This work was supported by the Georgia Cancer Coalition grant-R9028.

Rationale
Exploiting the use of Thermo Scientific Dharmacon oligos, 50 to 80-mers, here we present a procedure, based on a gene correction assay, by which oligos can transfer genetic information to genomic DNA of yeast cells, following annealing to the target chromosomal DNA. Successful targeting by oligos is scored by the appearance of yeast colonies displaying the expected phenotype. If the desired genetic modification is carried within one or more ribonucleotides incorporated in the oligo sequence, the genetic information flows directly from the RNA tract to the chromosomal target DNA, thus, an RNA/DNA hybrid forms at the chromosomal level during the targeting process. The ribonucleotide/s of a Thermo Scientific Dharmacon RNA-containing oligo can serve as template for gene targeting via DNA repair synthesis or can be embedded into the DNA and serve as template during DNA replication. In these experiments we use the yeast /Saccharomyces cerevisiae/ eukaryotic model system; however, a similar approach can also be applied in other organisms or cell types. In this video, we use a Thermo Scientific Dharmacon oligo designed with homology to a target yeast genomic locus and containing one ribonucleotide in the middle to correct a mutation in the target gene. After transformation with the RNA-containing oligo, we observe the correction of the targeted site at a certain frequency, which indicates that the RNA tract of the oligo could be incorporated into the chromosomal DNA and serve as template for DNA synthesis during DNA replication. The genetic information carried within the RNA tract is stably transmitted to the following cell generations. Here we describe the steps of yeast cell transformation by the Thermo Scientific Dharmacon RNA-containing oligo and the approach to detect the RNA information transferring into chromosomal DNA.
A. Design of the RNA-containing Oligo used in this Experiment
We have designed an RNA-containing oligo to correct a genetic defect in the yeast S. cerevisiae chromosomal DNA at the trp5 locus. The yeast strain used in the protocol contains a mutant trp5 gene with a two-base deletion and one nonsense mutation (Figure 1A). Such yeast strain is a tryptophan auxotrophic mutant and does not form colonies on media without tryptophan. The DNA sequence of the TRP5 gene is available at the Saccharomyces Genome Database (http://www.yeastgenome.org/). The Thermo Scientific Dharmacon RNA-containing oligo used in this protocol is a 65-mer with a 2-base DNA insertion, designed to correct the deletion mutation and one ribonucleotide designed to correct the nonsense mutation of the trp5 allele (Figure 1A). The sequence of the oligo is designed as follows: 
 5'-AAAAGGGTTTTGATGAAGCTGTCG-CG-GATCCCACATTCTG-rG-GAAGACTTCAAATCCTTGTATTCT. This oligo is synthesized by Thermo Scientific Dharmacon (Lafayette, CO) at 200 nM scale, and is desalted, deprotected and used without PAGE purification.
B. Preparation of the RNA-containing Oligo for Yeast Transformation (modified from Storici et al., 2007 1)
<ol>
 <li>Wipe materials that will be used for the experiment including oligo tubes, pipettes, vortex, racks, experimental area and gloves worn by the investigator with RNase decontamination solution to remove potential RNase contamination before everything starts. Use RNase-free water, chemical reagents, tubes and pipette tips in all steps. Every step in this experiment should be RNase-free.</li>
 <li>Resuspend the Thermo Scientific Dharmacon RNA-containing oligo received from the company to 250 pmoles/μl stock solution with RNase-free water and vortex vigorously to dissolve the pellet. Store at -80°C.</li>
 <li>Immediately before the transformation, thaw the RNA-containing oligo on ice and dilute to 50 pmoles/μl with RNase-free water in RNase-free tubes. Each transformation requires 1 nmole of the RNA-containing oligos.</li>
 <li>Denature a chosen amount of the RNA-containing oligos on the 100 °C heat block for 2 min to eliminate secondary structures of the oligos.</li>
 <li>Immediately after denaturation place the tube on ice. Keep on ice till transformation.</li>
 <li>As control in the experiment a corresponding DNA-only oligo is used. This oligo is thawed from -20 °C and prepared for the transformation as the RNA-containing oligo, as described above.</li>
</ol>
C. Transformation of Yeast Cells using the RNA-containing Oligo
<ol>
 <li>Inoculate 5 ml of rich YPD liquid medium with the trp5 mutant yeast cells and grow at 30 °C overnight (see Materials).</li>
 <li>Transfer 1.5 ml of the overnight culture into 50 ml of YPD liquid medium.</li>
 <li>Incubate cells in a 30 °C shaker (225 rpm) for 4h.</li>
 <li>Prepare Solution 1 and Solution 2 immediately before transformation in RNase-free tubes (see Materials).</li>
 <li>Transfer the cell culture to a 50 ml RNase-free tube and spin at 3000 rpm, corresponding to 1562 g, for 2 min. The pellet of the cell precipitation is approx. 0.3 cm3.</li>
 <li>Remove the supernatant and wash cells with 50 ml of RNase-free water and spin at 3000 rpm for 2 min.</li>
 <li>Repeat step 6 for 5 times to get rid of the culture medium and RNases that could be present in the medium as much as possible.</li>
 <li>Remove the supernatant and resuspend cells in 5 ml of Solution 1 and spin at 3000 rpm for 2 min.</li>
 <li>Remove supernatant and resuspend cells in 250 μl of Solution 1. This amount of cells is sufficient for 7-8 transformations. </li>
 <li>Aliquot 50 μl of the cell suspension in RNase-free microcentrifuge tubes, add 20 μl of RNA-containing oligo working solution (1 nmole), or 20 μl of DNA-only oligo working solution (1 nmole), or 20 μl of sterile water with no oligo for the negative control. Then add 300 μl of Solution 2 for each transformation reaction. There is no need to add salmon sperm DNA in the process of transformation, as the oligos act as carrier themselves. </li>
 <li>Vortex vigorously to mix components homogenously.</li>
 <li>Incubate transformation reactions at 30 °C for 30 min in a shaker.</li>
 <li>Heat shock at 42 °C for 15 min. </li>
 <li>Spin down cells at 5000 rpm, corresponding to 2236 g, for 4 min.</li>
 <li>Remove supernatant and resuspend cells in 100 μl of sterile water.</li>
 <li>Take an aliquot of this cell suspension and dilute it with sterile water by 100,000 fold and plate cells on one YPD plate using approx. 15 sterile glass beads and incubate at 30 °C for 2 days.</li>
 <li>Plate all resuspended cells from each transformation reaction on one Petri dish of synthetic complete solid medium without tryptophan (SC-Trp) using approx. 15 sterile glass beads and incubate at 30 °C for 4-5 days (Figure 2).</li>
</ol>
D. Analysis of Gene Correction by the RNA-containing Oligo
<ol>
 <li>Count the number of colonies grown on the selective medium (Figure 2) as well as on YPD medium to calculate the gene correction frequency for the RNA-containing oligo, the DNA-only oligo and for the no-oligo control. Compare the number obtained. Spontaneous reversion rate of the trp5 alleles with the two-base deletions and the nonsense mutation is less than 10-9 in the used yeast strain, thus we expect no colonies formation on the selective medium when no oligos are added to the cells.</li>
 <li>Streak out several randomly picked transformant colonies onto YPD medium to obtain single colony isolates. Wait two days for colony growth, then take several (at least 5) single colonies and make patches on YPD and on selective medium.</li>
 <li>Design a pair of primers to amplify the region (250-1,000 bp) targeted by the RNA-containing oligo by colony PCR (Figure 1B). The procedure for colony PCR is as follows (modified from Storici and Resnick, 2006 2).
 <ol>
 <li>Resuspend cells (approx. 1 mm3) taken from the individual patches in 50 μl of water and add 1 unit of lyticase. Incubate at room temperature for 10 min, followed by incubation in a heat block at 100 °C for 5 min to break the cells and release genomic DNA in solution.</li>
 <li>PCR conditions: The PCR reaction includes 10 μl of the cell resuspension solution, 50 pmoles each of forward and reverse primers, 1 μl of 10 mM dNTPs, 1 unit of Taq polymerase, 5 μl of 10x buffer and is adjusted with sterile water to a final volume of 50 μl. The PCR program is 3 min at 95 °C; 30 cycles of 30 s at 95°C, 30 s at 55 °C, and 1 min at 72 °C; a final extension time of 7 min at 72 °C; then samples are kept at 4 °C. An extension time of 1 min/kb is assumed for this reaction.</li>
 <li>Following PCR, samples are run on a 1% or 2% (depending on the expected size of the PCR product) agarose gel for observation of the PCR product (Figure 3). </li>
 </ol>
 </li>
 <li>If the genetic information transferred by the RNA-containing oligo generates a new restriction site in the yeast genomic target region (Figure 1B), it is possible to verify the correct transfer of information by digesting the PCR product with the appropriate restriction enzyme. If no restriction site is generated by the RNA-containing oligo, go to step 6. Digest PCR products using a specific restriction enzyme. The digestion reaction includes 6 μl of PCR product, buffer, BSA (may not be needed for some enzymes, see instruction for the enzyme used), 0.5 μl of restriction enzyme, and sterile water to 15 μl. Samples are incubated for 1 h at the temperature specific for the enzyme used. </li>
 <li>Run an undigested sample together with the digested samples on the same row of a 2% agarose gel to observe the genetic modification transferred by the RNA tract of the RNA-containing oligo (Figure 3). </li>
 <li>Purify PCR products by using a PCR purification kit and prepare them for DNA sequencing. Submit samples for sequencing with the same primers used to amplify the product.</li>
 <li>Analyze DNA sequencing results using software that allows alignment of multiple sequences with a chosen reference sequence (Figure 4).</li>
</ol>
E. Alkali Treatment of the RNA-containing oligo (Figure 5)
<ol>
 <li>For each reaction, add 1 nmole (4 μl of 250 pmoles/μl stock solution) of the RNA-containing oligo, or DNA-oligo into a 1.5 ml tube.</li>
 <li>Add 4 μl of 1 M NaOH for hydrolysis, or alternatively add 4 μl H2O as negative control, and incubate at 65° C in a water bath for 1 h. Then move from the water bath to ice.</li>
 <li>Neutralize with 2 μl of 1.2 M HCl, 4 μl of 1 M Tris-HCl, and 4 μl of H2O, or alternatively 6 μl of H2O and 4 μl of 1 M Tris-HCl for negative control. Keep on ice till transformation.</li>
</ol>
<img src="/files/ftp_upload/2152/2152fig1.jpg" alt="Gene editing process, RNA oligo guides correction of mutated genomic DNA, diagram showing sequence edits." data-altupdatedat="1747911167000" data-alt="Figure 1"> Figure 1. Schematic diagram of the defective trp5 gene and of the TRP5 allele corrected by the RNA-containing oligo. A) The trp5 mutant gene contains a 2-base deletion (black triangles) and 1-base nonsense mutation (asterisk). The single-strand RNA-containing oligo with 2-base insertion (blue loop) and 1-RNA base substitution (red rectangle) generating a Van91I restriction enzyme site (shown by the bracket) is transformed into yeast cells to correct the genetic defects of the trp5 gene. B) After the TRP5 gene is repaired by the RNA-containing oligo (corrected bases are indicated as blue rectangles), the Van91I site is generated into the TRP5 gene. A 278 bp fragment including only one Van91I restriction site in TRP5 gene is PCR amplified by a pair of primers (P1 and P2). The 177 bp and 101 bp fragments generated after digestion of the P1 and P2 PCR product by Van91I are also shown.
<img src="/files/ftp_upload/2152/2152fig2.jpg" alt="Bacterial colony growth on agar plates; microbial experiment results; comparative analysis." data-altupdatedat="1747911167000" data-alt="Figure 2"> Figure 2. Transformation result with the RNA-containing oligo. A) Yeast cells transformed with no oligo do not form any colony on the SC-Trp medium, see plates a-b. B) Plates c-g show yeast colonies growing on SC-Trp after cells are transformed with 1 nmole of the RNA-containing oligo. C) Plates h-l show yeast colonies growing on SC-Trp after cells are transformed with 1 nmole of the corresponding DNA-only control oligo.
<img src="/files/ftp_upload/2152/2152fig3.jpg" alt="Gel electrophoresis, DNA fragmentation analysis, 100-500 bp marker, molecular biology results." data-altupdatedat="1747911167000" data-alt="Figure 3"> Figure 3. Detection of genetic information transfer from the RNA-containing oligo to yeast chromosomal DNA by restriction digestion of the PCR product amplifying the targeted genomic region. 2% agarose gel electrophoresis of PCR samples amplified by P1 and P2 and digested with Van91I restriction enzyme. Lanes 1, 8 and 15, DNA ladder with sizes of 100, 200, 300, 400 and 500 bp indicated on the left; lane 2, PCR product of trp5 locus amplified from the genomic DNA of the trp5 mutant strain; lane 3, PCR product amplified from genomic DNA derived from a Trp+ colony targeted by the DNA-only oligo; lane 4-7, PCR products amplified from genomic DNA derived from Trp+ colonies targeted by the RNA-containing oligo; lane 9 to 14, Van91I restriction digestion of the PCR products from lanes 2 to 7. The presence of the uncut PCR product bands in lanes 10 to 14 can be explained by partial digestion by Van91I at the TRP5 locus (CCACATTCTGG). Considering that the cutting site for Van91I (CCANNNN NTGG) can have multiple sequences, the site generated in TRP5 may not be the most optimal target for the enzyme. In fact, following DNA sequencing of all the above PCR products we detect no additional changes beyond those carried by the oligos (see Figure 4). The 278 bp PCR band amplified by P1 and P2 primers and the digestion product bands by Van91I of 177 bp and 101 bp are shown by the arrows on the right.
<img src="/files/ftp_upload/2152/2152fig4tmb.jpg" border="0" alt="Gene editing sequence analysis, DNA-RNA oligo alignment, chromatogram diagram." data-altupdatedat="1747911167000" data-alt="Figure 4Thumb"> Figure 4. DNA sequencing results showing gene correction by the RNA-containing oligo. A) DNA electropherogram of the genomic region targeted by the RNA-containing oligo. The G in the DNA sequence (blue boxed) derives from the rG on the RNA-containing oligo. Also boxed is the insertion of the CG bases. Sequencing results from all other PCR products are also as clear as this one, with fluorescent signals well above the background. B) Sequences of the TRP5 region of the Trp+ transformants targeted by the DNA-only oligo (T1) and the RNA-containing oligo (T2-T5) match in the consensus sequence on the top and are compared with that of the trp5 mutant cells before targeting by the oligos (Genomic DNA, red shaded). The repair RNA-containing oligo on the bottom has two-base DNA insertion and one-base (G) RNA substitution marked in red. The regions boxed in blue with yellow shade show that the RNA-containing oligo as well as the DNA-only oligo precisely corrected the deletion mutation and nonsense mutation in all tested samples. The dashed lines mark the position of the RNA-containing oligo sequence. Please <a href="https://www.jove.com/files/ftp_upload/2152/2152fig4.jpg">click here</a> to see a larger version of figure 4.
<img src="/files/ftp_upload/2152/2152fig5.jpg" alt="Bar chart comparing transformation efficiency with and without NaOH treatment; biological analysis." data-altupdatedat="1747911167000" data-alt="Figure 5"> 
 Figure 5. Alkali treatment prevents gene correction by the RNA-containing oligo. The transformation frequency by the RNA-containing oligo (R) is shown in the red bar and that by the DNA-only oligo (D) is shown in the blue bars. The error bars represent the standard error of the mean for 3 independent transformations for each oligo. The DNA-only oligo displays similar transformation frequency without and with NaOH treatment. Differently, transformation frequency by the RNA-containing oligo drops to 0 following treatment with NaOH. Therefore, the preparation with the RNA-containing oligo is not contaminated with DNA-only oligo, thus, the observed transformation frequency is specific to the RNA-containing oligo.

<ol>
	<li>Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A., Resnick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-templated+DNA+repair.">RNA-templated DNA repair.</a> Nature. 447, 338-3341 (2007).</li><li>Storici, F., Resnick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+delitto+perfetto+approach+to+in+vivo+site-directed+mutagenesis+and+chromosome+rearrangements+with+synthetic+oligonucleotides+in+yeast.">The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.</a> Methods Enzymol. 409, 329-345 (2006).</li><li>Storici, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-mediated+DNA+modifications+and+RNA-templated+DNA+repair.">RNA-mediated DNA modifications and RNA-templated DNA repair.</a> Curr Opin Mol Ther. 10, 224-230 (2008).</li><li>Cerritelli, S. M., Crouch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonuclease+H:+the+enzymes+in+eukaryotes.">Ribonuclease H: the enzymes in eukaryotes.</a> FEBS J. 276, 1494-1505 (2009).</li></ol>

The video production for this article was sponsored by Thermo Fisher Scientific, which produces reagents and instrument used in this publication.

The fact that RNA can transfer genetic information directly to genomic DNA in cells was discovered exploiting the use of synthetic RNA-containing oligos (Dharmacon synthesized oligos) 1. It was the proof of principle that cells can use RNA-containing molecules or RNA-only sequences as templates for DNA synthesis. The use of RNA-containing oligos not only led to the demonstration that genetic information can be transferred directly from RNA to genomic DNA without the need of a reverse transcribed DNA copy intermediate, but also to the proof that RNA can be used as homologous template in the repair of a DNA damage 1,3. The oligos can be made of RNA-only or contain just a single ribonucleotide embedded in a DNA sequence, as in the example we presented here. The RNA-containing oligo has one embedded ribonucleotide designed to correct the nonsense mutation in the genomic defective trp5 allele. The transfer of genetic information from the ribonucleotide to the genomic DNA is revealed by a change in phenotype (cell growing on the medium lacking tryptophan) of the targeted cells. The frequency of gene correction obtained with the RNA-containing oligo is measured and compared with that of a control DNA-only oligo. By performing this gene correction assay in different cell backgrounds, where we mutate various yeast genes, we can detect which factor/s specifically affect targeting by the RNA-containing oligos. Thus, we can reveal mechanisms how cells regulate the stability of RNA/DNA hybrids. By simply designing different variants of the RNA-containing oligos we can determine the likelihood for a particular RNA tract to serve as template in DNA modification and we can determine the stability of specific RNA sequences embedded into DNA. Moreover, we can identify what are the preferred in vivo substrates for factors interacting with RNA/DNA hybrids.
While several in vitro studies, mainly utilizing short RNA-containing oligos, have been conducted to characterize the function of factors that can recognize RNA in a hybrid with DNA, such as the RNase H enzymes 4, the in vivo functions of RNases H as well as the identity of other proteins that could affect RNA/DNA hybrid stability remain mostly unknown. The possibility to utilize RNA-containing oligos of a significant length (50 to 80-mers) and optimal quality (such as Thermo Scientific Dharmacon RNA-containing oligos) has open the way to examining a wide range of molecular processes directly in vivo in the cells of interest. As shown in this video, transformation using RNA-containing oligos requires essentially only a few additional steps compared to transformation using DNA molecules, to prevent degradation by RNases. Thus, transformation using RNA-containing oligos is not limited to the yeast system, but can be applied to any organism or cell type where transformation by DNA oligos is proficient.
In conclusion, targeting cells by RNA-containing oligos provides the opportunity to generate RNA/DNA hybrids and ribonucleotides embedded in DNA in vivo in cells. The stability, function and consequences of these in vivo generated RNA/DNA hybrids can be analyzed and characterized, potentially uncovering unknown mechanisms of DNA repair and revealing novel strategies for gene targeting.

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;A. Transformation reagents and media (modified from Storici and Resnick, 2006 &lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt;YPD (Yeast Peptone Dextrose): For 1 L, 10 g yeast extract, 20 g soy peptone, 20 g dextrose. Add 15 g agar to make YPD solid media. Autoclave before use. Store at room temperature.&lt;/li&gt; &lt;li&gt;Solution 1: 0.1 M of lithium acetate. Prepare immediately before transformation. Solution 1 is a working solution, thus it is prepared directly from the powder. No stock solution is made. Keep at room temperature. LiAc increases the yeast cell wall permeability to DNA. &lt;/li&gt; &lt;li&gt;Solution 2: 0.1 M of lithium acetate and 50 % of polyethylene glycol 4000. Also solution 2 is a working solution and it is made directly from the powder. No stock solution is prepared. Keep at room temperature. PEG deposits oligos onto yeast cells.&lt;/li&gt; &lt;li&gt;RNA-containing oligos (Thermo Scientific Dharmacon), 50-80-mers, desalted, deprotected and non-purified. Resuspend to 250 pmoles/&amp;mu;l. Store at -80 &amp;deg;C.&lt;/li&gt; &lt;li&gt;DNA-only oligos, 50-80-mers, desalted and non-purified. Resuspend to 50 pmoles/&amp;mu;l. Store at -20 &amp;deg;C.&lt;/li&gt; &lt;li&gt;SC-Trp (Synthetic complete media lacking tryptophan) solid media.&lt;/li&gt; &lt;li&gt;0.5 cm diameter glass beads, sterilized by autoclaving. &lt;/li&gt; &lt;li&gt;RNase-off: RNase decontamination solution.&lt;/li&gt; &lt;li&gt;DNase/RNase-free, sterile centrifuge tubes.&lt;/li&gt; &lt;li&gt;DNase/RNase-free, sterile conical tubes.&lt;/li&gt; &lt;li&gt;DNase/RNase-free, sterile aerosol pipette tips with ZAP: 1-200 ml, 100-1000 ml.&lt;/li&gt; &lt;/ol&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;B. Colony PCR materials.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt;DNA primers, desalted and non-purified. Dissolve in sterile water to 50 pmoles/&amp;mu;l. Store at -20 &amp;deg;C.&lt;/li&gt; &lt;li&gt;Taq DNA polymerase, buffer, dNTPs.&lt;/li&gt; &lt;li&gt;PCR tubes.&lt;/li&gt; &lt;/ol&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;C. PCR purification.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt;PCR purification kit.&lt;/li&gt; &lt;/ol&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;D. Gel Electrophoresis.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt;Agarose.&lt;/li&gt; &lt;li&gt;1 x TBE running buffer (45 mM Tris-borate and 1 mM ethylenediamine tetraacetate) diluted from 10x TBE. &lt;/li&gt; &lt;li&gt;Prestained molecular weight marker.&lt;/li&gt; &lt;li&gt;DNA loading dye.&lt;/li&gt; &lt;/ol&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;E. Restriction digestion.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt;Restriction enzymes, 10x buffers, BSA.&lt;/li&gt; &lt;/ol&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;F. Alkali treatment for the RNA-containing oligo.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt;1 M of NaOH solution.&lt;/li&gt; &lt;li&gt;1.2 M of HCl solution.&lt;/li&gt; &lt;li&gt;1 M of Tris-HCl, pH 7.4 solution.&lt;/li&gt; &lt;/ol&gt;</td></tr></tbody></table>

generation of rna/dna hybrids in genomic dna by transformation using rna-containing oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

Watch this Scientific Journal Video about Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides at JoVE.com

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

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Biology

26.8K Views.  Georgia Institute of Technology. Dit werk laat zien hoe een RNA / DNA hybride vorm op de chromosomale niveau en onthullen de overdracht van genetische informatie van RNA naar DNA genoom in gistcellen.

Video: Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Genetics

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Immunology and Infection

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Medicine

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 ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Developmental Biology

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Chemistry

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Biochemistry

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

Environment

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Bioengineering

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-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&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

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