עבודה זו נתמכה על ידי הקואליציה סרטן גאורגיה מענק-R9028.

<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>

הייצור וידאו עבור מאמר זה נערך בחסות תרמו פישר סיינטיפיק, אשר מייצרת ריאגנטים מכשיר בשימוש בפרסום זה.

העובדה RNA יכולים להעביר מידע גנטי ישירות הדנ&quot;א הגנומי בתאים התגלה ניצול השימוש RNA המכיל oligos סינתטי (oligos Dharmacon מסונתז) 1. זה היה הוכחה של העיקרון כי ניתן להשתמש בתאים המכילים מולקולות RNA או RNA בלבד רצפים כתבניות עבור סינתזה של DNA. השימוש RNA המכיל oligos לא רק הוביל להפגנה כי המידע הגנטי ניתן להעביר ישירות מ-RNA ל-DNA הגנומי ללא צורך הפוכה עיבד DNA להעתיק ביניים, אלא גם הוכחה כי RNA יכול לשמש כתבנית הומולוגיים ב את התיקון של נזק לדנ&quot;א 1,3. Oligos יכול להתבצע של RNA בלבד או להכיל רק ribonucleotide בודד מוטבע ברצף ה-DNA, כמו בדוגמה שהצגנו כאן. RNA המכיל אוליגו יש אחד ribonucleotide מוטבע שנועדה לתקן את המוטציה שטויות את האלל גנומית trp5 פגום. העברת מידע גנטי ribonucleotide ל-DNA הגנומי מתגלה על ידי שינוי פנוטיפ (תא גדל על המדיום חסר טריפטופן) של התאים הממוקדים. התדירות של תיקון הגן שהושגו עם אוליגו RNA המכיל נמדד בהשוואה לזה של ה-DNA רק לשלוט אוליגו. על ידי ביצוע תיקון זה גן assay ברקע תאים שונים, כאשר אנו עוברים מוטציה גנים שמרים שונים, אנו יכולים לזהות מה גורם / ים משפיעים במיוחד על מיקוד על ידי RNA המכיל oligos. לפיכך, אנו יכולים לחשוף מנגנונים כיצד תאים מווסתים את היציבות של RNA / DNA כלאיים. פשוט על ידי תכנון גרסאות שונות של RNA המכיל oligos אנו יכולים לקבוע את הסבירות עבור מערכת RNA מסוים לשמש תבנית שינוי ה-DNA, ואנחנו יכולים לקבוע את יציבות מסוימת רצפי הרנ&quot;א מוטבע לתוך ה-DNA. יתר על כן, אנו יכולים לזהות מה הם העדיפו בשנת מצעים vivo גורמים אינטראקציה עם RNA / DNA כלאיים. בעוד מספר מחקרים במבחנה, בעיקר ניצול קצר RNA המכיל oligos, נערכו כדי לאפיין את הפונקציה של גורמים שיכולים לזהות רנ&quot;א היברידית עם ה-DNA, כגון אנזימים RNase H 4, בפונקציות vivo של RNases H כמו גם כזהות של חלבונים אחרים שיכולים להשפיע על RNA / DNA יציבות היברידית אינם ידועים ברובם. האפשרות לנצל RNA המכיל oligos באורך משמעותי (50-80, מרס) ובאיכות אופטימלית (כגון Thermo Scientific Dharmacon RNA המכיל oligos) יש לפתוח את הדרך בחינת מגוון רחב של תהליכים מולקולריים ישירות in vivo ב תאים של עניין. כפי שניתן לראות השינוי הזה וידאו, באמצעות RNA המכיל oligos דורש בעצם רק כמה צעדים נוספים לעומת השינוי באמצעות מולקולות ה-DNA, כדי למנוע השפלה ידי RNases. לכן, השינוי באמצעות RNA המכיל oligos אינו מוגבל למערכת שמרים, אבל יכול להיות מיושם על כל אורגניזם או סוג התא שבו שינוי ה-DNA על ידי oligos הוא מיומן. לסיכום, על ידי מיקוד תאים המכילים RNA oligos מספקת את ההזדמנות כדי לייצר RNA / DNA היברידיים ribonucleotides מוטבע ב DNA בתאי in vivo. היציבות, פונקצית וההשלכות של אלה in vivo שנוצר RNA / DNA כלאיים ניתן לנתח ומאופיינת, פוטנציאל חשיפת מנגנוני ידוע של תיקון DNA וגילוי אסטרטגיות חדשניות גן מיקוד.

א טרנספורמציה ריאגנטים המדיה (שונה מן Storici ו רזניק, 2006 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> YPD (שמרים Peptone דקסטרוז): עבור L 1, 10 גרם תמצית שמרים, 20 גרם סויה peptone, 20 גרם דקסטרוז. הוסף 15 גר &#39;אגר לעשות התקשורת מוצק YPD. החיטוי לפני השימוש. אחסן בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> פתרון 1: 0.1 M של ליתיום אצטט. הכן מיד לפני השינוי. פתרון 1 הוא פתרון של העבודה, ולכן הוא מוכן ישירות האבקה. פתרון ללא מניות הוא עשה. לשמור בטמפרטורת החדר. LiAc מגדיל את חדירות התא שמרים קיר ל-DNA. </li><li style=";text-align:right;direction:rtl"> פתרון 2: 0.1 M של ליתיום אצטט ו -50% פוליאתילן גליקול 4000. כמו כן פתרון 2 הוא פתרון עבודה הוא עשה ישירות האבקה. פתרון ללא מניות מוכנה. לשמור בטמפרטורת החדר. פיקדונות PEG oligos על תאים ושמרים. </li><li style=";text-align:right;direction:rtl"> RNA המכיל oligos (Thermo Scientific Dharmacon), 50-80, מרס, desalted, deprotected שאינם מטוהרים. Resuspend עד 250 pmoles / μl. חנות ב -80 ° C. </li><li style=";text-align:right;direction:rtl"> ה-DNA רק oligos, 50-80, מרס, desalted שאינם מטוהרים. Resuspend עד 50 pmoles / μl. חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> SC-TRP (התקשורת להשלים סינתטי חסר טריפטופן) מדיה מוצק. </li><li style=";text-align:right;direction:rtl"> בקוטר 0.5 ס&quot;מ חרוזי זכוכית, מעוקרים ידי מעוקר. </li><li style=";text-align:right;direction:rtl"> RNase-off: פתרון RNase טיהור. </li><li style=";text-align:right;direction:rtl"> DNase / RNase חופשי, מבחנות צנטריפוגה סטרילית. </li><li style=";text-align:right;direction:rtl"> DNase / RNase חינם, צינורות חרוטי סטרילית. </li><li style=";text-align:right;direction:rtl"> DNase / RNase חינם, פיפטה סטרילית אירוסול טיפים עם ZAP: 100-200 מ&quot;ל, 100-1000 מ&quot;ל. </li></ol> ב המושבה PCR חומרים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ה-DNA primers, desalted ולא מטוהרים. מתמוססים במים סטריליים עד 50 pmoles / μl. חנות ב -20 ° C. </li><li style=";text-align:right;direction:rtl"> תקי פולימראז ה-DNA, מאגר, dNTPs. </li><li style=";text-align:right;direction:rtl"> PCR צינורות. </li></ol> ג טיהור PCR. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> PCR ערכת טיהור. </li></ol> ד ג&#39;ל אלקטרופורזה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Agarose. </li><li style=";text-align:right;direction:rtl"> 1 x חיץ TBE ריצה (45 mM טריס-borate ו 1 ethylenediamine tetraacetate mM) מדוללת מ TBE 10x. </li><li style=";text-align:right;direction:rtl"> Prestained משקל מולקולרי הסמן. </li><li style=";text-align:right;direction:rtl"> ה-DNA לצבוע הטעינה. </li></ol> E. העיכול סייג. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגבלת אנזימים, מאגרים 10x, BSA. </li></ol> פ טיפול אלקליות עבור RNA המכיל אוליגו. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 1 M NaOH של פתרון. </li><li style=";text-align:right;direction:rtl"> 1.2 מ &#39;של פתרון HCl. </li><li style=";text-align:right;direction:rtl"> 1 M של טריס-HCl, pH 7.4 פתרון. </li></ol>

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

פולימרים סינתטיים קצר חומצות גרעין, oligonucleotides (oligos), הם כלי פונקציונלי ביותר נרחבת של הביולוגיה המולקולרית. Oligos יכול להיות מיוצר להכיל את כל ה-DNA הרצוי או רצף הרנ&quot;א יכול להיות מוכן לכלול מגוון רחב של הבסיס שינויים סוכר. יתר על כן, oligos ניתן שנועד לחקות שינויים ספציפיים חומצות גרעין, ולכן, יכול לשמש כלי חשוב לחקור את ההשפעות של נזק לדנ&quot;א מנגנוני התיקון. מצאנו כי Thermo Scientific Dharmacon RNA המכיל oligos עם אורך בין 50 ל 80 נוקלאוטידים יכול להיות מתאים במיוחד ללמוד, ב, פונקציות vivo וההשלכות של RNA כרומוזומליות / כלאיים דנ&quot;א של ribonucleotides מוטבע לתוך ה-DNA. RNA / DNA כלאיים יכול בקלות ליצור במהלך התיקון שכפול, שעתוק דנ&quot;א, לעומת זאת, מעט מאוד ידוע על היציבות של RNA / DNA כלאיים בתאים אשר במידה כלאיים אלה יכולים להשפיע על שלמות הגנטי של התאים. RNA המכיל oligos, אם כן, מייצגים וקטור המושלם להציג ribonucleotides לתוך הדנ&quot;א בכרומוזומים וליצור RNA / DNA כלאיים של אורך נבחר הרכב הבסיס. כאן אנו מציגים את הפרוטוקול עבור שילוב של ribonucleotides לתוך הגנום של שמרים מערכת אוקריוטים מודל / cerevisiae Saccharomyces /. עם זאת, במעבדה שלנו ניצלה Thermo Scientific Dharmacon RNA המכיל oligos לייצר RNA / DNA כלאיים ברמת כרומוזומליות במערכות תאים שונים, בין חיידקים לתאי האדם.

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

עבודה זו מראה כיצד ליצור כלאיים RNA / DNA ברמה כרומוזומליות ולחשוף העברת מידע גנטי מ-RNA ל-DNA הגנומי בתאי שמרים.

הדור של RNA / DNA כלאיים ב-DNA הגנום על ידי טרנספורמציה באמצעות RNA המכיל oligonucleotides

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

generation-rnadna-hybrids-genomic-dna-transformation-using-rna

Research

JoVE Journal

Biology

26.8K Views.  Georgia Institute of Technology. עבודה זו מראה כיצד ליצור כלאיים RNA / DNA ברמה כרומוזומליות ולחשוף העברת מידע גנטי מ-RNA ל-DNA הגנומי בתאי שמרים.

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.

מתודולוגיה סטנדרטי כדי לבדוק באתר המוטגניות כפונקציה של תיקון נקודת מוטציה על ידי CRISPR/Cas9 ו- SsODN בתאי אדם

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.

פיתוח נייד מסוג ספציפי נגד HIV gp120 aptamers למסירה siRNA

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.

וקטור מבוסס DNA התערבות RNA ללמוד לתפקד גנים סרטן

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.

בצפיפות גבוהה DNA ו-RNA מיקרומערכים-פוטוליתוגרפיה סינתזה, היברידיזציה והכנת ספריות גרעין גדול חומצות

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.

הדור של עכברים השתנה גנטית דרך Microinjection של ביציות

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.

Protocol עבור שלב מוצק סינתזה של אוליגומרים של רנ&quot;א המכיל 2 &#39;<em&gt; הו</em&gt; שינוי ואפיון של תיאופנילמתיל באמצעות דיכרואיזם מעגלי

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.

שימוש באלמנט Alu המכיל מיני-גנים לניתוח Rnas מעגלית

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.

הפרעות RNA בחפשיות מימיות ככלי רב עוצמה למניפולציה ביטוי גנים בנקודות זמן התפתחותיות ספציפיות

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.

RNA פולימראז קשור DNA לתכנות תמלול במבחנה וחישוב מולקולרי

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.

טריפוספורילציה כימית של אוליגונוקלאוטידים

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