Current Protocols in Nucleic Acid Chemistry最新文献

{"title":"Solid-Phase Synthesis of RNA 5′-Azides and Their Application for Labeling, Ligation, and Cyclization Via Click Chemistry","authors":"Marcin Warminski,&nbsp;Joanna Kowalska,&nbsp;Jacek Jemielity","doi":"10.1002/cpnc.112","DOIUrl":"10.1002/cpnc.112","url":null,"abstract":"<p>RNAs with 5′ functional groups have been gaining interest as molecular probes and reporter molecules. Copper-catalyzed azide-alkyne cycloaddition is one of the most straightforward methods to access such molecules; however, RNA functionalization with azide group has been posing a synthetic challenge. This article describes a simple and efficient protocol for azide functionalization of oligoribonucleotides 5′-end in solid-phase. An azide moiety is attached directly to the C5′-end in two steps: (i) -OH to -I conversion using methyltriphenoxyphosphonium iodide, and (ii) -I to -N₃ substitution using sodium azide. The reactivity of the resulting compounds is exemplified by fluorescent labeling using both copper(I)-catalyzed (CuAAC) and strain-promoted (SPAAC) azide-alkyne cycloaddition reactions, ligation of two RNA fragments, and cyclization of short bifunctionalized oligonucleotides. The protocol makes use of oligoribonucleotides synthesized by standard phosphoramidite approach on solid support, using commercially available 2′-<i>O</i>-PivOM-protected monomers. Such a protection strategy eliminates the interference between the iodination reagent and silyl protecting groups (TBDMS, TOM) commonly used in RNA synthesis by phosphoramidite approach. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Solid-phase synthesis of oligoribonucleotide 5′-azides</p><p><b>Basic Protocol 2</b>: CuAAC labeling of oligoribonucleotide 5′-azides in solution</p><p><b>Alternate Protocol 1</b>: CuAAC labeling of oligoribonucleotide 5′-azides on solid support</p><p><b>Basic Protocol 3</b>: SPAAC labeling of oligoribonucleotide 5′-azides</p><p><b>Basic Protocol 4</b>: CuAAC ligation of oligoribonucleotide 5′-azides</p><p><b>Basic Protocol 5</b>: CuAAC cyclization of oligoribonucleotide 5′-azides</p><p><b>Support Protocol</b>: HPLC Purification</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"82 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.112","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38205202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis","authors":"Shiyue Fang,&nbsp;Dhananjani Eriyagama,&nbsp;Yinan Yuan,&nbsp;Shahien Shahsavari,&nbsp;Jinsen Chen,&nbsp;Xi Lin,&nbsp;Bhaskar Halami","doi":"10.1002/cpnc.111","DOIUrl":"10.1002/cpnc.111","url":null,"abstract":"<p>This protocol provides details for the preparation of nucleoside phosphoramidites with 1,3-dithian-2-yl-methyl (Dim) and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) as protecting groups, and a linker with Dmoc as the cleavable function, then using them for solid phase synthesis of sensitive oligodeoxynucleotides (ODNs). Using these Dim-Dmoc phosphoramidites and Dmoc linker, ODN synthesis can be achieved under typical conditions using phosphoramidite chemistry with slight modifications, and ODN deprotection and cleavage can be achieved under mild conditions involving oxidation with sodium periodate at pH 4 followed by aniline at pH 8. Under the mild deprotection and cleavage conditions, many sensitive functional groups including but not limited to esters, thioesters, alkyl halides, <i>N-</i>aryl amides, and α-chloroamides—which cannot survive the basic and nucleophilic deprotection and cleavage conditions such as concentrated ammonium hydroxide and dilute potassium methoxide used in typical ODN synthesis technologies—can survive. Thus, it is expected that the Dim-Dmoc ODN synthesis technology will find applications in the synthesis of ODNs that contain a wide range of sensitive functional groups. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Synthesis, deprotection, cleavage, and purification of sensitive oligodeoxynucleotides</p><p><b>Support Protocol 1</b>: Synthesis of Dim-Dmoc nucleoside phosphoramidites</p><p><b>Support Protocol 2</b>: Preparation of CPG with a Dmoc linker</p><p><b>Support Protocol 3</b>: Synthesis of a phosphoramidite containing a sensitive alkyl ester group</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"82 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.111","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38121782","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of 9-(6-Deoxy-α-L-Talofuranosyl)-6-Methylpurine and 9-(6-Deoxy-β-D-Allofuranosyl)-6-Methylpurine Nucleosides.","authors":"Abdalla E A Hassan,&nbsp;Reham A I Abou-Elkhair,&nbsp;Hend M Maaroof,&nbsp;John A Secrist","doi":"10.1002/cpnc.105","DOIUrl":"https://doi.org/10.1002/cpnc.105","url":null,"abstract":"<p><p>6-Methylpurine (MeP) is a cytotoxic adenine analog that does not exhibit selectivity when administered systemically and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli purine nucleoside phosphorylase (PNP). 9-(6-Deoxy-β-D-allofuranosyl)-6-methylpurine [methyl(allo)-MePR, 18] and 9-(6-deoxy-α-L-talofuranosyl)-6-methylpurine [methyl(talo)-MePR, 21] were synthesized as potential prodrugs for MeP in the E. coli PNP/prodrug cancer gene therapy approach. The detailed syntheses of [methyl(allo)-MePR] and [methyl(talo)-MePR] are described. The glycosyl donors, 1,2-di-O-acetyl-3,5-di-O-benzyl-α-D-allofuranose (12) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-α-L-talofuranose (16) were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (4) in nine and eleven steps, respectively. Vorbrüggen coupling of the latter glycosyl donors with 6-methylpurine (3), followed by deprotection of the sugar hydroxyl groups, gave the title compounds in good overall yields. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of 6-methylpurine Basic Protocol 2: Preparation of the D-allofuranose derivative (12) Basic Protocol 3: Preparation of 6-deoxy-α-L-talofuranoside Basic Protocol 4: Preparation of methyl(allo)-MePR (18) Basic Protocol 5: Preparation of methyl(talo)-MePR (21).</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"81 1","pages":"e105"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37807658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of DNA and RNA Oligonucleotides Containing a Dual-Purpose Selenium-Modified Fluorescent Nucleoside Probe.","authors":"Ashok Nuthanakanti,&nbsp;Seergazhi G Srivatsan","doi":"10.1002/cpnc.106","DOIUrl":"https://doi.org/10.1002/cpnc.106","url":null,"abstract":"Development of efficient tools that would enable direct correlation of nucleic acid structure and recognition in solution and in solid state at atomic resolution is highly desired. In this context, we recently developed dual‐purpose nucleoside probes made of a 5‐selenophene‐modified uracil core, which serves both as a conformation‐sensitive fluorophore and as an X‐ray crystallography phasing agent. In this article, we provide a detailed synthetic procedure to synthesize the phosphoramidites of 5‐selenophene‐modified 2′‐deoxyuridine and 5‐selenophene‐modified uridine analogs. We also describe their site‐specific incorporation into therapeutically relevant DNA and RNA oligonucleotide motifs by an automated solid support synthesis protocol. The dual‐purpose and minimally invasive nature of the probes enables efficient analysis of the conformation and ligand binding abilities of bacterial decoding site RNA (A‐site) and G‐quadruplex structures of the human telomeric overhang in real time by fluorescence and in 3D by X‐ray crystallography. © 2020 by John Wiley & Sons, Inc.","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"81 1","pages":"e106"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37853599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of Azobenzene Derivative Phosphoramidites for Incorporation into Oligonucleotides.","authors":"Matthew L Hammill,&nbsp;Jean-Paul Desaulniers","doi":"10.1002/cpnc.107","DOIUrl":"https://doi.org/10.1002/cpnc.107","url":null,"abstract":"<p><p>This article contains the detailed synthesis and characterization protocols of azobenzene containing siRNAs, which have photoswitchable properties effectively controlled with light. First, the azobenzene scaffolds are synthesized via reduction of nitrophenyl alcohols in the presence of zinc. Next, the hydroxyl group of azobenzene derivatives are protected with a dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate azobenzene-containing oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxymethyl)-azobenzene phosphoramidite Basic Protocol 2: Synthesis of 4,4'-bis(hydroxyethyl)-azobenzene phosphoramidite Basic Protocol 3: Synthesis, purification and characterization of oligonucleotides containing azobenzene derivatives.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"81 1","pages":"e107"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37915237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid Synthesis of Nucleoside Triphosphates and Analogues.","authors":"Alexander Ripp,&nbsp;Jyoti Singh,&nbsp;Henning J Jessen","doi":"10.1002/cpnc.108","DOIUrl":"https://doi.org/10.1002/cpnc.108","url":null,"abstract":"<p><p>Nucleoside triphosphates (NTPs) are essential biomolecules involved in almost all biological processes, and their study is therefore critical to understanding cellular biology. Here, we describe a chemical synthesis suitable for obtaining both natural and highly modified NTPs, which can, for example, be used as surrogates to probe biological processes. The approach includes the preparation of a reagent that enables the facile introduction and modification of three phosphate units: cyclic pyrophosphoryl P-amidite (c-PyPA), derived from pyrophosphate (P^V ) and a reactive phosphoramidite (P^III ). By using non-hydrolyzable analogues of pyrophosphate, the reagent can be readily modified to obtain a family of non-hydrolyzable analogues containing CH₂ , CF₂ , CCl₂ , and NH that are stable in solution for several weeks if stored appropriately. They enable the synthesis of NTPs by reaction with nucleosides to give deoxycyclotriphosphate esters that are then oxidized to cyclotriphosphate (cyclo-TP) esters. The use of different oxidizing agents provides an opportunity for modification at P-α. Furthermore, terminal modifications at P-γ can be introduced by linearization of the cyclo-TP ester with various nucleophiles. © 2020 The Authors. Basic Protocol 1: Synthesis of cyclic pyrophosphoryl P-amidite (c-PyPA) and derivatives (c-Py_NH PA, c-Py_CH2 PA, c-Py_CCl2 PA, c-Py_CF2 PA) Basic Protocol 2: Synthesis of 3'-azidothymidine 5'-γ-P-propargylamido triphosphates and analogues Basic Protocol 3: Synthesis of 2'-deoxythymidine 5'-γ-P-propargylamido triphosphate (15) Basic Protocol 4: Synthesis of adenosine 5'-γ-P-amido triphosphate (19) and adenosine 5'-γ-P-propargylamido triphosphate (20) Basic Protocol 5: Synthesis of d4T 5'-γ-propargylamido β,γ-(difluoromethylene)triphosphate Support Protocol: Synthesis of diisopropylphosphoramidous dichloride.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"81 1","pages":"e108"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.108","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37922281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pegylation of RNA Spiegelmers by a Novel Widely Applicable Two-Step Process for the Conjugation of Carboxylic Acids to Amino-Modified Oligonucleotides.","authors":"Lucas Bethge,&nbsp;Stefan Vonhoff","doi":"10.1002/cpnc.109","DOIUrl":"https://doi.org/10.1002/cpnc.109","url":null,"abstract":"<p><p>The reaction between N-hydroxy succinimide (NHS) ester-activated carboxylic acids and amino-modified nucleic acids is commonly used for the post-synthetic modification of oligonucleotides. Here, we report a two-step variation of the method in which the NHS ester is replaced by the corresponding parent carboxylic acid. In the first step, the carboxylic acid is activated with a standard peptide coupling reagent like HBTU in an anhydrous water-miscible aprotic organic solvent. In the second step, the solution of the activated carboxylic acid is added to the amino-modified oligonucleotide in water. The method is demonstrated using 40-kDa polyethylene glycol (PEG) carboxylic acid and biotin as examples. Recycling of the carboxylic acid, which is typically used in molar excess over the nucleic acid, is shown for the conjugation with 40-kDa PEG carboxylic acid. This conjugation method is generally applicable to the conjugation of carboxylic acids to amino-modified oligonucleotides, thus enabling the attachment of small to large molecular entities such as dyes, tags, peptides, and other macromolecules. © 2020 Wiley Periodicals LLC. Basic Protocol 1: General protocol for the conjugation of an amino-modified oligonucleotide with a carboxylic acid, exemplified for 40-kDa PEG carboxylic acid Basic Protocol 2: Biotinylation of an amino-modified oligonucleotide using the general conjugation protocol Basic Protocol 3: Recycling of the carboxylic acid component from the conjugation reaction, demonstrated for 40-kDa PEG carboxylic acid using ultrafiltration Support Protocol 1: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with 40-kDa PEG carboxylic acid Support Protocol 2: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with biotin Support Protocol 3: Analytical IP-RP-HPLC method used as in-process control method to monitor the conjugation reaction Alternate Protocol: Separation of 40-kDa PEG carboxylic acid from unreacted and conjugated oligonucleotide by preparative AEX-HPLC.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"81 1","pages":"e109"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.109","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37960558","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Automated Synthesis and Purification of Guanidine-Backbone Oligonucleotides.","authors":"Kacper Skakuj,&nbsp;Katherine E Bujold,&nbsp;Chad A Mirkin","doi":"10.1002/cpnc.110","DOIUrl":"https://doi.org/10.1002/cpnc.110","url":null,"abstract":"<p><p>This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMade^TM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMade^TM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMade^TM synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMade^TM synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"81 1","pages":"e110"},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.110","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38042219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Issue Information","authors":"","doi":"10.1002/cpnc.88","DOIUrl":"https://doi.org/10.1002/cpnc.88","url":null,"abstract":"","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.88","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49082213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis of 2′-Deoxyuridine Modified with a 3,5-Difluoro-4-Methoxybenzylidene Imidazolinone Derivative for Incorporation into Oligonucleotide Probes for Detection of HER2 Breast Cancer Marker","authors":"Abed Saady,&nbsp;Noam Y. Steinman,&nbsp;Melissa Wojtyniak,&nbsp;Christian Ducho,&nbsp;Bilha Fischer","doi":"10.1002/cpnc.104","DOIUrl":"10.1002/cpnc.104","url":null,"abstract":"<p>Nucleoside intercalator conjugates (NICs) describe an innovative methodology developed in our research group for preparation of fluorescence turn-on DNA hybridization probes targeting specific mRNA sequences (e.g., breast cancer markers). In this methodology, we conjugate a non-fluorescent intercalator to the base of a nucleic acid (e.g., uracil) via a flexible spacer. This modified monomer can be incorporated into oligonucleotides by solid-phase synthesis and a large fluorescence enhancement is observed when the modified oligonucleotide is hybridized with its complementary strand due to intercalation of the fluorophore between the two strands. 5-(6-<i>p</i>-Methoxybenzylidene imidazolinone-1-hexene)-2′-deoxyuridine (dU^MBI) is a synthetic monomer to which 4-methoxybenzylidene imidazolinone (MBI), the fluorescent chromophore of green fluorescent protein (GFP), has been conjugated via a flexible spacer. The detection of human epidermal growth factor receptor 2 (HER2) mRNA by this probe has already been established by our group. The fluorescent intensity of the single-strand DNA can be considered as negligible due to the free rotation of the fluorophore. Upon hybridization, however, the flexible spacer allows for the intercalation of the fluorophore between the hybridized strands, giving rise to enhanced fluorescence and indicating the presence of target mRNA. 3,5-Difluoro-4-methoxybenzylidene (DFMBI) has enhanced photophysical properties compared to MBI fluorophore. This protocol describes a simple, reliable, efficient, and general method for the synthesis of improved derivative dU^DFMBI as a monomer of fluorescent turn-on DNA hybridization probe with application for detection of HER2 mRNA. © 2020 by John Wiley &amp; Sons, Inc.</p><p><b>Basic Protocol</b>: Synthesis of 5-[(6)-3,5-difluoro-4-methoxybenzylidene imidazolinone-1-hexene]-2′-deoxyuridine</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37622071","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}