- Email: [email protected]
Ribozyme mediated trans insertion-splicing of modified oligonucleotides into RNA
Archives of Biochemistry and Biophysics 478 (2008) 81–84
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y a b b i
Ribozyme mediated trans insertion-splicing of modified oligonucleotides into RNA Insertion-Splicing
P. Patrick Dotson II, Kristen N. Frommeyer, Stephen M. Testa * Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA Ke n t u ck y, 4 0 5 0 6
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 21 May 2008 and in revised form 10 July 2008 Available online 17 July 2008
The trans insertion-splicing reaction, catalyzed by a group I intron-derived from Pneumocystis carinii, was recently developed for the site-specific insertion of a segment of RNA into a separate RNA substrate. The molecular determinants of this reaction for binding and catalysis are reasonably well understood, making them easily and highly modifiable for altering substrate specificity. To demonstrate proof-of-con cept, we now report that the P. carinii ribozyme can except modified oligonucleotides as substrates for catalyzing the trans insertion-splicing reaction. Oligonucleotides that contain one or more sugar modifi cations (deoxy or methoxy substitution), a backbone modification (phosphorothioate substitution), or a base modification (2-aminopurine or 4-thiouridine) are effective substrates in this reaction. Apparently, trans insertion-splicing is a unique and viable reaction for the site-specific incorporation of modified oligonucleotides into RNAs. This is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of a modified oligonucleotide into RNA. © 2008 Elsevier Inc. All rights reserved. P n e u m o c yst i s
Keywords: Ribozyme RNA modifications Trans insertion-splicing
The trans insertion-splicing (TIS)1 reaction (Fig. 1) was recently developed to site-specifically insert a segment of RNA into a cen tral segment of a different RNA [1]. The TIS reaction, catalyzed by a group I intron-derived ribozyme from the fungal pathogen Pneumo cystis carinii (P. carinii), utilizes two RNA substrates and has been proposed [1] to proceed through three concerted chemical steps (Fig. 2). These include two consecutive cleavage steps followed by a single ligation step. Furthermore, the P. carinii ribozyme appears to utilize the same molecular recognition components for orient ing their substrates and intermediates as seen for other ribozymemediated reactions [2,3]. The molecular determinants of the TIS reaction for binding and catalysis are reasonably well understood, making them easily and highly modifiable for altering substrate specificity. Moreover, the reaction mechanism appears to be such that certain positions within the insertion substrate could be chem ically altered without undo consequence to the fidelity and effec tiveness of the overall insertion reaction. If true, this would indicate that the TIS reaction could be exploited for the sequence-specific insertion of small, chemically modified synthetic substrates into RNAs. To demonstrate proof-of-concept, we now report that oligonu cleotides that contain one or more sugar modifications (deoxy (TIS)
* Corresponding author. Fax: +1 859 323 1069. E-mail address: [email protected] (S.M. Testa). 1 Abbreviations used: TIS, trans insertion-splicing; TES, trans excision-splicing; RE1, recognition element 1; RE2, recognition element 2; RE3, recognition element 3; GBS, guanosine-binding site; HEPES, N-(2-hydroxylethyl) piperazine-N9-2-eth anesulfonic acid. 0003-9861/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.07.010
or methoxy substitution), a backbone modification (phosphoro thioate substitution), or a base modification (2-aminopurine or 4-thiouridine) are effective substrates in the TIS reaction. All five modified oligonucleotides in this report were shown to be inserted sequence-specifically and effectively into their intended target RNAs. Furthermore, it appears that certain positions within the insertion substrate can be chemically altered without undo conse quence to the fidelity and effectiveness of the overall TIS reaction. Lastly, this is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of modified oligonucleo tides into RNA. Materials and methods M et h o d s
Oligonucleotide synthesis and preparation RNA oligonucleotides (Table 1) were purchased from Dharma con Research, Inc. (Lafayette, CO) and were deprotected using the manufacturer’s recommended protocol. Nucleic acid concentra tions were calculated from UV absorption measurements using a Beckman DU 650 spectrophotometer (Beckman Coulter, Inc.; Fuller ton, CA). Oligonucleotides were 59-end radiolabeled with [c-32P] ATP (Amersham Pharmacia Biotech; Piscataway, NJ) as previously described [4]. 5’-end
Ribozyme synthesis Template plasmid [4] was linearized in a 50 lL reaction consist ing of 16 lg of PC plasmid, 50 units XbaXbaI (Invitrogen; Grand I
82
P.P. Dotson II et al. / Archives of Biochemistry and Biophysics 478 (2008) 81–84
mize the binding and subsequent reactivity of the tri-component system during the original proof-of-concept report [1]. Therefore, the same ribozyme and substrate sequences were used in this report, except for the nucleotide analog substitutions at positions 6–8 (see Figure S1 in Supplementary material ) within the donor substrate (see Table 1). These positions were chosen because they are expected to have little, if any, structural or functional role in the TIS reaction, and so their substitution is least likely to have a deleterious effect on the reaction (Fig. 2). To determine the gen erality of the method, nucleotides that contain sugar, phosphate, and nucleobase modifications were analyzed. These include deoxy ribose (deoxycytosine, dC or deoxyguanosine, dG), methoxyribose (mU), phosphorothioate (SH), 2-aminopurine (2AP), and 4-thiouri dine (4SU) substitutions (Table 1). Note that for each of the donor substrates, the same sized TIS product is expected to form, which is the same size as that with the previously sequenced unmodified substrate [1].
Trans Insertion-Splicing TIS Acceptor Substrate
TIS Donor Substrate
6-8
M a te r i a l
5’ Exon
3’ Exon
5’ Exon
Insert
Ribozyme
TIS Product 5’ Exon
3’ Exon
Insert
+ 5’ Exon Fig. 1. The trans insertion-splicing reaction. The ribozyme (not shown) catalyzes the insertion of a portion of the donor substrate into a central region of the accep tor substrate.
Island, NY), and REACT 2 buffer at 37 °C for 2 h. The resulting plasmid was purified using the QIAquick PCR purification kit (Qiagen; Valencia, CA) using the manufacturers recommended pro tocol. Run-off transcription was performed for 2 h in a 50 lL reac tion consisting of 1 lg linearized DNA template, 50 units of T7 RNA polymerase (New England Biolabs; Beverly, MA), 40 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 5 mM DTT, 5 mM spermidine, and 1 mM rNTPs. The P. carinii (rPC) ribozyme was subsequently purified using a Qiagen Plasmid Midi Kit (Qiagen), as previously described [5]. Ribozyme concentrations were calculated from UV absorption measurements using a Beckman DU 650 spectrophotometer (Beck man Coulter, Inc). Tr i s - H C l
P n e u m o c yst i s
TIS insertion of modified oligonucleotides I n s e r t i o n
M o di f i e d O l i g o n u c l eot i d e s
TIS reactions were conducted as previously described [1], under previously optimized reaction conditions (see Fig. 3 legend). Sub strates, intermediates, and products of each of the individual TIS reactions were separated and visualized via polyacrylamide gel electrophoresis. The initial reactions focused on modifications only at position 7, as this was expected to be least disruptive to the reaction. As shown in Fig. 3, each of the four substrates modified at position 7 resulted in the expected 18-mer product band (lanes F–J). Note that the relatively small size differences between the TIS products are due to different modifications within the substrates. Apparently, position 7 of the donor substrate is readily modifiable; resulting in the effective insertion of modified RNAs into other RNAs (see reaction yields in Table 2). At position 7, the deoxy (Fig. 3, lane G) and phosphothioate ( Fig. 3, lane H) substitutions were essentially as effective as the non-modified substrate ( Fig. 3, lane F). Lower, although comparable, yields (Table 2) were obtained for the two nucleobase modifications (Fig. 3, lanes I and J). This result suggests that base modifications do hinder TIS product formation, most probably through interrupting structural elements within the rPC ribozyme utilized during the course of the reaction. It was then tested whether more than one modification could be added to the donor substrate. We simultaneously tested whether positions 6 and 8 could be modified (in addition to position 7). In addition different modifications were chosen to broaden the potential applicability of the method (Table 1). As shown in Fig. 3 (lane K), the doubly-modified donor substrate is as effective a TIS substrate as its non-modified counterpart. Apparently, the 29 position of the ribose at positions 6 and 8 (in addition to position 7) of the donor substrate are sites that can be readily modified. This result shows that the TIS reaction is an effective strategy for the insertion of multiple modifications within an RNA. Taken together, these results demonstrate that the TIS reaction can be exploited for inserting non-native, chemically modified oligonucle otides into RNAs in trans. (Lanes F-J).
[ Fi g u re 4 ,
G]
[
H]
[
TIS reactions TIS reactions were conducted using the rPC ribozyme under previously optimized reaction conditions [1]. Briefly, 240 nM rPC in H10Mg buffer (50 mM HEPES (25 mM Na+). About 135 mM KCl, and 10 mM MgCl2 at pH 7.5) was pre-incubated at 60 °C for 5 min in a reaction volume of 5.0 lL. The reaction was then slow cooled to 44 °C. Separately, 6 nM 59-end radiolabeled acceptor substrate and 30 lM donor substrate was pre-incubated in H10Mg at 44 °C. The reaction was initiated by the addition of 1.0 lL of the substrate solution to the 5.0 lL ribozyme solution. Final nucleic acid con centrations were 200 nM rPC ribozyme, 1.0 lM donor substrate, and 1.0 nM acceptor substrate. Reactions were incubated for 2 h at 44 °C, at which time the reaction was terminated by addition of an equal volume (6 lL) of a 2£ stop buffer (10 M urea, 3 mM EDTA, and 0.1£ TBE). The reaction mixture was denatured for 1 min at 90 °C and separated on a 12% polyacrylamide/8 M urea gel. The gel was transferred to chromatography paper and dried under vacuum. The bands were visualized and quantified on a Molecular Dynamics Storm 860 Phosphorimager. 5’-end
2X
0 .1 X
(Lane
F].
d o u b ly m o di f i e d
2’
Future directions D i re c t i o n s
Results and discussion D is c u s s i o n
Design of TIS model test system M o d e l Te s t S ys te m
We have previously shown that a truncated group I intron from P. carinii could catalyze the insertion reaction shown in Fig. 2 [1]. For these previous studies, the sequence of the intron was kept intact, albeit truncated, and the donor and acceptor substrates closely mimicked the intron’s native 59 and 39 exon sequences. Maintaining these sequences were important in order to maxi 5’
wa s
3’
Modified RNAs are routinely utilized in a number of experimen tal applications, including those that utilize fluorescent probes, cross linking agents, affinity tags, and a myriad of functional group substi tutions [6–8]. It is envisioned that the TIS reaction could be exploited for the synthesis of large, site-specifically modified RNAs. This could be accomplished using TIS to insert a small, chemically synthesized RNA (acting as the donor molecule, modified at a position correspond ing to positions 6, 7, or 8 in Fig. 2) into a full length RNA transcript (acting as the acceptor substrate). Note that in terms of ribozyme
P.P. Dotson II et al. / Archives of Biochemistry and Biophysics 478 (2008) 81–84
5’
g1 c2 u3 c4 u5 c6 g7 u8 g9
G (RE3) A G G G U (RE1) C A U
TIS Donor Substrate (9-mer) Binding
5’
G P10 A (RE3) G G P1i G (RE1) U C A U
g9 u8 g7 c6 u5 c4 u3 c2 g1
3’
G336 U (RE2) A
3’
G (RE3) A G G G P1 U (RE1) C A U
Nucleophilic attack by 3’ terminal guanosine
c G u c g U P9.0 u A (RE2) g 3’
u 3’ a c a a a u c a g u a
g1 c2 u3
Nucleophilic attack by 3’ terminal guanosine
Second Step (5’ Cleavage Reaction) u 3’ a c a a a g u g c u c
5’
5’
c G u c U P9.0 g u A (RE2) g 3’
5’
5’
Dissociation
G (RE3) A G G G U (RE1) C A U
5’
5’
5’
5’
Binding
Ribozyme
u c g
5’ augacuaaacau3’ TIS Acceptor Substrate (12-mer)
3’
First Step (5’ Cleavage Reaction)
G (RE3) A G G P1i G (RE1) U C A U
c G u c g U P9.0 u A (RE2) g 3’
5’
3’
G P10 A (RE3) G G G P1 U (RE1) C A U
3’
c G u c g U P9.0 u A (RE2) g 3’
83
u c a g u a
U (RE2) A
G
5’
Third Step (Exon Ligation)
Nucleophilic attack by terminal uridine
5’
a u g a c u c4 u5 c6 g7 u8 g9 a a a c a u
3’
TIS product (18-mer) Fig. 2. The recognition elements of the rPC ribozyme (RE1, RE2, and RE3 which form the P1, P9.0, and P10 helices, respectively) are designated with black uppercase lettering, and the rest of the ribozyme is shown as a simple black line. The 9-mer TIS donor substrate is shown in lowercase lettering with a gray background, except for the 39 terminal guanosine (called xGi), which has white lettering with a black background. The 12-mer TIS acceptor substrate is shown in black lowercase lettering. The TIS donor substrate forms the P1i and P10 helices by base pairing with RE1 and RE3 of the ribozyme. Note that for simplicity, the TIS donor and substrate sequences are shown in uppercase lettering throughout the text. In the proposed TIS mechanism, the 39 terminal guanosine (G336) of the ribozyme (in bold) performs a nucleophilic attack upon the 59 splice site of the TIS donor substrate, resulting in the covalent attachment of the insert region of the donor substrate to the 39-end of the ribozyme. The 59-half of the TIS donor sub strate then dissociates from the ribozyme and the TIS acceptor substrate binds through a second P1 helix interaction. The xGi (aligned via P9.0 helix formation) then takes part in a nucleophilic attack upon the 59 splice site of the acceptor substrate, resulting in the covalent attachment of the 39-half of the acceptor substrate to the 39-end of the ribozyme. Exon ligation then proceeds via nucleophilic attack of the uridine at the 59-half of the TIS substrate performing a nucleophilic attack upon G336 of the ribozyme, resulting in the final TIS product.
design, all of the ribozyme and substrate sequences that make up the molecular recognition elements can be altered as desired to create
appropriate target-substrate combinations, as long as the substrateribozyme base pairs shown in Fig. 2 are maintained.
84
P.P. Dotson II et al. / Archives of Biochemistry and Biophysics 478 (2008) 81–84 Table 2 Percent TIS product formation for individual modified oligonucleotides
18-mer
( 5’AUGACUCUCGUGAAACAU3’)
12-mer
( 5’AUGACUAAACAU3’)
6-mer
( 5’AUGACU3’)
A
B
C
D E F G
(Size Controls)
(-)
H
I
J
K
(-) (N) (dG) (SH) (2AP) (4SU) (DM)
Fig. 3. Polyacrylamide gel showing substrates, intermediates, and products of the trans insertion-splicing reaction. Reactions were conducted with 200 nM ribozyme, 1 nM acceptor substrate, and 1 lM donor substrate in H10Mg at 44 °C for 2 h. Lanes A, B, and C contain 59-end radiolabeled 18-mer, 12-mer, and 6-mer size controls, respectively. Negative control lanes (-) consist of reactions run in H0Mg buffer (lane D) or without rPC ribozyme (lane E). Lane F contains the TIS reaction with the unmodified donor substrate (N), and serves as a positive control. Lanes G, H, I, J, and K contain the TIS reaction with deoxy (dG), phosphothioate (SH), 2-ami nopurine (2AP), 4-thiouridine (4SU), and doubly-modified (DM) substituted donor substrates, respectively.
Table 1 TIS starting material and insert substrate sequences
a
Predicted TIS product (18-mer) for TIS reactions conducted with the TIS acceptor substrate (12-mer) and either unmodified or modified TIS donor substrates. The sequences highlighted in gray represent the insert region of the TIS donor mole cule. TIS reactions were conducted under previously optimized reaction conditions (200 nM ribozyme, 10 mM MgCl2, 1 lM insert at 44 °C for 2 h). The results are the average of two independent experiments.
TIS reaction. In contrast, nucleobase modifications at position 7 do reduce TIS yields, although not by prohibitive amounts (approximately 15% reduction). This reduction is likely due to a disruption of the P9.0 interaction between this nucleobase and the ribozyme during the second TIS reaction step (see Fig. 2). Nevertheless, the TIS reaction is relatively effective for synthe sizing RNAs that contain sugar-phosphate backbone and nucle obase modifications. Like that at position 7, ribose modifications at donor substrate positions 6 and 8 also do not reduce TIS yields. Apparently, the 29 OH groups at positions 6, 7, and 8 are expendable for the TIS reac tion, which enhances the flexibility of using TIS as a synthetic tool. In addition, that the TIS reaction conducted with the doubly-modi fied donor substrate was successful demonstrates that the TIS reac tion is effective for the insertion of multiple modified nucleotides within a given RNA sequence. 2’
Acknowledgments The sequences for the TIS acceptor substrate (12-mer), normal (unmodified) TIS donor substrate (9-mer), and modified TIS donor substrates (9-mer) are shown for the standard TES reaction. For both the unmodified and modified TIS donor sub strates the sequence to be inserted is highlighted in gray. For each modified TIS donor substrate the modified nucleotide (located at either position C6, G7, or U8) is shown in parentheses. For the doubly-modified TIS donor substrate, the substrate contains a deoxycytosine (dC) and methoxyuridine (mU) modification.
This research was supported by grants from the Kentucky Lung Cancer Research Program and The Lexington Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.abb.2008.07.010.
Conclusion In this report, we demonstrate that the trans insertionsplicing ribozyme from P. carinii can utilize substrates that contain functional group modifications, and that this activity can be exploited to generate RNAs with one or more internal modifications. This is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of a mod ified oligonucleotide into RNA. We also show that functional group modifications within the sugar-phosphate backbone at position 7 of the donor substrate (see Fig. 2) do not hinder the
References
Pneumocystis
sugar phosphate
[1] A.J. Johnson, J. Sinha, S.M. Testa, Biochemistry 44 (2005) 10702–10710. [2] P.P. Dotson II, S.M. Testa, Recent Dev. Nucleic Acids Res. 2 (2006) 307–324. [3] T.R. Cech, Ann. Rev. Biochem. 59 (1990) 543–568. [4] S.M. Testa, C.G. Haidaris, F. Gigliotti, D.H. Turner, Biochemistry 36 (1997) 15303– 15314. [5] M.A. Bell, A.J. Johnson, S.M. Testa, Biochemistry 41 (2002) 15327–15333. [6] J.A. Grasby, M.J. Gait, Biochimie 76 (1994) 1223–1234. [7] S. Verma, F. Eckstein, Ann. Rev. Biochem. 67 (1998) 99–134. [8] S.R. Das, R. Fong, J.A. Piccirilli, Curr. Opin. Chem. Biol. 9 (2005) 1–9.
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y a b b i
Ribozyme mediated trans insertion-splicing of modified oligonucleotides into RNA Insertion-Splicing
P. Patrick Dotson II, Kristen N. Frommeyer, Stephen M. Testa * Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA Ke n t u ck y, 4 0 5 0 6
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 21 May 2008 and in revised form 10 July 2008 Available online 17 July 2008
The trans insertion-splicing reaction, catalyzed by a group I intron-derived from Pneumocystis carinii, was recently developed for the site-specific insertion of a segment of RNA into a separate RNA substrate. The molecular determinants of this reaction for binding and catalysis are reasonably well understood, making them easily and highly modifiable for altering substrate specificity. To demonstrate proof-of-con cept, we now report that the P. carinii ribozyme can except modified oligonucleotides as substrates for catalyzing the trans insertion-splicing reaction. Oligonucleotides that contain one or more sugar modifi cations (deoxy or methoxy substitution), a backbone modification (phosphorothioate substitution), or a base modification (2-aminopurine or 4-thiouridine) are effective substrates in this reaction. Apparently, trans insertion-splicing is a unique and viable reaction for the site-specific incorporation of modified oligonucleotides into RNAs. This is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of a modified oligonucleotide into RNA. © 2008 Elsevier Inc. All rights reserved. P n e u m o c yst i s
Keywords: Ribozyme RNA modifications Trans insertion-splicing
The trans insertion-splicing (TIS)1 reaction (Fig. 1) was recently developed to site-specifically insert a segment of RNA into a cen tral segment of a different RNA [1]. The TIS reaction, catalyzed by a group I intron-derived ribozyme from the fungal pathogen Pneumo cystis carinii (P. carinii), utilizes two RNA substrates and has been proposed [1] to proceed through three concerted chemical steps (Fig. 2). These include two consecutive cleavage steps followed by a single ligation step. Furthermore, the P. carinii ribozyme appears to utilize the same molecular recognition components for orient ing their substrates and intermediates as seen for other ribozymemediated reactions [2,3]. The molecular determinants of the TIS reaction for binding and catalysis are reasonably well understood, making them easily and highly modifiable for altering substrate specificity. Moreover, the reaction mechanism appears to be such that certain positions within the insertion substrate could be chem ically altered without undo consequence to the fidelity and effec tiveness of the overall insertion reaction. If true, this would indicate that the TIS reaction could be exploited for the sequence-specific insertion of small, chemically modified synthetic substrates into RNAs. To demonstrate proof-of-concept, we now report that oligonu cleotides that contain one or more sugar modifications (deoxy (TIS)
* Corresponding author. Fax: +1 859 323 1069. E-mail address: [email protected] (S.M. Testa). 1 Abbreviations used: TIS, trans insertion-splicing; TES, trans excision-splicing; RE1, recognition element 1; RE2, recognition element 2; RE3, recognition element 3; GBS, guanosine-binding site; HEPES, N-(2-hydroxylethyl) piperazine-N9-2-eth anesulfonic acid. 0003-9861/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.07.010
or methoxy substitution), a backbone modification (phosphoro thioate substitution), or a base modification (2-aminopurine or 4-thiouridine) are effective substrates in the TIS reaction. All five modified oligonucleotides in this report were shown to be inserted sequence-specifically and effectively into their intended target RNAs. Furthermore, it appears that certain positions within the insertion substrate can be chemically altered without undo conse quence to the fidelity and effectiveness of the overall TIS reaction. Lastly, this is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of modified oligonucleo tides into RNA. Materials and methods M et h o d s
Oligonucleotide synthesis and preparation RNA oligonucleotides (Table 1) were purchased from Dharma con Research, Inc. (Lafayette, CO) and were deprotected using the manufacturer’s recommended protocol. Nucleic acid concentra tions were calculated from UV absorption measurements using a Beckman DU 650 spectrophotometer (Beckman Coulter, Inc.; Fuller ton, CA). Oligonucleotides were 59-end radiolabeled with [c-32P] ATP (Amersham Pharmacia Biotech; Piscataway, NJ) as previously described [4]. 5’-end
Ribozyme synthesis Template plasmid [4] was linearized in a 50 lL reaction consist ing of 16 lg of PC plasmid, 50 units XbaXbaI (Invitrogen; Grand I
82
P.P. Dotson II et al. / Archives of Biochemistry and Biophysics 478 (2008) 81–84
mize the binding and subsequent reactivity of the tri-component system during the original proof-of-concept report [1]. Therefore, the same ribozyme and substrate sequences were used in this report, except for the nucleotide analog substitutions at positions 6–8 (see Figure S1 in Supplementary material ) within the donor substrate (see Table 1). These positions were chosen because they are expected to have little, if any, structural or functional role in the TIS reaction, and so their substitution is least likely to have a deleterious effect on the reaction (Fig. 2). To determine the gen erality of the method, nucleotides that contain sugar, phosphate, and nucleobase modifications were analyzed. These include deoxy ribose (deoxycytosine, dC or deoxyguanosine, dG), methoxyribose (mU), phosphorothioate (SH), 2-aminopurine (2AP), and 4-thiouri dine (4SU) substitutions (Table 1). Note that for each of the donor substrates, the same sized TIS product is expected to form, which is the same size as that with the previously sequenced unmodified substrate [1].
Trans Insertion-Splicing TIS Acceptor Substrate
TIS Donor Substrate
6-8
M a te r i a l
5’ Exon
3’ Exon
5’ Exon
Insert
Ribozyme
TIS Product 5’ Exon
3’ Exon
Insert
+ 5’ Exon Fig. 1. The trans insertion-splicing reaction. The ribozyme (not shown) catalyzes the insertion of a portion of the donor substrate into a central region of the accep tor substrate.
Island, NY), and REACT 2 buffer at 37 °C for 2 h. The resulting plasmid was purified using the QIAquick PCR purification kit (Qiagen; Valencia, CA) using the manufacturers recommended pro tocol. Run-off transcription was performed for 2 h in a 50 lL reac tion consisting of 1 lg linearized DNA template, 50 units of T7 RNA polymerase (New England Biolabs; Beverly, MA), 40 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 5 mM DTT, 5 mM spermidine, and 1 mM rNTPs. The P. carinii (rPC) ribozyme was subsequently purified using a Qiagen Plasmid Midi Kit (Qiagen), as previously described [5]. Ribozyme concentrations were calculated from UV absorption measurements using a Beckman DU 650 spectrophotometer (Beck man Coulter, Inc). Tr i s - H C l
P n e u m o c yst i s
TIS insertion of modified oligonucleotides I n s e r t i o n
M o di f i e d O l i g o n u c l eot i d e s
TIS reactions were conducted as previously described [1], under previously optimized reaction conditions (see Fig. 3 legend). Sub strates, intermediates, and products of each of the individual TIS reactions were separated and visualized via polyacrylamide gel electrophoresis. The initial reactions focused on modifications only at position 7, as this was expected to be least disruptive to the reaction. As shown in Fig. 3, each of the four substrates modified at position 7 resulted in the expected 18-mer product band (lanes F–J). Note that the relatively small size differences between the TIS products are due to different modifications within the substrates. Apparently, position 7 of the donor substrate is readily modifiable; resulting in the effective insertion of modified RNAs into other RNAs (see reaction yields in Table 2). At position 7, the deoxy (Fig. 3, lane G) and phosphothioate ( Fig. 3, lane H) substitutions were essentially as effective as the non-modified substrate ( Fig. 3, lane F). Lower, although comparable, yields (Table 2) were obtained for the two nucleobase modifications (Fig. 3, lanes I and J). This result suggests that base modifications do hinder TIS product formation, most probably through interrupting structural elements within the rPC ribozyme utilized during the course of the reaction. It was then tested whether more than one modification could be added to the donor substrate. We simultaneously tested whether positions 6 and 8 could be modified (in addition to position 7). In addition different modifications were chosen to broaden the potential applicability of the method (Table 1). As shown in Fig. 3 (lane K), the doubly-modified donor substrate is as effective a TIS substrate as its non-modified counterpart. Apparently, the 29 position of the ribose at positions 6 and 8 (in addition to position 7) of the donor substrate are sites that can be readily modified. This result shows that the TIS reaction is an effective strategy for the insertion of multiple modifications within an RNA. Taken together, these results demonstrate that the TIS reaction can be exploited for inserting non-native, chemically modified oligonucle otides into RNAs in trans. (Lanes F-J).
[ Fi g u re 4 ,
G]
[
H]
[
TIS reactions TIS reactions were conducted using the rPC ribozyme under previously optimized reaction conditions [1]. Briefly, 240 nM rPC in H10Mg buffer (50 mM HEPES (25 mM Na+). About 135 mM KCl, and 10 mM MgCl2 at pH 7.5) was pre-incubated at 60 °C for 5 min in a reaction volume of 5.0 lL. The reaction was then slow cooled to 44 °C. Separately, 6 nM 59-end radiolabeled acceptor substrate and 30 lM donor substrate was pre-incubated in H10Mg at 44 °C. The reaction was initiated by the addition of 1.0 lL of the substrate solution to the 5.0 lL ribozyme solution. Final nucleic acid con centrations were 200 nM rPC ribozyme, 1.0 lM donor substrate, and 1.0 nM acceptor substrate. Reactions were incubated for 2 h at 44 °C, at which time the reaction was terminated by addition of an equal volume (6 lL) of a 2£ stop buffer (10 M urea, 3 mM EDTA, and 0.1£ TBE). The reaction mixture was denatured for 1 min at 90 °C and separated on a 12% polyacrylamide/8 M urea gel. The gel was transferred to chromatography paper and dried under vacuum. The bands were visualized and quantified on a Molecular Dynamics Storm 860 Phosphorimager. 5’-end
2X
0 .1 X
(Lane
F].
d o u b ly m o di f i e d
2’
Future directions D i re c t i o n s
Results and discussion D is c u s s i o n
Design of TIS model test system M o d e l Te s t S ys te m
We have previously shown that a truncated group I intron from P. carinii could catalyze the insertion reaction shown in Fig. 2 [1]. For these previous studies, the sequence of the intron was kept intact, albeit truncated, and the donor and acceptor substrates closely mimicked the intron’s native 59 and 39 exon sequences. Maintaining these sequences were important in order to maxi 5’
wa s
3’
Modified RNAs are routinely utilized in a number of experimen tal applications, including those that utilize fluorescent probes, cross linking agents, affinity tags, and a myriad of functional group substi tutions [6–8]. It is envisioned that the TIS reaction could be exploited for the synthesis of large, site-specifically modified RNAs. This could be accomplished using TIS to insert a small, chemically synthesized RNA (acting as the donor molecule, modified at a position correspond ing to positions 6, 7, or 8 in Fig. 2) into a full length RNA transcript (acting as the acceptor substrate). Note that in terms of ribozyme
P.P. Dotson II et al. / Archives of Biochemistry and Biophysics 478 (2008) 81–84
5’
g1 c2 u3 c4 u5 c6 g7 u8 g9
G (RE3) A G G G U (RE1) C A U
TIS Donor Substrate (9-mer) Binding
5’
G P10 A (RE3) G G P1i G (RE1) U C A U
g9 u8 g7 c6 u5 c4 u3 c2 g1
3’
G336 U (RE2) A
3’
G (RE3) A G G G P1 U (RE1) C A U
Nucleophilic attack by 3’ terminal guanosine
c G u c g U P9.0 u A (RE2) g 3’
u 3’ a c a a a u c a g u a
g1 c2 u3
Nucleophilic attack by 3’ terminal guanosine
Second Step (5’ Cleavage Reaction) u 3’ a c a a a g u g c u c
5’
5’
c G u c U P9.0 g u A (RE2) g 3’
5’
5’
Dissociation
G (RE3) A G G G U (RE1) C A U
5’
5’
5’
5’
Binding
Ribozyme
u c g
5’ augacuaaacau3’ TIS Acceptor Substrate (12-mer)
3’
First Step (5’ Cleavage Reaction)
G (RE3) A G G P1i G (RE1) U C A U
c G u c g U P9.0 u A (RE2) g 3’
5’
3’
G P10 A (RE3) G G G P1 U (RE1) C A U
3’
c G u c g U P9.0 u A (RE2) g 3’
83
u c a g u a
U (RE2) A
G
5’
Third Step (Exon Ligation)
Nucleophilic attack by terminal uridine
5’
a u g a c u c4 u5 c6 g7 u8 g9 a a a c a u
3’
TIS product (18-mer) Fig. 2. The recognition elements of the rPC ribozyme (RE1, RE2, and RE3 which form the P1, P9.0, and P10 helices, respectively) are designated with black uppercase lettering, and the rest of the ribozyme is shown as a simple black line. The 9-mer TIS donor substrate is shown in lowercase lettering with a gray background, except for the 39 terminal guanosine (called xGi), which has white lettering with a black background. The 12-mer TIS acceptor substrate is shown in black lowercase lettering. The TIS donor substrate forms the P1i and P10 helices by base pairing with RE1 and RE3 of the ribozyme. Note that for simplicity, the TIS donor and substrate sequences are shown in uppercase lettering throughout the text. In the proposed TIS mechanism, the 39 terminal guanosine (G336) of the ribozyme (in bold) performs a nucleophilic attack upon the 59 splice site of the TIS donor substrate, resulting in the covalent attachment of the insert region of the donor substrate to the 39-end of the ribozyme. The 59-half of the TIS donor sub strate then dissociates from the ribozyme and the TIS acceptor substrate binds through a second P1 helix interaction. The xGi (aligned via P9.0 helix formation) then takes part in a nucleophilic attack upon the 59 splice site of the acceptor substrate, resulting in the covalent attachment of the 39-half of the acceptor substrate to the 39-end of the ribozyme. Exon ligation then proceeds via nucleophilic attack of the uridine at the 59-half of the TIS substrate performing a nucleophilic attack upon G336 of the ribozyme, resulting in the final TIS product.
design, all of the ribozyme and substrate sequences that make up the molecular recognition elements can be altered as desired to create
appropriate target-substrate combinations, as long as the substrateribozyme base pairs shown in Fig. 2 are maintained.
84
P.P. Dotson II et al. / Archives of Biochemistry and Biophysics 478 (2008) 81–84 Table 2 Percent TIS product formation for individual modified oligonucleotides
18-mer
( 5’AUGACUCUCGUGAAACAU3’)
12-mer
( 5’AUGACUAAACAU3’)
6-mer
( 5’AUGACU3’)
A
B
C
D E F G
(Size Controls)
(-)
H
I
J
K
(-) (N) (dG) (SH) (2AP) (4SU) (DM)
Fig. 3. Polyacrylamide gel showing substrates, intermediates, and products of the trans insertion-splicing reaction. Reactions were conducted with 200 nM ribozyme, 1 nM acceptor substrate, and 1 lM donor substrate in H10Mg at 44 °C for 2 h. Lanes A, B, and C contain 59-end radiolabeled 18-mer, 12-mer, and 6-mer size controls, respectively. Negative control lanes (-) consist of reactions run in H0Mg buffer (lane D) or without rPC ribozyme (lane E). Lane F contains the TIS reaction with the unmodified donor substrate (N), and serves as a positive control. Lanes G, H, I, J, and K contain the TIS reaction with deoxy (dG), phosphothioate (SH), 2-ami nopurine (2AP), 4-thiouridine (4SU), and doubly-modified (DM) substituted donor substrates, respectively.
Table 1 TIS starting material and insert substrate sequences
a
Predicted TIS product (18-mer) for TIS reactions conducted with the TIS acceptor substrate (12-mer) and either unmodified or modified TIS donor substrates. The sequences highlighted in gray represent the insert region of the TIS donor mole cule. TIS reactions were conducted under previously optimized reaction conditions (200 nM ribozyme, 10 mM MgCl2, 1 lM insert at 44 °C for 2 h). The results are the average of two independent experiments.
TIS reaction. In contrast, nucleobase modifications at position 7 do reduce TIS yields, although not by prohibitive amounts (approximately 15% reduction). This reduction is likely due to a disruption of the P9.0 interaction between this nucleobase and the ribozyme during the second TIS reaction step (see Fig. 2). Nevertheless, the TIS reaction is relatively effective for synthe sizing RNAs that contain sugar-phosphate backbone and nucle obase modifications. Like that at position 7, ribose modifications at donor substrate positions 6 and 8 also do not reduce TIS yields. Apparently, the 29 OH groups at positions 6, 7, and 8 are expendable for the TIS reac tion, which enhances the flexibility of using TIS as a synthetic tool. In addition, that the TIS reaction conducted with the doubly-modi fied donor substrate was successful demonstrates that the TIS reac tion is effective for the insertion of multiple modified nucleotides within a given RNA sequence. 2’
Acknowledgments The sequences for the TIS acceptor substrate (12-mer), normal (unmodified) TIS donor substrate (9-mer), and modified TIS donor substrates (9-mer) are shown for the standard TES reaction. For both the unmodified and modified TIS donor sub strates the sequence to be inserted is highlighted in gray. For each modified TIS donor substrate the modified nucleotide (located at either position C6, G7, or U8) is shown in parentheses. For the doubly-modified TIS donor substrate, the substrate contains a deoxycytosine (dC) and methoxyuridine (mU) modification.
This research was supported by grants from the Kentucky Lung Cancer Research Program and The Lexington Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.abb.2008.07.010.
Conclusion In this report, we demonstrate that the trans insertionsplicing ribozyme from P. carinii can utilize substrates that contain functional group modifications, and that this activity can be exploited to generate RNAs with one or more internal modifications. This is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of a mod ified oligonucleotide into RNA. We also show that functional group modifications within the sugar-phosphate backbone at position 7 of the donor substrate (see Fig. 2) do not hinder the
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