Skip to main content
SpringerLink
Log in

Synthesis of Site-Specifically Modified Long-mer RNAs

  • Chapter
  • First Online:
Chemical Biology of Nucleic Acids

Part of the book series: RNA Technologies ((RNATECHN))

Abstract

Site-specific functionalization of RNA and DNA molecules has become a major task in nucleic acid chemistry. Synthetic RNAs are required for a large number of applications. Beyond synthetic RNA for antisense, aptamer, ribozyme, and siRNA technologies, oligoribonucleotides carrying site-specific modifications for structure and function studies are needed. A wide variety of monomer-building blocks is commercially available to be used for site-specific incorporation by solid-phase RNA synthesis. However, the efficient chemical preparation of RNA is limited to oligomers of about 60–70 nucleotides. Therefore, efficient strategies for fragment ligation or other alternative novel protocols are required. We here provide an overview on our work on the synthesis of site-specifically modified long-mer RNAs, focusing on enzymatic and chemical ligation of synthetic RNA fragments and on ribozyme-mediated sequence exchanges.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Aalto AP, Pasquinelli AE (2012) Small non-coding RNAs mount a silent revolution in gene expression. Curr Opin Cell Biol 24:333–340

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Aravin A, Tusch T (2005) Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett 579:5830–5840

    Article  CAS  PubMed  Google Scholar 

  • Bain JD, Switzer C (1992) Regioselective ligation of oligoribonucleotides using DNA splints. Nucleic Acids Res 20:4372

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bullard DR, Bowater RP (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem J 398:135–144

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Burrows CJ, Muller JG (1998) Oxidative nucleobase modifications leading to strand scission. Chem Rev 98:1109–1151

    Article  CAS  PubMed  Google Scholar 

  • Chan TR, Hilgraf R, Sharpless KB et al (2004) Polytriazoles as copper(I)-stabilizing ligands in catalysis. Org Lett 6:2853–2855

    Article  CAS  PubMed  Google Scholar 

  • Davies MJ, Shah A, Bruce IJ (2000) Synthesis of fluorescently labeled oligonucleotides and nucleic acids. Chem Soc Rev 29:97–107

    Article  CAS  Google Scholar 

  • Dolinnaya NG, Sokolova NI, Grayaznova OI et al (1988) Site-directed modification of DNA duplexes by chemical ligation. Nucleic Acids Res 16:3721–3738

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Drude I, Vauléon S, Müller S (2007) Twin ribozyme mediated removal of nucleotides from an internal RNA site. Biochem Biophys Res Commun 363:24–29

    Article  CAS  PubMed  Google Scholar 

  • Drude I, Strahl A, Galla D et al (2011) Design of hairpin ribozyme variants with improved activity for poorly processed substrates. FEBS J 278:622–633

    Article  CAS  PubMed  Google Scholar 

  • Edwards JB, Delort J, Mallet J (1991) Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5′-ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic Acids Res 19:5227–5232

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Eisenführ A, Arora PS, Sengle G et al (2003) A ribozyme with michaelase activity: synthesis of the substrate precursors. Bioorg Med Chem 11:235–249

    Article  PubMed  Google Scholar 

  • El-Sagheer AH, Brown T (2009) Synthesis and polymerase chain reaction amplification of DNA strands containing an unnatural triazol linkage. J Am Chem Soc 131:3958–3964

    Article  CAS  PubMed  Google Scholar 

  • El-Sagheer AH, Brown T (2010) New strategy for the synthesis of chemically modified RNA constructs exemplified by hairpin and hammerhead ribozymes. Proc Natl Acad Sci USA 35:15329–15334

    Article  Google Scholar 

  • England TE, Uhlenbeck OC (1978a) Enzymatic oligoribonucleotide synthesis with T4 RNA ligase. Biochemistry 17:2069–2076

    Article  CAS  PubMed  Google Scholar 

  • England TE, Uhlenbeck OC (1978b) 3′-terminal labelling of RNA with T4 RNA ligase. Nature 275:560–561

    Article  CAS  PubMed  Google Scholar 

  • Gasparutto D, Livache T, Bazin H et al (1992) Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucleic Acids Res 20:5159–5166

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hein JE, Fokin VV (2010) Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and beyond: new reactivity of copper(I) acetylides. Chem Soc Rev 39:1302–1315

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hermanson GT (2008) Nucleic acid and oligonucleotide modification and conjugation. In: Hermanson GT (ed) Bioconjugate techniques, 2nd edn. Academic, San Diego, pp 969–1002

    Chapter  Google Scholar 

  • Herrlein MK, Nelson JS, Letsinger RL (1995) A covalent lock for self-assembled oligonucleotide conjugates. J Am Chem Soc 117:10151–10152

    Article  CAS  Google Scholar 

  • Ho CK, Shuman S (2002) Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains. Proc Natl Acad Sci USA 99:12709–12714

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Igloi GL (1996) Nonradioactive labeling of RNA. Anal Biochem 233:124–129

    Article  CAS  PubMed  Google Scholar 

  • Isobe H, Fujino T, Yamazaki N et al (2008) Triazole-linked analogue of deoxyribonucleic acid (TLDNA): design, synthesis, and double-strand formation with natural DNA. Org Lett 17:3729–3732

    Article  Google Scholar 

  • Ivanov SA, Vauléon S, Müller S (2005) Efficient RNA ligation by reverse-joined hairpin ribozymes and engineering of twin ribozymes consisting of conventional and reverse-joined hairpin ribozyme units. FEBS J 272:4464–4474

    Article  CAS  PubMed  Google Scholar 

  • Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed Engl 40:2004–2021

    Article  CAS  PubMed  Google Scholar 

  • Kool ET, Xu Y (1997) A novel 5′-iodonucleoside allows efficient nonenzymatic of single-stranded and duplex DNAs. Tetrahedron Lett 38:5595–5598

    Article  PubMed Central  PubMed  Google Scholar 

  • Kumar R, El-Sagheer AH, Tumpane J et al (2007) Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry. J Am Chem Soc 129:6859–6864

    Article  CAS  PubMed  Google Scholar 

  • Kurschat WC, Müller J, Wombacher R et al (2005) Optimizing splinted ligation of highly structured small RNAs. RNA 11:1909–1914

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lai Y, DeStefano JJ (2011) A primer-free method that selects high-affinity single-stranded DNA aptamers using thermostable RNA ligase. Anal Biochem 414:246–253

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lang K, Micura R (2008) Preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments. Nat Protoc 8:1457–1466

    Article  Google Scholar 

  • Malaprade L (1928) Action of polyalcohols on periodic acid. Analytical application. Bull Soc Chim (France) 43:683–696

    CAS  Google Scholar 

  • Marchán V, Ortega S, Pulido D et al (2006) Diels-Alder cycloaddition in water for the straightforward preparation of peptide-oligonucleotide conjugates. Nucleic Acids Res 34:e24/1–e24/9

    Article  Google Scholar 

  • Marinetti G, Rouser G (1955) The periodate oxidation of ribose-5-phosphate in acid and alkaline solution. J Am Chem Soc 77:5345–5349

    Article  CAS  Google Scholar 

  • Martin G, Keller W (1998) Tailing and 3′-end labeling of RNA with yeast poly(A) polymerase and various nucleotides. RNA 4:226–230

    CAS  PubMed Central  PubMed  Google Scholar 

  • Middleton T, Herlihy WC, Schimmel PR et al (1985) Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography. Anal Biochem 144:110–117

    Article  CAS  PubMed  Google Scholar 

  • Miller ES, Kutter E, Mosig G et al (2003) Bacteriophage T4 genome. Microbiol Mol Biol Rev 67:86–156

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 polymerase. Methods Enzymol 180:51–63

    Article  CAS  PubMed  Google Scholar 

  • Moran VA, Perera RJ, Khalil AM (2012) Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs. Nucleic Acids Res 40:6391–6400

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Müller S, Wolf J, Ivanov SA (2004) Current strategies for the synthesis of RNA. Curr Org Synth 1:293–307

    Article  Google Scholar 

  • Nandakuman J, Shuman S (2004) How an RNA ligase discriminates RNA versus DNA damage. Mol Cell 16:211–221

    Article  Google Scholar 

  • Nuzzi A, Massi A, Dondoni A (2007) Model studies toward the synthesis of thymidine oligonucleotides with triazole internucleosidic linkages via iterative Cu(I)-promoted azide-alkyne ligation chemistry. QSAR Comb Sci 26:1191–1199

    Article  CAS  Google Scholar 

  • Oh BK, Pace NR (1994) Interaction of the 3′-end of tRNA with ribonuclease P RNA. Nucleic Acids Res 22:4087–4094

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ohtsuki T, Kawai G, Watanabe K (1998) Stable isotope-edited NMR analysis of Ascaris suum mitochondrial tRNAMet having a TV-replacement loop. J Biochem 124:28–34

    Article  CAS  PubMed  Google Scholar 

  • Paredes E, Das SR (2011) Click chemistry for rapid labeling and ligation of RNA. Chembiochem 12:125–131

    Article  CAS  PubMed  Google Scholar 

  • Paredes E, Evans M, Das SR (2011) RNA labeling, conjugation and ligation. Methods 54:251–259

    Article  CAS  PubMed  Google Scholar 

  • Persson T, Cuzic S, Hartmann RK (2003) Catalysis by RNase P RNA: unique features and unprecedented active site plasticity. J Biol Chem 278:43394–43401

    Article  CAS  PubMed  Google Scholar 

  • Pfeffer S, Sewer A, Lagos-Quintana M et al (2005) Identification of microRNAs of the herpesvirus family. Nat Methods 2:269–276

    Article  CAS  PubMed  Google Scholar 

  • Pheiffer BH, Zimmerman SB (1983) Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribo-oligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res 11:7853–7871

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Piekielska K, Gębala M, Gwiazda S et al (2011) Impedimetric detection of hairpin ribozyme activity. Electroanalysis 23:37–42

    Article  CAS  Google Scholar 

  • Rader SD, Stark MR, Pleiss JA et al (2006) An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs. RNA 12:2014–2019

    Article  PubMed Central  PubMed  Google Scholar 

  • Remaut E, Tsao H, Fiers W (1983) Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene 22:103–113

    Article  CAS  PubMed  Google Scholar 

  • Rigden JE, Rezaian MA (1992) In vitro synthesis of an infectious viroid: analysis of the infectivity of monomeric linear CEV. Virology 186:201–206

    Article  CAS  PubMed  Google Scholar 

  • Romaniuk E, McLaughlin LW, Neilson T et al (1982) The effect of acceptor oligoribonucleotide sequence on the T4 RNA ligase reaction. Eur J Biochem 125:639–643

    Article  CAS  PubMed  Google Scholar 

  • Rosemeyer V, Laubrock A, Seibl R (1995) Nonradioactive 3′-end-labeling of RNA molecules of different lengths by terminal deoxynucleotidyltransferase. Anal Biochem 224:446–449

    Article  CAS  PubMed  Google Scholar 

  • Rossi R, Montecucco A, Ciarrocchi G et al (1997) Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action. Nucleic Acids Res 25:2106–2113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rostovtsev VV, Green LG, Fokin VV et al (2002) A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective ligation of azides and terminal alkynes. Angew Chem Int Ed Engl 41:2596–2299

    Article  CAS  PubMed  Google Scholar 

  • Rublack N, Nguyen H, Appel B et al (2011) Synthesis of specifically modified oligonucleotides for application in structural and functional analysis of RNA. J Nucleic Acids 2011:1–19

    Article  Google Scholar 

  • Sampson JR, Uhlenbeck OC (1988) Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci USA 85:1033–1037

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sanchez A, Pedroso E, Grandas A (2013) Oligonucleotide cyclization: the thiol-maleimide reaction revisited. Chem Commun 49:309–311

    Article  CAS  Google Scholar 

  • Seela F, Sirivolu VR (2008) Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne “click”reaction. Org Biomol Chem 6:1674–1687

    Article  CAS  PubMed  Google Scholar 

  • Seelig B, Jäschke A (1999) Ternary conjugates of guanosine monophosphate as Initiator nucleotides for the enzymatic synthesis of 5′-modified RNAs. Bioconjug Chem 10:371–378

    Article  CAS  PubMed  Google Scholar 

  • Serganov A, Patel DJ (2012) Molecular recognition and function of riboswitches. Curr Opin Struct Biol 22:279–286

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sindbert S, Kalinin S, Nguyen H et al (2011) Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity. J Am Chem Soc 133:2463–2480

    Article  CAS  PubMed  Google Scholar 

  • Sokolova NI, Ashirbekova DT, Dolinnaya NG et al (1988) Chemical reactions within DNA duplexes: cyanogen bromide as an effective oligodeoxyribonucleotide coupling agent. FEBS Lett 232:153–155

    Article  CAS  PubMed  Google Scholar 

  • Sontheimer EJ (1994) Site-specific RNA crosslinking with 4-thiouridine. Mol Biol Rep 20:35–44

    Article  CAS  PubMed  Google Scholar 

  • Tessier DC, Brousseau R, Vernet T (1986) Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA Ligase. Anal Biochem 158:171–178

    Article  CAS  PubMed  Google Scholar 

  • Tornøe CW, Christensen C, Meldal M (2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem 67:3057–3064

    Article  PubMed  Google Scholar 

  • Turunen JJ, Pavlova LV, Hengesbach M et al (2013) RNA ligation. In: Hartmann R, Bindereif A, Schön A, Westhof E (eds) Handbook of RNA biochemistry, 2nd edn. Wiley, Weinheim

    Google Scholar 

  • Vauléon S, Ivanov SA, Gwiazda S et al (2005) Site-specific fluorescent and affinity labelling of RNA by using a small engineered twin ribozyme. ChemBioChem 6:2158–2162

    Article  PubMed  Google Scholar 

  • Welz R, Müller S (2002) 5-(Benzylmercapto)-1H-tetrazole as activator for 2′-O-TBDMS phosphoramidite building blocks in RNA synthesis. Tetrahedron Lett 43:795–797

    Article  CAS  Google Scholar 

  • Welz R, Bossmann K, Klug C et al (2003) Site-directed alteration of RNA sequence mediated by an engineered twin ribozyme. Angew Chem Int Ed Engl 42:2424–2427

    Article  CAS  PubMed  Google Scholar 

  • Willkomm DK, Hartmann RK (2009) 3′-terminal attachment of fluorescent dyes and biotin. In: Hartmann RK, Bindereif A, Schön A, Westhof E (eds) Handbook of RNA biochemistry. Wiley-VCH, Weinheim, pp 86–94

    Google Scholar 

  • Wincott F, DiRenzo A, Shaffer C et al (1995) Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res 23:2677–2684

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Winkler WC, Cohen-Chalamish S, Breaker RR (2002) An mRNA structure that controls gene expression by binding FMN. Proc Natl Acad Sci USA 99:15908–15913

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Winston SE, Fuller SA, Evelegh MJ et al (2001) Conjugation of enzymes to antibodies. Curr Protoc Mol Biol 11(11.1)

    Google Scholar 

  • Winz ML, Samanta A, Benzinger D et al (2012) Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry. Nucleic Acids Res 40:e78

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wolf J, Dombos V, Appel B et al (2008) Synthesis of guanosine 5′-conjugates and their use as initiator molecules for transcription priming. Org Biomol Chem 6:899–907

    Article  CAS  PubMed  Google Scholar 

  • Wunnicke D, Strohbach D, Weigand JE et al (2011) Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR. RNA 17:182–188

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yu YT (1999) Construction of 4-thiouridine site-specifically substituted RNAs for cross-linking studies. Methods 18:13–21

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Biochemie, Ernst-Moritz-Arndt Universität Greifswald, Felix-Hausdorff Str. 4, 17487, Greifswald, Germany

    Darko Balke, Jennifer Frommer, Nico Rublack, Danilo Springstubbe, Bettina Appel & Sabine Müller

Authors
  1. Darko Balke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennifer Frommer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nico Rublack
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Danilo Springstubbe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bettina Appel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sabine Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sabine Müller .

Editor information

Editors and Affiliations

  1. Institute of Chemistry/Biochemistry, Free University Berlin, Berlin, Germany

    Volker A. Erdmann

  2. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

    Wojciech T. Markiewicz

  3. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

    Jan Barciszewski

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Balke, D., Frommer, J., Rublack, N., Springstubbe, D., Appel, B., Müller, S. (2014). Synthesis of Site-Specifically Modified Long-mer RNAs. In: Erdmann, V., Markiewicz, W., Barciszewski, J. (eds) Chemical Biology of Nucleic Acids. RNA Technologies. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-54452-1_26

Download citation

Publish with us

Policies and ethics

Search

Navigation