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 Alternatively, our unpublished data suggest that Cy3 dye-labeled RNAs can be used in non-denaturing PAGE experiments to measure the tight binding events.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

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 ...

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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Biochemistry

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Chemistry

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

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

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

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.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Immunology and Infection

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

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

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5&#39;-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

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.

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

Author Spotlight: Advancements in DNA Nanosensors &#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

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-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...