Re: [ccp4bb] Synthetic RNA for Crystallization
Hi folks, crystallized a lot of SRP RNAs - never had success with synthetic RNA, even with small 30mers T7 RNA pol in vitro transcribed was always extremely better - moreover: I always use a 3' hammerhead which yields in the end 100% pure material in mg amounts - Kiyoshi Nagais protocol is a perfect basis. Greetings Klemens Martin Hällberg wrote: Hi, I'll second Israels's comment. Since the yield per coupling in synthesis is lower for RNA than for DNA it gets really expensive over 30-35 nucleotides. However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint). Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice. For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft. See: Batey, R.T. Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389. You are going to need milligrams of T7 RNA polymerase in the end so forget about transcription kits, make your own. Cheers, Martin On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote: Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps J Mol Biol. 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SR, Ito N, Oubridge C, Avis JM, Nagai K. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi Michael, If the problem is the absence of a purine on the 5' end, try using the Marc Dreyfus' mutant T7 RNA polymerase. It can accommodate any nucleotide at the 5' end. Guillerez, J., Lopez, P. J., Proux, F., Launay, H., and Dreyfus, M. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance, Proceedings of the National Academy of Sciences of the United States of America 102, 5958-5963. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrievedb=PubMeddopt=Citationlist_uids=15831591 With best regards, Eric -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Martin Hällberg Sent: Sunday, March 13, 2011 9:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Synthetic RNA for Crystallization Hi, I'll second Israels's comment. Since the yield per coupling in synthesis is lower for RNA than for DNA it gets really expensive over 30-35 nucleotides. However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint). Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice. For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft. See: Batey, R.T. Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389. You are going to need milligrams of T7 RNA polymerase in the end so forget about transcription kits, make your own. Cheers, Martin On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote: Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps J Mol Biol. 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SR, Ito N, Oubridge C, Avis JM, Nagai K. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate
[ccp4bb] Synthetic RNA for Crystallization
Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps javascript:AL_get(this,%20'jour',%20'J%20Mol%20Biol.'); J Mol Biol. javascript:AL_get(this,%20'jour',%20'J%20Mol%20Biol.'); 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SRhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Price%20SR%22%5BAuthor%5D, Ito N http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ito%20N%22%5BAuthor%5D, Oubridge C http://www.ncbi.nlm.nih.gov/pubmed?term=%22Oubridge%20C%22%5BAuthor%5D, Avis JM http://www.ncbi.nlm.nih.gov/pubmed?term=%22Avis%20JM%22%5BAuthor%5D, Nagai K http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nagai%20K%22%5BAuthor%5D. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] Synthetic RNA for Crystallization
1) Make your own with in vitro transcription (straight T7 r T7+RDRP like in Finnzymes kit) 2) Buy from IDTDNA - they are very good Long RNA tends to be expensive. Consider RNA ligase if two or more pieces can be stitched together. Artem On Sun, Mar 13, 2011 at 3:39 AM, Michael Thompson mi...@chem.ucla.eduwrote: Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi Mike, For long RNAs ( 40bases), in vitro transcription is the method of choice. You might want to take a look at this introductory page from Ambion: http://www.ambion.com/techlib/basics/transcription/index.html For structural studies you will have to scale up to milliliter scale. For that you might want to produce your own T7 RNA polymerase. Another thing to consider is the method of purification of your RNA. I believe purification from denaturing gel is the most widely used method, but you should consider native chromatographic purification as well. The following references should get you started on the subject: http://www.ncbi.nlm.nih.gov/pubmed/20946782 http://www.ncbi.nlm.nih.gov/pubmed/17272845 If you absolutely want to go with chemical synthesis, I believe you will have to buy shorter RNA pieces (40 bases or less) and ligate them later using a T4 RNA ligase. There are many good references out there that deal with production, purification and crystallization or RNA. Good luck, Mario Sanches On Sun, Mar 13, 2011 at 4:39 AM, Michael Thompson mi...@chem.ucla.eduwrote: Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi, I'll second Israels's comment. Since the yield per coupling in synthesis is lower for RNA than for DNA it gets really expensive over 30-35 nucleotides. However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint). Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice. For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft. See: Batey, R.T. Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389. You are going to need milligrams of T7 RNA polymerase in the end so forget about transcription kits, make your own. Cheers, Martin On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote: Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps J Mol Biol. 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SR, Ito N, Oubridge C, Avis JM, Nagai K. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
