Solid‐Phase Synthesis of Branched Oligonucleotides

Sandra Carriero1, Masad J. Damha1

1 McGill University, Montreal, Canada
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.14
DOI:  10.1002/0471142700.nc0414s09
Online Posting Date:  August, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Synthesis and Characterization of the Adenosine Branching Synthon N6‐Benzoyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐ Adenosine‐2′,3′‐Bis‐O‐(2‐Cyanoethyl‐N,N‐Diisopropyl) Phosphoramidite
  • Basic Protocol 2: Convergent Synthesis of Symmetrical Branched Nucleic Acids
  • Basic Protocol 3: Regiospecific Synthesis of Branched Nucleic Acids
  • Support Protocol 1: Preparation of LCAA‐CPG Supports with High Nucleoside Loadings
  • Support Protocol 2: Complete Deprotection of Branched Oligonucleotides (DNA and RNA)
  • Support Protocol 3: Analysis and Purification of Branched Oligonucleotides Using Anion‐Exchange HPLC
  • Support Protocol 4: Analysis and Purification of Branched Oligonucleotides by Denaturing Page
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Synthesis and Characterization of the Adenosine Branching Synthon N6‐Benzoyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐ Adenosine‐2′,3′‐Bis‐O‐(2‐Cyanoethyl‐N,N‐Diisopropyl) Phosphoramidite

  Materials
  • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)adenosine (S.1; ChemGenes)
  • 4‐Dimethylaminopyridine (DMAP; 99%; Aldrich)
  • Nitrogen or argon gas, dry
  • Anhydrous THF (see recipe) in a septum‐sealed distillation collection bulb
  • Anhydrous DIPEA (see recipe)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (S.2; ChemGenes)
  • 1:1 (v/v) dichloromethane/diethyl ether
  • 20% (v/v) sulfuric acid (optional)
  • Ethyl acetate prewashed with 5% (w/v) NaHCO 3
  • NaCl solution, saturated
  • Sodium sulfate (Na 2SO 4), anhydrous
  • 50:47:3 (v/v/v) CH 2Cl 2/hexanes/triethylamine
  • Silica gel (230‐ to 400‐mesh) in 50:47:3 CH 2Cl 2/hexanes/triethylamine
  • 95% (v/v) ethanol
  • Diethyl ether
  • 50‐mL oven‐ or flame‐dried round‐bottom flask with rubber septum
  • Glass syringe and needle, oven dried
  • 2 × 5 cm silica‐coated thin‐layer chromatography (TLC) plate with fluorescent indicator (e.g., Kieselgel 60 F 254 aluminum sheets)
  • 254‐nm UV light source
  • 500‐mL separatory funnel
  • Gravity filtration device and filter paper
  • 250‐ and 500‐mL round‐bottom flasks
  • Rotary evaporator with a water aspirator
  • 5 × 25–cm glass chromatography column with solvent reservoir bulb
  • Additional reagents and materials for thin‐layer chromatography (TLC; appendix 3D), column chromatography ( appendix 3E), 31P‐NMR (unit 7.2), and mass spectrometry (units 10.1 & 10.2)

Basic Protocol 2: Convergent Synthesis of Symmetrical Branched Nucleic Acids

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N‐protected‐2′‐deoxyribonucleoside‐ or ‐ribonucleoside‐derivatized succinyl‐LCAA‐CPG (ChemGenes; also see protocol 4)
  • Cap A and B capping reagents (see reciperecipes)
  • DNA and/or RNA 3′‐phosphoramidites (S.4ad and S.5a‐d; Fig. )
  • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)adenosine‐2′,3′‐bis‐O‐(2‐cyanoethyl‐N,N‐ diisopropyl)phosphoramidite (BIS‐A; S.3; see protocol 1)
  • Anhydrous acetonitrile (see recipe)
  • Activator solution: 0.5 M 1H‐tetrazole (sublimed) in anhydrous acetonitrile
  • Oxidant solution (see recipe)
  • Detritylation solution (see recipe)
  • Nitrogen or argon gas (optional)
  • Synthesis columns for 1‐µmol scale synthesis, with seals and filters (PE Biosystems) and 13‐mm aluminum seals (Chromatographic Specialties)
  • ABI 381A automated DNA synthesizer (PE Biosystems)
  • Synthesizer bottles for phosphoramidites, oven dried
  • Fraction collector and 15‐mL test tubes
  • 50‐mL buret
  • Quartz cuvettes
  • UV‐Vis spectrophotometer
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C), cleaving and deprotecting oligonucleotides (see protocol 5), and anion‐exchange HPLC (see protocol 6) or denaturing PAGE (see protocol 7)
NOTE: 1H‐Tetrazole is no longer commercially available in crystalline form. Solutions of 0.45 M 1H‐tetrazole in anhydrous acetonitrile may be purchased from ChemGenes. For a list of supplementary phosphoramidite activating reagents, see unit 3.5.CAUTION: All solutions for the DNA/RNA synthesizer should be prepared in a well‐ventilated fume hood.

Basic Protocol 3: Regiospecific Synthesis of Branched Nucleic Acids

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N‐protected‐2′‐deoxyribonucleoside‐derivatized succinyl‐LCAA‐CPG (ChemGenes; also see protocol 4)
  • Cap A and B capping reagents (see reciperecipes)
  • DNA 3′‐phosphoramidites (S.4ad; Chem Genes; Fig. )
  • Anhydrous acetonitrile (see recipe)
  • RNA 3′‐phosphoramidite (S.5a‐d; ChemGenes; Fig. )
  • Activator solution: 0.5 M 1H‐tetrazole (sublimed) in anhydrous acetonitrile
  • Oxidant solution (see recipe)
  • Detritylation solution (see recipe)
  • 4:6 (v/v) triethylamine/acetonitrile (see recipe)
  • Anhydrous THF (see recipe)
  • 1 M tetra‐n‐butylammonium fluoride (TBAF; Aldrich) in THF, fresh
  • Inverted DNA 5′‐phosphoramidites (S.6a‐d; ChemGenes; Fig. )
  • Argon or nitrogen gas (optional)
  • Synthesis columns for 1 µmol scale synthesis, with seals and filters (PE Biosystems) and 13‐mm aluminum seals (Chromatographic Specialties)
  • ABI 381A automated DNA synthesizer (PE Biosystems)
  • External fraction collector and 15‐mL test tubes
  • Empty DNA synthesizer bottles, oven‐dried
  • 10‐ and 1‐mL disposable syringes
  • 25‐mL glass syringe
  • Additional reagents and equipment for cleaving and deprotecting the oligonucleotide (see protocol 5), anion‐exchange HPLC (see protocol 6) or denaturing PAGE (see protocol 7), and measuring coupling efficiency by trityl color analysis (see protocol 5)
CAUTION: All solutions required for bNA solid‐phase synthesis should be prepared in a well‐ventilated fume hood.

Support Protocol 1: Preparation of LCAA‐CPG Supports with High Nucleoside Loadings

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N‐protected‐ribonucleoside or ‐2′‐deoxyribonucleoside (ChemGenes)
  • O‐(7‐Azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HATU; PE Biosystems)
  • 4‐Dimethylaminopyridine (DMAP; 99%, Aldrich)
  • Succinylated long‐chain‐aminoalkyl controlled‐pore glass (succinyl‐LCAA‐CPG; unit 3.2)
  • Anhydrous acetonitrile (see recipe)
  • Dichloromethane, reagent grade (Fisher)
  • Methanol, reagent grade (Fisher)
  • Cap A and B capping reagents (see reciperecipes)
  • 15‐mL glass bottle with septum
  • 1‐mL syringe and needle
  • Wrist‐action shaker
  • Sintered glass funnel or Buchner funnel with filter paper
  • Additional reagents and equipment for quantitation of released trityl groups (unit 3.2) and acetylation of support (unit 3.2; optional)

Support Protocol 2: Complete Deprotection of Branched Oligonucleotides (DNA and RNA)

  Materials
  • Branched oligonucleotide attached to CPG (bNA‐CPG; see protocol 2Basic Protocols 2 and protocol 33)
  • 29% ammonium hydroxide, 4°C (store up to 1 month at 4°C)
  • 70% and 100% (v/v) ethanol, former at 4°C
  • DEPC‐treated water (optional; appendix 2A)
  • Autoclaved water
  • Triethylammonium trihydrofluoride (TREAT‐HF; 98%; Aldrich)
  • 3 M sodium acetate, pH 5.5 ( appendix 2A)
  • 1‐Butanol, analytical grade, 4°C
  • 1.5‐mL screw‐cap microcentrifuge tubes with O‐ring seals (preferred)
  • Wrist‐action shaker
  • Speedvac evaporator (Savant)
  • Double‐beam UV spectrophotometer, calibrated

Support Protocol 3: Analysis and Purification of Branched Oligonucleotides Using Anion‐Exchange HPLC

  Materials
  • Deprotected branched oligonucleotide (see protocol 5)
  • Autoclaved water
  • 1 M LiClO 4 (see recipe)
  • Reagent‐grade 1‐propanol, 4°C (>5‐mers; Fisher)
  • Sephadex G‐25 columns (Amersham Pharmacia Biotech) or Sep‐Pak cartridges (Waters Chromatography) for <5‐mers
  • Nuclease P1 buffer (see recipe)
  • 0.3 U/µL Penicillium citrinum nuclease P1 (NP1)
  • 9 U/µL alkaline phosphatase (AP)
  • 0.1 M triethylammonium acetate (TEAA), pH 7.0
  • Acetonitrile, HPLC grade
  • 50°C water bath or heating block
  • High‐performance liquid chromatograph (HPLC) with:
  •  Anion‐exchange column (7.5 × 75–mm Waters Protein‐Pak DEAE‐5PW)
  •  UV detector with adjustable range or dual‐wavelength detection
  •  Sample loop
  •  Column heater
  •  Reversed‐phase C18 column (e.g., Whatman Partisil ODS‐2, 10‐µm, 4.6 × 250–mm; Chromatographic Specialties)
  • Syringe
  • Speedvac evaporator (Savant)
  • Additional reagents and equipment for reversed‐phase chromatography (units 10.1 & 10.6) and quantitation of bNAs by UV spectrophotometry (see 4.14)

Support Protocol 4: Analysis and Purification of Branched Oligonucleotides by Denaturing Page

  Materials
  • 20% (w/v) denaturing acrylamide gel solution (see recipe)
  • Deprotected branched oligonucleotide sample, dry (see protocol 5)
  • Formamide loading buffer (see recipe)
  • Gel extraction buffer (see recipe)
  • Running dye (see recipe)
  • Gel electrophoresis equipment ( appendix 3B) with:
  •  16 × 18–cm glass plates
  •  Spacers: 0.75 mm (analysis) or 1.5 mm (purification)
  •  Gel combs: 0.75 mm thick with 12 to 20 wells (analysis) or 1.5 mm thick with one to three large wells (purification)
  • Sonicator or ∼50°C water bath (optional)
  • 20 × 20–cm silica‐coated thin‐layer chromatography (TLC) plate with fluorescent indicator (e.g., Kieselgel 60 F 254 aluminum sheets)
  • Handheld UV lamp (254 nm)
  • Camera equipped with UV filter (optional)
  • 90°C water bath or heating block (optional)
  • UV shadow box
  • Wrist‐action shaker (optional)
  • Speedvac evaporator (Savant)
  • Sephadex G‐25 (Amersham Pharmacia Biotech) or reversed‐phase cartridges (e.g., Sep‐Pak cartridges; Waters Chromatography)
  • Additional reagents and equipment for denaturing PAGE (unit 10.4 and appendix 3B), reversed‐phase chromatography using Sep‐Pak cartridges (unit 10.1), UV spectrophotometry (see protocol 5), MALDI‐TOF‐MS (unit 10.1), and enzymatic digestion (see protocol 6)
CAUTION: Acrylamide and N,N′‐methylenebisacrylamide are neurotoxins. Prepare all solutions containing these two reagents in a fume hood. Minimize exposure and contact to both crystalline forms and solutions by conducting all handling (including weighing) in a well‐ventilated area and wearing disposable gloves at all times.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Braich, R.S. and Damha, M.J. 1997. Regiospecific solid‐phase synthesis of branched oligonucleotides. Effect of vicinal 2′,5′‐ (or 2′,3′‐) and 3′,5′‐phosphosdiester linkages on the formation of hairpin DNA. Bioconjugate Chem. 8:370‐377.
   Carriero, S., Braich, R.S., Hudson, R.H.E., Anglin, D., Friesen, J.D., and Damha, M.J. 2001. Inhibition of in vitro pre‐mRNA splicing in S. cerevisiae by branched oligonucleotides. Nucleosides, Nucleotides, and Nucl. Acids. 20:873‐877.
   Chapman, K.B. and Boeke, J.D. 1991. Isolation and characterization of the gene encoding yeast debranching enzyme. Cell 65:483‐492.
   Chen, Z. and Ruffner, D.E. 1996. Modified crush‐and‐soak method for recovering oligodeoxynucleotides from polyacrylamide gel. BioTechniques 21:820‐822.
   Damha, M.J., and Braich, R.S. 1998. Synthesis of branched DNA/RNA chimera similar to the msDNA molecule of Myxoccus xanthus. Tetrahedron lett. 39:3907‐3910.
   Damha, M.J. and Ogilvie, K.K. 1988. Synthesis and spectroscopic analysis of branched RNA fragments: Messenger RNA splicing intermediates. J. Org. Chem. 53:3710‐3722.
   Damha, M.J. and Zabarylo, S.V. 1989. Automated solid‐phase synthesis of branched oligonucleotides. Tetrahedron Lett . 30:6295‐6298.
   Damha, M.J., Pon, R.T., and Ogilvie, K.K. 1985. Chemical synthesis of branched RNA: Novel trinucleoside diphosphates containing vicinal 2′‐5′ and 3′‐5′ phosphodiester linkages. Tetrahedron Lett. 26:4839‐4842.
   Damha, M.J., Giannaris, P.A., and Zabarylo, S.V. 1990. An improved procedure for derivatization of controlled‐pore glass beads for solid‐phase oligonucleotide synthesis. Nucl. Acids Res. 18:3813‐3821.
   Damha, M.J., Ganeshan, K., Hudson, R.H.E., and Zabarylo, S.V. 1992. Solid‐phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates. Nucl. Acids Res . 20:6565‐6573.
   Eadie, J.S., McBride, L.J., Efcavitch, J.W., Hoff, L.B., and Cathcart, R. 1987. High‐performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition. Anal. Biochem. 165:442‐447.
   Ganeshan, K., Tadey, T., Nam, K., Braich, R., Purdy, W.C., Boeke, J.D., and Damha, M.J. 1995. Novel approaches to the synthesis and analysis of branched RNA. Nucleosides & Nucleotides 14:1009‐1013.
   Gasparutto, D., Livache, T., Bazin, H., Duplaa, A.M., Guy, A., Khorlin, A., Molko, D., Roget, A., and Teoule, R. 1992. Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucl. Acids Res . 20:5159‐5166.
   Hakimelahi, G.H., Proba, Z.A., and Ogilvie, K.K. 1982. New catalysts and procedures for the dimethoxytritylation and selective silylation of ribonucleosides. Can. J. Chem. 60:1106‐1113.
   Hudson, R.H.E. and Damha, M.J. 1993. Nucleic acid dendrimers: Novel biopolymer structures. J. Am. Chem. Soc. 115:2119‐2124.
   Hudson, R.H.E., Uddin, A.H., and Damha, M.J. 1995. Association of branched nucleic acids: Structural and physicochemical analysis of antiparallel TAT triple‐helical DNA. J. Am. Chem. Soc. 117:12470‐12477.
   Hudson, R.H.E., Robidoux, S., and Damha, M.J. 1998. Divergent synthesis of nucleic acid dendrimers. Tetrahedron Lett. 32:1299‐1302.
   Inouye, S., Furuichi, T., Dhundle, A., and Inouye, M. 1987. Molecular Biology of RNA: New Perspectives (M. Inouye and B.S. Dudock, eds.) pp. 271. Academic Press, San Diego.
   Kierzek, R., Kopp, D.W., Edmonds, M., and Caruthers, M.H. 1986. Chemical synthesis of branched RNA. Nucl. Acids Res. 14:4751‐4764.
   Lecchi, P., Le, H.M.T., and Pannell, L.K. 1995. 6‐Aza‐2‐thiothymine: A matrix for MALDI spectra of oligonucleotides. Nucl. Acids Res. 23:1276‐1277.
   Lyttle, M.H., Adams, H., Hudson, D., and Cook, R.M. 1997. Versatile linker chemistry for synthesis of 3′‐modified DNA. Bioconjugate Chem. 8:193‐198.
   Nam, K., Hudson, R.H.E., Chapman, K.B., Ganeshan, K., Damha, M.J., and Boeke, J.D. 1994. Yeast lariat debranching enzyme: Substrate and sequence specificity. J. Biol. Chem. 269:20613‐20621.
   Ooi, S.L., Dann, C. III, Nam, K., Leahy, D., Damha, M.J., and Boeke, J.D. 2001. Ribonucleases part A: Functional roles and mechanisms. Methods Enzymol. 342:233‐250.
   Pon, R.T., Yu, S., and Sanghvi, Y.S. 1999. Rapid esterification of nucleosides to solid‐phase supports for oligonucleotide synthesis using uronium and phosphonium coupling reagents. Bioconjugate Chem. 10:1051‐1057.
   Robidoux, S., Klinck, R., Gehring, K., and Damha, M.J. 1997. Association of branched oligonucleotides into the i‐motif. J. Biomol. Struct. Dyn. 15:517‐527.
   Rousse, B., Puri, N., Viswanadham, G., Agback, P., Glemarec, C., Sandstroem, A., Sund, C., and Chattopadhyaya, J. 1994. Solution conformation of hexameric and heptameric lariat‐RNAs and their self‐cleavage reactions which give products mimicking those from some catalytic RNAs (ribozymes). Tetrahedron 50:1777‐1810.
   Ruskin, B. and Green, M. 1985. An RNA processing activity that debranches RNA lariats. Science 229:135‐140.
   Ruskin, B., Krainer, A.R., Maniatis, T., and Green, M.R. 1984. Excision of an intact intron as a novel lariat structure during pre‐mRNA spicing in vitro. Cell 38:317‐331.
   Sproat, B.S., Beijer, B., Grotli, M., Ryder, U., Morand, K.L., and Lamond, A.I. 1994. Novel solid‐phase synthesis of branched oligoribonucleotides including a substrate for the RNA debranching enzyme. J. Am. Chem. Soc. Perkin. Trans. 1:419‐431.
   Sproat, B., Colonna, F., Mullah, B., Tsou, D., Andrus, A., Hampel, A., and Vinayak, R. 1995. An efficient method for the isolation and purification of oligoribonucleotides. Nucleosides & Nucleotides 14:255‐273.
   Still, W.C., Kahn, M., and Mitra, A. 1978. Rapid chromatographic technique for preparative separations with moderate resolution. J. Org. Chem. 43:2923‐2925.
   Ti, G.S., Gaffney, B.L., and Jones, R.A. 1982. Transient protection: Efficient one‐flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319.
   Uddin, A.H., Piunno, P.A.E., Hudson, R.H.E., Damha, M.J., and Krull, U.J. 1997. A fiber optic biosensor for fluorimetric detection of triple‐helical DNA. Nucl. Acids Res. 25:4139‐4146.
   Urdea, M.S., Horn, T., Fultz, T.J., Anderson, M., Running, J.A., Hamren, S., Ahle, D., and Chang, C.A. 1991. Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses. Nucl. Acids Symp. Series. 24:197‐200.
   Wallace, J.C. and Edmonds, M. 1983. Polyadenylated nuclear RNA contains branches. Proc. Natl. Acad. Sci. U.S.A. 80:950‐954.
   Wincott, F., Di Renzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. 1995. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucl. Acids. Res. 23:2677‐2684.
   Wu, T., Ogilvie, K.K., and Pon, R.T. 1989. Prevention of chain cleavage in the chemical synthesis of 2′‐silylated oligoribonucleotides. Nucl. Acids Res. 17:3501‐3517.
   Yee, T., Furuichi, T., Inouye, S., and Inouye, M. 1984. Multicopy single‐stranded DNA isolated from a Gram‐negative bacterium, Myxococcus xanthus. Cell 38:203‐209.
Key References
   Damha and Zabarylo, 1989. See above.
  Reports on the first general procedure for the convergent solid‐phase synthesis of branched oligonucleotides via an adenosine bisphosphoramidite.
   Damha et al., 1992. See above.
  Reports on the convergent synthesis of branched RNA oligonucleotides using the standard silyl‐phosphoramidite RNA synthesis methodology. The bNAs synthesized are related to the splicing intermediates derived from S. cerevisiae.
   Nam et al., 1994. See above.
  Reports on the substrate and sequence specificity of the yeast lariat debranching enzyme (yDBR), a unique 2′,5′‐phosphodiesterase. The enzyme accepts a variety of substrates including group II intron lariats, msDNA, and synthetic bNAs.
   Padgett, R.A., Konarska, M.M., Grabowski, P.J., Hardy, S.F., and Sharp, P.A. 1984. Lariat RNA's as intermediates and products in the splicing of messenger precursors. Science 225:898‐903.
  This report provides evidence that the branched lariat structure is an intermediate of splicing of an adenovirus ML2 RNA transcript. Specifically demonstrated is that the excised intron contains an unusual nuclease‐resistant core consisting of a branched trinucleotide structure with vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages.
   Sharp, P.A. 1994. Split genes and RNA splicing. Cell 77:805‐815.
  A Nobel lecture. A paramount review describing the splicing of introns from nascent RNA, the evolutionary significance of introns, and the plethora of factors involved in post‐transcriptional processing.
   Wallace and Edmonds, 1983. See above.
  A first account demonstrating the occurrence of a branched nuclear polyadenylated RNA containing vicinal 2′,5′‐ and 3′,5′‐phosphodiester bonds. Such molecules were absent from cytoplasmic polyadenylated RNA, implicating these structures as intermediates during mRNA processing.
Internet Resources
   http://paris.chem.yale.edu/extinct.html
  A useful site for the calculation of molecular weights of oligonucleotides and peptides as well as the determination of extinction coefficients (ɛ).
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library