T7 transcription is the standard method of producingRNA longer than 10 or 20 bases. It is easy, quick, and produces relativelygood quality RNA, when compared to solid phase synthesis. RNA can be bodylabeled with 32P, but unusual bases cannot be inserted intothe middle of the sequence. It is routine to produce any amount up to manymilligrams.
T7 Polymerase is one of a group of very active phage polymerases, thatuse a short double-stranded promoter sequence to initiate transcriptionat a specific base in the template, and then transcribe single or doublestranded DNA with good fidelity until the end of the DNA bottom strandis reached. At this point, T7 will sometimes add one or more bases, somewhatrandomly, and with a frequency that is dependent in an unknown manner onthe sequence, but can reach 50% or more. T7 strongly prefers to start witha G (GG is better still), but otherwise will transcribe virtually any sequence.Certain sequences can cause problems, however, such as strings of eightor more A or U (Milligan & Uhlenbeck, 1989). Too many A or U basesin the first six nucleotides can reduce yield substantially.
Template oligos can be from any source that provides the double strandedpromoter region followed by the transcribed region. The most common methodis to make a stock of synthetic top strand (that can be used for any template),then synthesize any desired bottom strand with a complementary region forthe top strand. The top strand sequence I use has a 17-base section containingthe promoter, preceded by three bases than help stabilize it, and followedby the first (can be first two) bases that will be transcribed (usually"G").
leader | promoter sequence |first base
Top Strand: 5' ATG TAA TAC GAC TCA CTA TA G 3'
Template: 3' TAC ATT ATG CTG AGT GAT AT Cxxx(template)
Synthetic oligos produce useable RNA, but appear to cause enough mistakesthat reactions involving ribozymes, for instance, never go to completion(Milligan & Uhlenbeck, 1989).
Basic Protocol: For 1 ml of transcription,mix together:
|200 ml||5X NTPs (25 mM each)||1X (5 mM each)|
|100 ml **||40 mM GTP||4 mM extra GTP|
|25 ml||1 M MgCl2||25 mM|
|80 ml||0.5 M TRIS pH ~7.8||40 mM|
|10 ml||0.25 M Spermidine||2.5 mM|
|10 ml||1% Triton X||0.01 %|
|10 ml||1 M DTT (keep stock frozen)||10 mM|
|5 ml **||RNAsin (Promega, 40 u/ml)||200 units / ml|
|10 ml **||inorganic pyrophosphatase||1 unit / ml|
|1-10 ml **||a-32PUTP||if body labeling desired|
|50 ml dep.on conc.||T7 polymerase||want ~ 20,000 units / ml|
|Template||to a final concentration of ~0.5 mM|
|balance||H2O||to make 1 ml.|
Bold indicates ingredients whose concentrations are most criticalto yield, and may be varied to optimize yield.
A 10X stock of the boxed ingredients can be made in advance, aliquoted,and stored at
** These ingredients are optional, and are not used by everyone. Note:RNAsin requires DTT.
Incubate at 37° C for 1 - 12 hours. I recommend the airincubator, so there is no temperature differential, and water will notevaporate off and condense on the top (which changes the salt concentrationsin the reaction mix). If inorganic pyrophosphatase is used, solution willremain clear. If not, solution will develop white precipitate which willsettle over several hours. Although incubation overnight is common practicein the lab, under the conditions described above transcription should becomplete within 4 hours, and longer incubation only increases the chancesof degradation and 5' dephosphorylation.
Add EDTA to bind up the amount of Mg2+ used. e.g.if 25 mM Mg is used in a 1 ml reaction, add EDTA to 25 mM (or slightlyhigher) - 60 ml of 0.5 M EDTA is sufficient.
Some people treat with DNAse (I know nothing about this -- add it ifyou want?)
Prepare for Gel Purification: Phenol/chloroform extract, desaltby gel filtration, EtOH precipitate, as desired, to prepare for gel purification.For fast results (probably just as good), you can just add 1 ml 2X urealoading buffer to your 1 ml transcription (after EDTA) and load onto gel.If you intend to load an entire 1 ml transcription on a single 1.5 mm thickgel (see Gel Purify, below), dissolve solid urea into transcription,to a final concentration of 5 or 6 molar. The advantage of desalting/precipitatingis that you can load a much smaller volume onto the gel, which resultsin somewhat better separation.
Expect up to 2-3 mg/ml of transcript, though very much sequence dependent,so usually yield is <Å 1 mg/ml.
Gel purify (see related protocol). 1 ml of transcription canbe purified on a 1.5 mm thick prep-sized gel (plates Å19 x 20 cm)if yield is moderate, or on a 3 mm gel (or 2 1.5 mm gels) if yield is good.
Advanced techniques: Refinements and explanationsof above protocol.
Optional ingredients: GTP is the universal starting pointfor transcription, but T7 frequently falls off early in transcription.As a result, the GTP is preferentially depleted in the reaction mixture.Adding extra GTP can prevent this from being a limiting factor. RNAsinis useful to prevent RNA degradation by RNases. I have always usedit, and have never had RNAse problems. But some people do not use it, andyou can experiment if you want! Inorganic Pyrophosphatase will breakup and solubilize the pyrophosphate that forms the precipitate. This hasin some cases increased yields, in other cases apparently had little effect.It is unlikely to decrease yield. It may operate by keeping more magnesiumfree, therefore stabilizing the Mg++ concentration. Hot UTPcan be used to body label the RNA if desired. 10 mCi(1 ml) per 100 mlof transcript will make quite hot RNA.
Yield: The yield is dependent on many factors, some controllableand some not. Sequence of template is very important, and several thingscan be controlled. While the first nucleotide must be G, having the secondalso G apparently increases yield. Also, avoiding the first U for as longas possible (for the first 8 - 14 bases) has been reported to be helpful.Once the polymerase is 'rolling along' it seems to be fine, though bandingof purification gels suggests that it tends to fall off at particular places.The other factors that interactively affect yield are template concentration,T7 concentration, and the ratio of NTP to MgCl2 concentrations.If yield is vital (as in isotopically labeled NMR preparations) pilot studiesshould be performed to optimize the concentrations of all these components.More extensive discussion can be found in Puglisi and Wyatt (1995).
Purification considerations: T7 generally adds one or more basesto the 3' end of the RNA a significant percentage of the time (up to oreven greater than 50% is common). If the 3' end must be uniform, then thecorrect species can be separated from the +1/+2 versions on a long gel,if the RNA is short (<Å 50 bases). Above that length, it mustbe enzymatically cleaved. Many methods have been proposed, but I like hammerheadbest. It can be designed to cleave if the sequence can be designed to accommodateit. (see related protocol).
5' phosphates: Theoretically, the first G that is used is a GTP,and retains its phosphates. In practice, it has been observed that thisphosphate can be hydrolyzed over the time of transcription, such that ina 12 hour transcription, Å 50 % is no longer triphosphate. If itis vital to have that triphosphate, it is best to increase concentrationsof T7 and perhaps other components in the reaction mixture (to speed uptranscription), then shorten the duration to one or a few hours. If insteadone wished a uniform species with a monophosphate on the 5' end, that canbe achieved by spiking the reaction mix not with extra GTP but with GMP.All NTP (and MgCl2) concentrations can be adjusted to make theNTP:GMP ratio whatever desired. It has been reported (Puglisi & Wyattagain) that GMP is actually preferred over GTP for a starting nucleotide,so it is possible to achieve a fairly uniform 5' end this way. Apparentlyguanosine can also be used, but it is rather insoluble, so it is not easy.Guanosine derivitives (such as thio-phosphate species) can also be incorporatedinto the 5' end.
Reagents: T7 and pyrophosphatase are inthe enzyme freezer: pyrophosphatase is not stable long term, so is aliquotedand stored at -80 in the bottom shelf. I recommend purchasing 1 M MgCl2from Sigma in 1 ml aliquots; it is cheap and avoids risk of unseen contamination.People tend to purchase their own stocks of DTT and spermidine. The inorganicphosphatase is purchased from Sigma, c/n I 2267, and dissolved in a 50%glycerol buffer containing 20 mM TRIS (pH 8), 0.1 mM EDTA, 1 mM DTT, and100 mM KCl.
References: The following references are all informative discussionsof T7 RNA production.
Milligan, J.F., Groebe, D.R., Witherell, G.W., & Uhlenbeck, O.C.(1987) Nucleic Acids Research 15:21 Pgs. 8783-8798.
Milligan, J.F. & Uhlenbeck, O.C. (1989) Chapter in Methods inEnzymology , Vol. 180. Pgs. 51-62.
Puglisi, J.D. & Wyatt, J.R. (1995) Chapter in Methods in Enzymology,Vol. 261. Pgs. 323-349.