Uniform

1
Site-specific modification of RNA molecules
Uniform labeling
Site-specific labeling
2
Site-specific modification of RNA molecules
Site-specifically placed 32P group
Crosslinkers (with or without 32P)
Modified nucleotides
3
(No Transcript)
4
T4 DNA ligase
Complementary DNA oligonucleotide (splint)
5
Preparation of the RNA pieces for ligation

• T7/T3/SP6 transcription or chemical RNA
  synthesis
• Make at least 10x more of each transcript that
  is needed in the final experiments
• 250-500 ml reaction will yield at least several
  nmoles of the transcript
• gt 6 mM rNTP
• gt 8 mM MgCl2
• gt 3 hr reaction
• Gel purify in a denaturing acrylamide gel
• gt detect the transcripts using UV shadow
  technique
• (enhancing screeen or fluorecent TLC)
• elute o/n in 50mM Tris-HCl pH 7.0, 300 mM NaCl,
  0.5 mM EDTA, 0.1 SDS
• - EtOH precipitate, measure concentration
  (nearest neighbour formula)

6

• Requirements for the successful ligation
• Perfect match between the two RNA pieces
• and the splint DNA oligo
• 5 piece 3-OH group
• 3 piece 5-(mono)phophate

RNA pieces ...CUACCpGGCACTG... DNA splint
...GATGG-CCGTGAC...
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RNA oligos

• ACE/TOM chemistry (Dharmacon, Xeragon) with RNA
  synthesis
• gt easy 2-OH deprotection (10 minutes)
• gt a good collection of various modified
  nucleotides available
• gt extremely good quality ? No need for gel
  purification?
• 5 phosphates chemical synthesis vs. kinasing

DNA splint

• Typically 20 nt surrounding the junction point is
  sufficient
• ? for two pice ligation 40 nt oligo is OK
• ? for three-piece ligation about 60 nt oligo is
  typically OK
• Sometimes it may be necessary to use a second
  disruptor
• oligo to melt a strong secondary structure on
  the targer RNA

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How to make a 5 fragment with a perfect 3 end

• - T7/T3/SP6 transcript
• Template by PCR -gt Use proof-reading DNA
  polymerases
• (Pfu, Vent) which do not add non-template
  nucleotides to
• the 3 end of the DNA molecule (extra
  nucleotides may cause the T7 RNA pol to
• loop back to make dsRNA)
• - Optional The two 5 nucleotides of the
  downstream PCR oligo can be
• 2-O-me RNA - will prevent nontemplate
  nucleotide addition by T7 RNA polymerase
• Kao, C., Zheng, M., and Rüdisser, S. (1999). A
  simple and efficient method to reduce
  nontemplated nucleotide
• addition at the 3 terminus of RNAs transcribed
  by T7 RNA polymerase. RNA 5, 1268-1272.)

Upstream oligo
T7
Downstream oligo
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How to make a 3 fragment with a perfect 5 end
I. The ligation junction has two G residues
Upstream oligo
T7
CCNNNNNNN
PCR
GGNNNNNNN
Downstream oligo
T7 transcription
pppGGNNNNNNNNNN....

• Large amounts of CIAP (50-100 U) are often needed
  for dephosphorylation
• RNA secondary structure may affect the
  efficiency of dephophorylation
• Dephophorylation at 50oC

Phosphatase
GGNNNNNNNNNN....
Kinase
pGGNNNNNNNNNN....
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How to make a 3 fragment with a perfect 5 end
II. No G residues at the ligation junction
Site-specific cutting with RNase H and 2-O-me
RNA/DNA oligonucleotide
2-O-Me 13 nt
2-O-Me 3 nt
DNA 4 nt
3
5
Target RNA
5
3
3
5
5
3

• Cleavage sites
• a. Pharmacia and USB RNase H
• b. Boehringer RNase H

b
a
5
-OH3
5-P-
-OH3
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Site-specific cutting of the RNA molecules
An example 20 ml RNA (350 pmol total) 1.0 ml
2omeRNA/DNA oligo (500 pmol total) 0.5 ml 1M
KCl 5.0 ml H2O -gt Anneal with a PCR machine
(heat at 95oC 5 min, slowly (15 min) to RT) 10
ml 5xbuff USB 10 ml RNase H (5U/ml USB) 1.0
ml 250 mM DTT 2.5 ml RNasin (40 U/ml
Promega) 37 oC, 3hr Phenol extract, EtOH
precipitate, purify in 5 DPAGE (detect bands by
UV shadow)
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RNA ligationI. Annealing II. The actual
ligation III. Purification and quantitation
Three fragment ligation -be very careful with
the molar amouts -1x 5 fragment -1x 3
fragment -3x RNA oligo -0.9x DNA
splint trace 32P-RNA oligo (105 cpm) in TE
buffer, 50 mM KCl Typically I use 100 500
pmol of each 5 and 3 fragments. The final
volume is 5-10 ml
Annealing PCR program 95oC 3 min. 87oC 10
sec. -0.1oC/sec ? 37oC 15oC for ever
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RNA ligationI. Annealing II. The actual
ligation III. Purification and quantitation

• Ligation buffer
• 50 mM Tris-HCl pH 7.5
• 10 mM MgCl2
• 20 mM DTT
• 1 mM ATP
• 2 Polyvinyl alcohol (PVA)
• (3-5 PEG 6000 is probably OK)
• 40 U RNasin
• 20-30 U T4 DNA ligase (Fermentas)

• Ligation
• The final volume is 20-30 ml
• o/n at room temperature (20oC)
• Alternatively 3-5 hr at 37oC
• After ligation
• Phenol extract
• EtOH precipitate
• Gel purify the ligated product
• (Urea-PAGE)

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RNA ligationI. Annealing II. The actual
ligation III. Purification and quantitation

• Separate the ligated RNAs from the unligated
  stuff in a denaturing PAGE
• Expose the wet gel to autoradiography film. Use
  fluorecent tape
• to aling the film with the gel. Typical
  exposure time 1 hr o/n.
• - The efficiency is anywhere between 5-50

Miracle
Typical purification gel
Ligated product
Ligated product
3 fragment
3 fragment
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RNA ligationI. Annealing II. The actual
ligation III. Purification and quantitation

• One of the RNAs should have trace label (32P)
• The final yield is easy to calculate
• (Amount of cold RNA fragment) pmol x (Recovered
  radioactivity) cpm
• (Total radioactivity in the reaction)
  cpm

? pmoles of ligated RNA
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Suppliers
RNA oligos Dharmacon research www.dharmacon.com
Xeragon www.xeragon.com 2ome/DNA
oligos Dharmacon research Yale Keck
facility info.med.yale.edu/wmkeck/oligos/ T4
ligase (HC) Fermentas, USB Fluorecent
tape Diversified biotech 4-thio-UTP USB 365 nm
UV lamp UV products (UVP)