Figure S3: Unspecific cleavage in SL4. (a) Scheme of the ... cleavage occurs in SL4. The cleavage site in SL4 is indicated by scissors. The 3'-RNA product.
A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
Supporting Information Olivier Duss, Christophe Maris, Christine von Schroetter and Frédéric H.-T. Allain
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Figures and Tables
a Chimeras for sequence-specific RNase H cleavage chim_12 5’-Gm Cm Um Gm | T G T C Um Am Um Cm Cm Gm-3’
chim_23 5’-Um Gm Um | C C T G Am Cm Cm Am Um Cm Gm Um Cm-3’
chim_34 5’-A A A A A A Cm Cm Um Gm Am Um Gm | A A T C Gm Cm Um Um Cm Cm Um-3’
chim_2 5’-Cm Cm Um Gm Am Cm Cm Am Um Cm Gm | T C C T Um Gm Am Um Gm Gm Cm Um Gm-3’
Splints for T4 DNA ligase mediated ligation splint_12 5’-C T T G A T G G C T G | T G T C T A T C C G-3’
splint_23 5’-C T G C G A T G T | C C T G A C C A T C-3’
splint_34 5’- T C A T C G T C C T G A T G | A A T C G C T T C -3’
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b
Figure S1: Overview of 2’-O-methyl-RNA/ DNA chimeras and DNA splints used for sequence-specific RNase H cleavage or splinted T4 DNA mediated ligation, respectively. (a) Sequences of the chimeras and the DNA splints. The nucleotides are colored according to the different SLs of the 72 nts target RsmZ RNA they are hybridizing to. The cleavage or ligation site on the target RNA is on the phosphodiester linkage opposite the red bar indicated on the chimera or the splint, respectively. The names of both chimeras and splints are indicating, which stemloops are involved in cleavage or ligation, respectively. The nucleotides in the chimeras are either DNA (A, C, G, T) or 2’-O-methyl-RNA (Am, Cm, Gm, Um). (b) Secondary structures of the target RsmZ RNA. Nucleotides hybridizing with the chimeras or DNA splints are highlighted. Black: RNA part hybridizing with 2’-O-methyl-RNA; grey: RNA part hybridizing with DNA. SL1: magenta, SL2: green, SL3: orange, SL4: cyan.
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29 nts
16 nts
43 nts
24 nts
18 nts
RNA fragment
5'-Terminus
3'-Terminus
29 nts 43 nts 16 nts (SL1) 24 nts (SL2) 18 nts (SL3) 14 nts (SL4)
hydroxyl phosphate hydroxyl phosphate phosphate phosphate
hydroxyl cyclic phosphate hydroxyl hydroxyl hydroxyl cyclic phosphate
14 nts
Calculated mass 9352.8 14080.6 5144.2 7831.8 5856.6 4632.8
Measured mass 9352.5 14082.0 5143.8 7833.6 5858.1 4633.8
Figure S2: Electrospray ionization mass spectra of unlabeled fragments obtained by RNase H cleavage. The RNA fragments with their corresponding ESI-MS spectrum are shown. Several lines spaced by 23 Da in most spectra are due to Na+ ions associated with the RNA. The lowest line corresponds to the Na+ ion free RNA form.
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Figure S3: Unspecific cleavage in SL4. (a) Scheme of the RNase H cleavage reactions when cleavage occurs in SL4. The cleavage site in SL4 is indicated by scissors. The 3’-RNA product (43 nts) is cleaved into a 37 nts and a 6 nts fragment, whereas the 72 nts full-length RNA is cleaved into a 66 nts and 6 nts fragment. (b) Scheme of the RNase H cleavage reaction of a construct missing SL4. The 40 nts fragment containing only SL1 and SL2 is cleaved into a 29 nts and a 11 nts fragment. The sequence of the chimera chim_2 is shown in the Figure S1. The
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unique cleavage site is indicated by scissors. (c) 16% PAGE gel showing the RNase H cleavage reaction of the construct missing SL4 (40 nts, see (b)). The cleavage reaction was complete after 1h at 37º C yielding 100% products and no unspecific cleavage or degradation. (d) Secondary structures of SL2 and SL4 are shown; the nucleotides hybridizing with the 2’-O-methyl-RNA/ DNA chimera chim_2 for RNase H cleavage are highlighted in color.
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Figure S4: Preparative scale (120 nmol) denaturing anion-exchange HPLC profiles of fragments obtained by site-specific RNase H cleavage of SL12 or SL34. (a) RNase H cleavage of SL12 (40 nts) to obtain SL1 (16 nts) and SL2 (24 nts) using only 5 % chim_12 chimera (14 nts). The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. (b) RNase H cleavage of SL34 (32 nts) to obtain SL3 (18 nts) and SL4 (14 nts) using 50 % chim_34 chimera (24 nts).
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Figure 5: Proposed scheme for fast and efficient incorporation of modified nucleotides into longer RNAs for single molecule fluorescence spectroscopy. The black fragments (A-E) are produced by one co-transcriptional ribozyme cleavage reaction followed by site-specific RNase H cleavage (3-5 days) (reaction on the top). If the fragments differ in length such that they can be separated by denaturing anion-exchange HPLC, a single four-site RNase H cleavage reaction (using 4 chimeras) should be sufficient to produce all homogenous fragments with correct termini for subsequent successful religation. As smaller scales are required for single molecule spectroscopy good separation of fragments with denaturing anion exchange HPLC should also be achieved for larger RNA fragments (>200 nts). After separation of the fragments A-E, the large fragments (A, C, E) are ligated with short synthetically produced fragments (corresponding to fragments B and D) containing a modified nucleotide (red fragments in the figure). Ligation of all five fragments in one step should be feasible when splinted ligation is used (1-2 days). Using this
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approach a RNA containing modified nucleotides at two defined positions could be obtained within 4-7 days.
Figure S6: Scheme of DNA plasmid used in this study. Black: Promoter for T7 polymerase, red: hammerhead ribozyme, which can be preceded by e.g. an MS-2 stem-loop sequence to increase the length if separation from the target RNA during denaturing HPLC is a problem, blue: target RNA, green: VS stem-loop sequence required for VS ribozyme cleavage in trans. The restriction sites are indicated by black bars.
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Method
Xu et al. 19961
Tzakos et al. 20072
Nelissen et al. 20083
Duss et al. [this paper]
Principle
Two labeled fragments are produced from the same RNA by site-specific RNase H cleavage, which allows direct cross religation with the corresponding unlabeled fragments, which are also obtained from only one transcription reaction.
Transcription of two fragments from the same plasmid: 3'-fragment is followed by a HH ribozyme connected to a linker preceding a HH ribozyme and the 5'fragment. Priming reaction with GMP leads to two labeled fragments with correct termini for ligation with the corresponding unlabeled fragments also obtained from only one transcription reaction.
Three fragments (e.g. two unlabeled and one isotopically labeled) are transcribed separately. Segmentation sites are chosen in such a way that the 3 segments fold into target or target-like structure.
One labeled and one unlabeled full-length RNA are obtained by co-transcriptional ribozyme cleavage (5’-HH and 3'-VS ribozyme). Subsequent sequence-specific RNase H cleavage(s) yield(s) homogenous fragments with correct termini for further ligation using either T4 RNA or DNA ligase.
Sequence requirements
Correct fragment termini (1)
Fragment homogeneity (2)
# of labeled transcription reactions required (3)
Multiple segmental isotope labeling possible
Yes
No
No
One
Yes
Yes
(3’ segment must start with G and requires U at penultimate position)
Yes
4 - 50 nmol
Time needed (6)
14-17d
(dependent on the 5’- sequence of full-length RNA)
(G at 5’ of RNA)
Yes
Yield (5) and dependence of yield on sequence of fragments to be ligated
No
One
No
(Non-native nucleotides can be introduced at ligation site)
No
(all segments must start with G and must be able to form target-like structure for proper ligation)
No
20/ 22 nmol
12-14d
(dependent on sequence 3’ to ligation site)
Several
Yes
(Non-native nucleotides can be introduced at ligation site)
(one per segment to be labeled)
(not feasible for >3 segments) (4)
Yes
One
Yes
< 30 nmol (2 fragments)
9-11d
60 ml transcription reaction (at a concentration of 4.5 mM for each NTP). To obtain two segmentally labeled samples, in which a different segment is labeled, 120 ml transcription reaction would be required to obtain 173 nmol of each sample. Therefore, they would obtain < 30 nmol of two segmentally isotope labeled RNAs from one 20 ml labeled transcription reaction. For our approach (Duss et al.) we differentiate the yield we obtain when producing two segmental isotope labeled RNAs, in which only one of both fragments is labeled, from the yield we obtained when producing four segmentally isotope labeled RNAs, in which only one of the four fragments is isotopically labeled. (6) The time requirement is without cloning and template plasmid production, however it includes the time needed for small scale optimization reactions required for a new system under study. If not mentioned by the authors, 1 day for each small scale optimization (transcription, RNase H cleavage or ligation) and 3-4 days for PAGE purification is assumed.
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Supplementary References
(1) (2) (3) (4) (5) (6)
Xu, J.; Lapham, J.; Crothers, D. M. Proc Natl Acad Sci U S A 1996, 93, 44. Tzakos, A. G.; Easton, L. E.; Lukavsky, P. J. Nat Protoc 2007, 2, 2139. Nelissen, F. H.; van Gammeren, A. J.; Tessari, M.; Girard, F. C.; Heus, H. A.; Wijmenga, S. S. Nucleic Acids Res 2008, 36, e89. Kim, I.; Lukavsky, P. J.; Puglisi, J. D. J Am Chem Soc 2002, 124, 9338. Ohtsuki, T.; Kawai, G.; Watanabe, K. J Biochem 1998, 124, 28. Kurata, S.; Ohtsuki, T.; Suzuki, T.; Watanabe, K. Nucleic Acids Res 2003, 31, e145.
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