RNA and DNA Aptamers in Cytomics Analysis

Henning Ulrich1, Antonio Henrique B. Martins2, João Bosco Pesquero2

1 Universidade de São Paulo, São Paulo, 2 Universidade Federal de São Paulo, São Paulo
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.28
DOI:  10.1002/0471142956.cy0728s33
Online Posting Date:  August, 2005
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Abstract

Using systematic evolution of ligands by exponential enrichment (SELEX), RNA or DNA molecules are selected from a combinatorial oligonucleotide library by their ability to bind their targets, i.e., cell surface antigens, with affinity and specificity similar to that of monoclonal antibodies. The generation of these high‐affinity binders, also denominated aptamers, is carried out in vitro and does not involve animals. Therefore, aptamers can be developed against almost every molecule of biological importance, including toxins and nonimmunogenic targets, against which antibodies cannot be raised. The incorporation of modified pyrimidines resulting in nuclease‐resistant RNA aptamers makes them promising candidates for studying protein interactions in vitro and in vivo. DNA aptamers do not need modifications for most applications. The protocols in this unit can be used for the development of fluorescent‐tagged RNA or DNA aptamers for any cell surface protein in cytomics analysis.

Keywords: SELEX; RNA and DNA aptamers; fluorescent‐tagged aptamers; cell surface protein binding; cytometry applications

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Target Preparation
  • Basic Protocol 2: Random DNA Pool Preparation, Second‐Strand Synthesis, and Amplification
  • Basic Protocol 3: Large‐Scale Second‐Strand Synthesis and Amplification of the Double‐Stranded DNA Using Error‐Prone PCR
  • Basic Protocol 4: Transcription and Purification of RNA Pools
  • Basic Protocol 5: Reiterative in Vitro Selection Cycles for Enrichment of Target Binders in the Selex Pool
  • Alternate Protocol 1: Gel‐Shift (Gel‐Retardation) Selection Experiments for Selection of Target‐Binding RNA Molecules
  • Basic Protocol 6: Negative Counterselection to Eliminate RNA Molecules Binding Nonspecifically to Nitrocellulose Membrane
  • Basic Protocol 7: Negative Counterselection to Eliminate RNA Molecules Binding to Proteins Other than the Target
  • Basic Protocol 8: Reverse Transcription and PCR Amplification
  • Basic Protocol 9: Binding Studies
  • Basic Protocol 10: Cloning and Identification of High‐Affinity Aptamers
  • Basic Protocol 11: Screening for RNA Aptamers with Desired Function
  • Basic Protocol 12: Fluorescence Labeling of Aptamers for Cytometry Applications
  • Basic Protocol 13: Imaging Using Biotinylated RNA Aptamers
  • Alternate Protocol 2: Development of a Fluorescent‐Tagged DNA Aptamer
  • Alternate Protocol 3: DNA Aptamer for Cytometry Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Target Preparation

  Materials
  • Cell culture expressing the target protein (i.e., wild‐type or recombinant membrane‐bound receptor), growing in 75‐cm2 culture flasks
  • Cell culture that does not express the target protein, growing in 75‐cm2 culture flasks
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • PBS ( appendix 2A) containing 2 mM EDTA
  • Buffer A (see recipe), ice‐cold
  • 1 M NaCl
  • 1 mg/ml MgSO 4
  • Buffer B (see recipe), ice‐cold
  • Bradford Protein Assay kit (Bio‐Rad)
  • Cell scrapers
  • Probe sonicator (e.g., Branson)
  • Low‐speed refrigerated centrifuge
  • Ultracentrifuge
  • Spectrophotometer

Basic Protocol 2: Random DNA Pool Preparation, Second‐Strand Synthesis, and Amplification

  Materials
  • Primer P40: 5′‐GTA‐ATA‐CGA‐CTC‐ACT‐ATA‐GGG‐AGA‐ATT‐CAA‐CTG‐CCA‐TCT‐A‐3′ (order HPLC‐purified from oligonucleotide synthesis facility)
  • 10 U/µl T4 polynucleotide kinase with 5× forward labeling buffer (Invitrogen)
  • 10 µCi/µl [γ‐32P]ATP (5000 Ci/mmol; Amersham Biosciences)
  • 16% denaturing urea PAGE gel (Ellington and Pollard, )
  • Partial random ssDNA template, gel‐purified (108 bases; synthesized by Biosource International): 5′‐ACC‐GAG‐TCC‐AGA‐AGC‐TTG‐TAG‐TAC‐N40‐G‐CCT‐AGA‐TGG‐CAG‐TTG‐AAT‐TCT‐CCC‐TAT‐AGT‐GAG‐TCG‐TAT‐TAC‐3′
  • 10 mM stock solution for each dNTP (Ambion)
  • 50 mM MgCl 2
  • 5 U/µl Taq DNA polymerase with 10 × PCR buffer (Invitrogen)
  • 8% nondenaturing polyacrylamide gel (Chory and Pollard, ; also see recipe)
  • 65°C water bath
  • S‐10 spin column (Sigma)
  • Thin‐walled PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (Ellington and Pollard, ), autoradiography (Voytas and Ke, ), and nondenaturing polyacrylamide gel electrophoresis (Chory and Pollard, )

Basic Protocol 3: Large‐Scale Second‐Strand Synthesis and Amplification of the Double‐Stranded DNA Using Error‐Prone PCR

  Materials
  • ssDNA (see protocol 2)
  • 10 mM stock solution for each dNTP (Ambion)
  • 1 M MnCl 2
  • MgCl 2
  • Primer P40: 5′‐GTA‐ATA‐CGA‐CTC‐ACT‐ATA‐GGG‐AGA‐ATT‐CAA‐CTG‐CCA‐TCT‐A‐3′ (order HPLC‐purified from oligonucleotide synthesis facility)
  • 5 U/µl Taq DNA polymerase (Invitrogen)
  • Primer P22: 5′‐ACC‐GAG‐TCC‐AGA‐AGC‐TTG‐TAG‐T‐3′ (order HPLC‐purified from oligonucleotide synthesis facility)
  • 8% nondenaturing polyacrylamide gel (Chory and Pollard, )
  • Buffered phenol, pH 7.4 (Invitrogen)
  • Chloroform
  • 3 M sodium acetate, pH 7.4
  • 5 mg/ml linear acrylamide (Ambion)
  • 99% and 80% ethanol
  • 96‐well PCR plates or thin‐walled PCR tubes in 96‐tube format
  • Refrigerated centrifuge
  • UV spectrophotometer
  • Additional reagents and equipment for nondenaturing polyacrylamide gel electrophoresis (Chory and Pollard, )

Basic Protocol 4: Transcription and Purification of RNA Pools

  Materials
  • DEPC‐treated H 2O (unit 8.9)
  • Double‐stranded DNA pool (see protocol 3)
  • 200 U/µl T7 RNA polymerase and 10× buffer (Ambion)
  • 10 mM ATP (Ambion)
  • 10 mM GTP (Ambion)
  • 10 mM 2′‐fluoro‐2′‐deoxycytidine‐5′‐triphosphate (2′‐F‐dCTP; Trilink Technologies)
  • 10 mM 2′‐fluoro‐2′‐deoxyuridine‐5′‐triphosphate (2′‐F‐dUTP; Trilink Technologies)
  • 10 µCi/µl [α‐32P]ATP (3000 Ci/mmol; Amersham Biosciences)
  • RNase‐free DNase (Ambion)
  • 8% denaturing polyacrylamide gel (see recipe; also see Ellington and Pollard, ) with 2‐mm gel spacers
  • 2× formamide loading buffer (see recipe)
  • RNA ladder (size range, 100 to 500 bases; RNA Century Markers, Ambion)
  • 3 M sodium acetate, pH 5.5, prepared with DEPC‐treated H 2O (see unit 8.9 for DEPC‐treated H 2O)
  • 5 mg/ml linear acrylamide
  • Buffered phenol, pH 5.5 (see recipe)
  • Chloroform
  • 65°C water bath
  • UV transilluminator
  • Fluorescent thin‐layer chromatography plate (Merck)
  • Transparent plastic bag
  • Sterile tweezer and razor blade for gel purification
  • Orbital shaker
  • UV spectrophotometer
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (Ellington and Pollard, )

Basic Protocol 5: Reiterative in Vitro Selection Cycles for Enrichment of Target Binders in the Selex Pool

  Materials
  • Selection pool consisting of random 32P‐labeled 2′‐F‐pyrimidine‐modified RNA generated as in protocol 4 (3 nmol; exact concentration depends on amount of available target‐binding sites and progress of selection)
  • Selection buffer (see recipe)
  • Membrane preparation containing the target protein (see protocol 1)
  • Yeast transfer RNA (Sigma)
  • Antagonist capable of displacing RNA from target receptors
  • Buffered phenol, pH 5.5 (see recipe)
  • Chloroform
  • DEPC‐treated H 2O (unit 8.9)
  • 3 M sodium acetate, pH 5.5
  • 99% and 80% ethanol
  • 65°C water bath
  • Vacuum dot‐blot manifold (Schleicher & Schuell) and vacuum pump
  • Nitrocellulose membrane (Schleicher & Schuell BA‐85)
  • 0.5‐ml, 1.5‐ml, and 2‐ml microcentrifuge tubes, autoclaved
  • UV spectrophotometer
NOTE: Use aerosol‐barrier pipet tips and autoclaved glass/plasticware for all steps.

Alternate Protocol 1: Gel‐Shift (Gel‐Retardation) Selection Experiments for Selection of Target‐Binding RNA Molecules

  • 10× TBE buffer, pH 7.4 ( appendix 2A)
  • 4× nondenaturing loading buffer, pH 7.4
  • 0.3 M sodium acetate pH 5.5
  • Buffered phenol, pH 5.5 (see recipe)
  • Scintillation fluid
  • Vertical gel electrophoresis apparatus (Chory and Pollard, )
  • Platform shaker
  • 0.2 µm Nylon 66 syringe filter (Whatman)
  • Additional reagents and equipment for nondenaturing gel electrophoresis (Chory and Pollard, ), phosphor imaging (Voytas and Ke, ), and reverse transcription and amplification (see protocol 9)

Basic Protocol 6: Negative Counterselection to Eliminate RNA Molecules Binding Nonspecifically to Nitrocellulose Membrane

  Materials
  • 32P‐labeled RNA pool from large‐scale transcription (see protocol 4)
  • Selection buffer (see recipe)
  • 65°C water bath
  • Nitrocellulose membrane (Schleicher & Schuell BA‐85)
  • Filter holder for immobilizing nitrocellulose filter
  • 3‐ml syringe
  • Additional reagents and equipment for ethanol precipitation of RNA (see protocol 4)

Basic Protocol 7: Negative Counterselection to Eliminate RNA Molecules Binding to Proteins Other than the Target

  Materials
  • 32P‐labeled RNA pool from large scale transcription (see protocol 4)
  • Selection buffer (see recipe)
  • Membrane preparation without target protein (see protocol 1)
  • DEPC‐treated H 2O (unit 8.9)
  • Buffered phenol, pH 5.5 (see recipe)
  • Chloroform
  • 3 M ammonium acetate, pH 5.5, ∼DEPC‐H 2O (see recipe)
  • 5 mg/ml acrylamide (Ambion)
  • 99% and 80% ethanol
  • 65°C water bath
  • Filter holder for immobilizing nitrocellulose filter
  • 3‐ml syringe
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation of RNA (see protocol 4)

Basic Protocol 8: Reverse Transcription and PCR Amplification

  Materials
  • Eluted [32P]RNA from SELEX cycle (see protocol 5)
  • 100 pmol/µl Primer P22: 5′‐ACC‐GAG‐TCC‐AGA‐AGC‐TTG‐TAG‐T‐3′ (order HPLC‐purified from oligonucleotide synthesis facility)
  • 10 mM stock solution for each dNTP (Ambion)
  • DEPC‐treated H 2O (unit 8.9)
  • 10× AMV buffer (supplied with enzyme)
  • AMV reverse transcriptase (Life Sciences)
  • 10 U/µl RNase H (Invitrogen)
  • 50 mM MgCl 2
  • 5 U/µl Taq DNA polymerase (Invitrogen)
  • 100 pmol/µl Primer P40: 5′‐GTA‐ATA‐CGA‐CTC‐ACT‐ATA‐GGG‐AGA‐ATT‐CAA‐CTG‐CCA‐TCT‐A‐3′ (order HPLC‐purified from oligonucleotide synthesis facility)
  • 3 M sodium acetate, pH 7.4
  • 5 mg/ml linear acrylamide (Ambion)
  • 8% nondenaturing polyacrylamide gel (Chory and Pollard, ; also see recipe)
  • Thin‐walled PCR tubes
  • Thermal cycler (e.g., Eppendorf Master‐Cycler)
  • Additional reagents and equipment for nondenaturing polyacrylamide gel electrophoresis (Chory and Pollard, )

Basic Protocol 9: Binding Studies

  Materials
  • dsDNA (from different SELEX pools; see protocol 5)
  • 10 mM GTP (Ambion)
  • 10 mM 2′‐fluoro‐2′‐deoxycytidine‐5′‐triphosphate (2′‐F‐dCTP; Trilink Technologies)
  • 10 mM 2′‐fluoro‐2′‐deoxyuridine‐5′‐triphosphate (2′‐F‐dUTP; Trilink Technologies)
  • 0.5 mM ATP (Ambion), prepared with DEPC‐treated H 2O (see unit 8.9 for DEPC‐treated H 2O)
  • 10 µCi/µl [α‐32P]ATP (3000 Ci/mmol; Amersham Biosciences)
  • 200 U/µl T7 RNA polymerase with 10× T7 RNA polymerase buffer (Ambion)
  • DEPC‐treated H 2O (unit 8.9)
  • 50 mM EDTA
  • RNase‐free DNase (Ambion)
  • 8% denaturing polyacrylamide gel (Chory and Pollard, ; also see recipe)
  • Scintillation fluid
  • Protein preparation containing the target protein (see protocol 1)
  • Yeast transfer RNA (Sigma)
  • Selection buffer (see recipe)
  • Competitor: 10 mM stock solution of, e.g., Lys‐des‐arg9‐leu8‐bradykinin (antagonist used to displace RNA bound to the kinin B1 receptor used as target)
  • 65°C water bath
  • S‐30 spin columns (Sigma)
  • Nitrocellulose membrane (Schleicher & Schuell BA‐85)
  • Filter paper GB002, 102 × 133 mm (Schleicher & Schuell)
  • Vacuum dot‐blot manifold (Schleicher & Schuell) and vacuum pump
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (Chory and Pollard, )

Basic Protocol 10: Cloning and Identification of High‐Affinity Aptamers

  Materials
  • Selected DNA sequences (final RNA pool; see protocol 5)
  • EcoRI and HindIII restriction enzymes (Invitrogen)
  • Restriction buffer (see recipe)
  • 1% and 1.5% preparative agarose gels (Voytas, )
  • Liquid N 2 or dry‐ice/ethanol bath
  • T4 DNA ligase and 10× ligation buffer (Invitrogen)
  • pGEM‐3Z vector (cloning vector that allows highly efficient synthesis of RNA in vitro; Promega)
  • Transformation‐competent E. coli cells (strains JM 29 or DH5α; Promega)
  • Plasmid miniprep kit
  • 16°C temperature‐controlled water bath
  • Additional reagents and equipment for reverse transcription and amplification (see protocol 9), agarose gel electrophoresis (Voytas, ), and transformation of E. coli (Seidman et al., )

Basic Protocol 11: Screening for RNA Aptamers with Desired Function

  Materials
  • Purified plasmid containing inserts suitable for aptamer (see protocol 11)
  • Primer P40: 5′‐GTA‐ATA‐CGA‐CTC‐ACT‐ATA‐GGG‐AGA‐ATT‐CAA‐CTG‐CCA‐TCT‐A‐3′ (order HPLC‐purified from oligonucleotide synthesis facility)
  • P22 pGEM: 5′‐GAA‐TAC‐TCA‐AGC‐TTG‐TAG‐TAC‐T‐3′ (order HPLC‐purified from oligonucleotide synthesis facility)
  • 5 U/µl Taq DNA polymerase with 10× PCR buffer (Invitrogen)
  • 10 mM stock solution for each dNTP (Ambion)
  • 50 mM MgCl 2
  • Buffered phenol, pH 7.4 (Invitrogen)
  • Chloroform
  • 3 M sodium acetate, pH 7.4
  • 5 mg/ml linear acrylamide (Ambion)
  • 99% and 80% ethanol
  • 1.0% agarose gel (Voytas, )
  • Thin‐walled PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 12: Fluorescence Labeling of Aptamers for Cytometry Applications

  Materials
  • Double‐stranded DNA of interest
  • 0.5 mM ATP (Ambion)
  • 10 mM GTP (Ambion)
  • Guanosine 5′‐O‐(2‐thio‐diphosphate) (GDP‐β‐S; Sigma)
  • 10 mM 2′‐fluoro‐2′‐deoxyuridine‐5′‐triphosphate (2′‐F‐dUTP; Trilink Technologies)
  • 10 mM 2′‐fluoro‐2′‐deoxycytidine‐5′‐triphosphate (2′‐F‐dCTP; Trilink Technologies)
  • 200 U/µl T7 RNA polymerase and 10× buffer (Ambion)
  • Biotin‐labeling buffer (see recipe)
  • 4 mM iodoacetyl‐LC‐biotin (Pierce) in N,N‐dimethylformamide (DMF)
  • 2 N NaOH
  • DEPC‐treated water (unit 8.9)
  • Buffered phenol, pH 5.5 (Invitrogen; see recipe)
  • Chloroform
  • 3 M sodium acetate, pH 5.5, ∼DEPC‐treated water
  • 5 mg/ml linear acrylamide (Ambion)
  • 99% and 80% ethanol
  • Fluorescent thin‐layer chromatography plates (Merck)
  • UV spectrophotometer
  • S‐30 spin columns (Sigma)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (Ellington and Pollard, ), isolation and purification of RNA (see protocol 4), and determining RNA concentration (see protocol 4)

Basic Protocol 13: Imaging Using Biotinylated RNA Aptamers

  Materials
  • Cell line expressing the target protein
  • 70% ethanol
  • Biotinylated RNA aptamers (see protocol 13)
  • 2% bovine serum albumin (BSA)/50 µg/ml propidium iodide in selection buffer (see recipe for buffer)
  • Selection buffer (see recipe)
  • Fluorochrome‐conjugated streptavidin (e.g., streptavidin‐Cy2, streptavidin fluorescein)
  • Lab‐Tek chamber slides (Nunc)
  • 65°C water bath
  • Fluorescence microscope with filters for excitation at 488 nm and emission at 525 nm (for fluorescein emission) and above 690 nm (for propidium iodide emission)

Alternate Protocol 2: Development of a Fluorescent‐Tagged DNA Aptamer

  Materials
  • Single‐stranded random DNA template: 5′‐GCCTGTTGTTGAGCCTCCT‐N34‐CGCT TATTCTTGTCTCCC‐3′; Operon Technologies)
  • Triple‐biotinylated forward primer: 5′‐BBB‐GCCTGTTGTGAGCCTCCT‐3′
  • Nonbiotinylated forward primer: 5′‐GCCTGTTGTGAGCCTCCT‐3′
  • Reverse primer: 5′‐ GGGAGACAAGAATAAGCG‐3′
  • Taq DNA polymerase with 10× PCR buffer
  • 50 mM MgCl 2
  • 10 mM stock solution for each dNTP (Ambion)
  • 10µCi/µl [α‐32P]dCTP (6000 Ci/mmol; Amersham Biosciences)
  • TE buffer ( appendix 2A)
  • 8% nondenaturing polyacrylamide gel (Chory and Pollard, ; also see recipe)
  • 2× denaturing (formamide) loading buffer (see recipe)
  • 0.5 M and 3 M sodium acetate, pH 7.4
  • Streptavidin (Pierce)
  • Transformation‐competent E. coli cells (JM29 or DH5α; Promega)
  • Buffered phenol, pH 7.4 (Invitrogen)
  • 99% and 80% ethanol
  • 5 mg/ml linear acrylamide (Ambion)
  • Liquid nitrogen or dry ice/ethanol bath
  • pGEM‐T Easy vector (Promega)
  • Plasmid miniprep kit
  • Thermal cycler
  • Sterile razor blade
  • Additional reagents and equipment for nondenaturing polyacrylamide gel electrophoresis (Chory and Pollard, ), denaturing gel electrophoresis (Ellington and Pollard, ), autoradiography and phosphor imaging (Voytas and Ke, ), isolation and purification of DNA (see protocol 2), end‐labeling of DNA (see protocol 2), binding assays (see protocol 10), and screening for DNA aptamers (see protocol 12)

Alternate Protocol 3: DNA Aptamer for Cytometry Analysis

  Materials
  • Selected DNA sequences (see protocol 15)
  • Biotinylated forward primer: 5′‐BBB‐GCCTGTTGTGAGCCTCCT‐3′
  • Taq DNA polymerase and 10× buffer
  • Fluorescein isothiocyanate‐C‐18‐labeled reverse primer (FITC‐18C‐GGGAGACA AGA ATAAGCG‐3′; 18C denotes an 18‐carbon ethylene glycol spacer incorporated into the aptamer strand to minimize interaction during PCR amplification and target binding)
  • 50 mM MgCl 2
  • 10 mM stock solution for each dNTP (Ambion)
  • 8% nondenaturing polyacrylamide gel (Chory and Pollard, ; also see recipe)
  • 2 × denaturing loading buffer
  • 3 M sodium acetate, pH 7.4
  • Buffered phenol, pH 7.4 (Invitrogen)
  • Chloroform
  • 99% and 80% ethanol
  • 5 mg/ml linear acrylamide (Ambion)
  • 1 N NaOH
  • Thermal cycler
  • Streptavidin magnetic beads and magnetic stand (Dynal)
  • Additional reagents and equipment for nondenaturing polyacrylamide gel electrophoresis (Chory and Pollard, ) and phenol/chloroform extraction and ethanol precipitation of DNA (see protocol 2)
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Literature Cited

Literature Cited
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