preheated heat block for 10 minutes.After allowing the samples to cool to room temperature, incubate them at four degrees Celsius overnight. Following this, transfer 70 microliters of each sample into a one centimeter square cuvette. Place the cuvette in a fluorescence spectrophotometer and measure the emission over wavelength range of 330 to 550 nanometers.Once the measurement is complete, remove the buffer from the cuvette. Rinse the cuvette with distilled water and dry with nitrogen gas. After repeating the measurement for all the samples, plot the fluorescence intensity against the wavelength.Transfer the appropriate quantities of single-stranded RNA to separate 1.5 milliliter tubes. Then transfer the appropriate volumes of targeted PNA from the main stock to the respective tubes containing the single-stranded RNA. After drying the samples, add 130 microliters of incubation buffer to each of the tubes and mix thoroughly.Subject each sample to annealing by placing each tube into a preheated heat block for 10 minutes. After allowing the samples to cool to room temperature, incubate them at four degrees Celsius overnight. Now, transfer 130 microliters of each sample to an eight microcell cuvette with one cell containing incubation buffer.Using a UV-Vis spectrophotometer, measure the absorbance of each sample at 260 nanometers. Measure at increasing temperature from 15 to 95 degrees Celsius followed by decreasing temperature from 95 to 15 degrees Celsius at a ramp rate of 0.5 degrees Celsius per minute. PNA oligomers were obtained after two reverse phase HPLC purifications.The identity of the PNAs can be confirmed by MALDI/TOF analysis. The nondenaturing PAGE data suggest that the Q and L modified PNA can only recognize the double-stranded RNA region with a C-G pair. This specific and enhanced recognition is through the T-A-U-L-G-C and the Q-C-G PNA RNA two based triple formation.A 2-aminopurine fluorescence titration study showed that a Q and L modified PNA binds to a targeted double-stranded RNA but not single-stranded RNA. PNAs containing Q residues do not show thermal melting transitions suggesting an absence of binding to single-stranded RNA due to the steric clash in the Watson Watson-Crick like Q-G pair. Compared to unmodified PNA P1, PNAs P4 and P5 containing L residues show a lower melting temperature for the corresponding RNA PNA duplexes due to the steric clash in the 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 strongly bind to single-stranded RNA. Incorporating a Q base is more destabilizing than an L base as a Q base has a more significant steric clash in the formation of a Watson-Crick like Q-G pair. Once mastered, the various experiments can be done in a week if it is performed properly.Following this procedure, other methods such as probing and targeting RNA structures in cells can be performed in order to answer additional questions like whether we can detect RNA structures and regulate the RNA functions by using double-stranded RNA binding PNAs. After its development, this technique paved the way for researchers in the field of RNA biology and disease to explore RNA structure targeted therapeutic development.

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

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Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

Oligonucleotide 5′-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5′-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

Chemical Triphosphorylation of Oligonucleotides

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

Learn protocols for synthesizing and characterizing modified Peptide Nucleic Acids that selectively recognize double-stranded RNA structures. This method enables precise RNA targeting for chemical biology research.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids (Video) | JoVE Journal

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

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