Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after
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《核酸研究医学期刊》
Institute for Medical Physics and Biophysics and 1 Institute for Biochemistry, University of Münster, Münster, Germany and 2 Department of Chemistry, Washington University, St Louis, MO 63130, USA
* To whom correspondence should be addressed. Tel: +49 251 83 55103; Fax: +49 251 83 55121; Email: hillenk@uni-muenster.de
ABSTRACT
Several DNA/RNA sequencing strategies have been developed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In the reverse Sanger sequencing approach -thiophosphate-containing NTPs are employed. Sequencing ladders are produced by the subsequent exonuclease cleavage, which is inhibited by the -S-NTP at the 3' terminus. Here the reverse Sanger sequencing of RNA is described. The stability of RNA during the UV-MALDI process is higher relative to DNA, and RNA can be easily synthesized by transcription using bacteriophage RNA polymerase. -S-rNTP was added to the reaction in a ratio of 1:3 to the native rNTPs and was incorporated statistically by the RNA polymerase. Four separate sequence ladders were produced, to avoid the problem of the only 1u mass difference between uridine and cytidine. However, it was shown that RNA transcription does not produce homogeneous transcripts. Therefore isolation of the full-length transcript is required to attain a non-ambiguous interpretation of cleavage spectra. This is achieved by the exclusive immobilization of the full-length transcript on a solid phase. The full-length transcripts were hybridized to magnetic beads, coated with short universal sequences, complementary to the in vitro RNA. After purification and isolation the RNA full-length transcript is cleaved by snake venom phosphodiesterase (SVP) and the obtained sequence ladder is analyzed by MALDI-MS.
INTRODUCTION
With the draft sequence of the human genome in place (1), new genomic applications are expected in clinical diagnostics and forensics which require high-throughput analysis of short nucleic acids sequences. Mass spectrometry (MS) offers advantages over gel electrophoretic separation and various forms of hybridization, particularly for automated, rapid and large-scale DNA/RNA screening. The use of electrophoretic mobility or fluorescence of a label are only indirect measures, but mass is an intrinsic property of the molecule. As a result, mass spectrometric assays are intrinsically more accurate. The development of the ‘soft’ ionization technique, matrix-assisted laser desorption/ionization (MALDI) (2), in the late 1980s enabled the analysis of large biomolecules (3) and has been applied to the analysis of DNA and RNA sequencing (4,5). However, base losses and metastable backbone fragmentation (6) as well as a dramatic decrease of ion yield with increasing fragment length limit the accessible mass range for DNA analysis to essentially less than 15 kDa (7,8) This problem is especially marked for Sanger sequencing ladders because of the intrinsic bias of ladder production to yield higher concentrations of shorter termination products. This adds to the suppression of longer termination products by MALDI-MS (9). Nonetheless, application of MALDI-MS for the sequencing by chain termination principle for RNA was demonstrated by Kwon et al. (10). Reverse Sanger sequencing as an alternative approach also uses four separate assays for the four different nucleotides but employs -thiophosphate containing dNTPs (-S-dNTPs) instead of using ddNTPS (11). Exonuclease activity is almost completely inhibited by the -S-dNTPs and therefore a sequence ladder is produced by cleavage reactions of full-length products. This approach is especially suitable for MALDI-TOF-MS analysis since the longer species are more abundant in the mixture. Additionally it is not necessary to acquire mass spectra at different time points of cleavage reaction to achieve a complete sequence coverage. So far, the combination of reverse Sanger sequencing and MALDI-MS has only been reported for DNA (12,13). RNA, however, shows higher sensitivity and is far more stable than DNA under UV-MALDI conditions due to the stabilizing effect of the electronegative 2'-hydroxyl group. Therefore, several strategies have been devised for comparative sequence analysis using MALDI-MS after base-specific endonucleolytic or exonucleolytic cleavage reaction (14–17). Moreover, RNA can be rapidly generated by in vitro transcription using bacteriophage RNA polymerases (18,19) with an intrinsic amplification factor. However, it is well known that RNA transcription does not produce homogeneous transcripts (20–22). Here it is shown that this interferes strongly with mass spectrometric assays of such transcripts. Therefore a procedure for the purification and isolation of the full-length transcripts has been developed.
MATERIALS AND METHODS
In vitro transcription
RNA was synthesized by runoff transcription from a linearized plasmid DNA vector pGEM 3Zf (Promega, Mannheim, Germany) carrying two promotors directed at each other from opposite ends of a multiple cloning site. The total reaction volume of 20 μl containing T7 transcription buffer , 1.5 μg DNA template, three unmodified NTPs (5 mM each) and 25 or 100% -S—NTP, respectively, of the fourth nucleotide (5 mM total) as Sp-stereoisomer as well as 25 U T7 RNA polymerase (New England Biolabs, Frankfurt, Germany). The transcription reaction was incubated at 37°C for 1 h, followed by addition of another 25 U polymerase and incubation for 1 h.
Isolation and purification
Method 1. RNA transcripts were purified by an Ethanol precipitation (23). The precipitate was dissolved in 200 mM TEAA, pH 7, for reversed phase purification using ZipTips-C18 (Millipore, Bedford, MA) (9,24). ZipTips were washed twice with 10 μl 200 mM TEAA, pH 7.0 and twice with 10 μl 20 mM TEAA, pH 7.0 followed by MALDI-MS.
Method 2. RNA in vitro transcripts were immobilized to 100–200 μl suspension of magnetic beads, coated with the vectors (+1–16) universal complementary sequence No. 1 to capture RNA transcripts at 5' end or with the vectors (+24–42) universal complementary sequence No. 2 to capture at 3' end. The magnetic beads were supplied by Dynal at a concentration of 10 mg/ml and a functionality of 150–200 pmol oligonucleotide load per milligram. The immobilization of the transcripts with magnetic beads No. 1 or No. 2 was obtained by adding 20 μl 1 M TEAA, pH 7.0 to the transcription reaction volume and keeping it at 40–50°C for 5 min. Using magnetic beads No. 1 the hybridization temperature is critical in order to discriminate against early quittings which are only 12 nt in length or less. The temperature was calculated by a home-made software using nearest-neighbor parameters as described by Sugimoto (25). After washing twice with 200 mM TEAA, pH 7.0 followed by cold 10 mM Tris–HCl, pH 7.5 the full-length transcripts were released by heating to 80°C for 5 min in 8 μl H2Obidest. The magnetic beads can be regenerated several times by washing twice with 0.1 N NaOH and storage buffer (10 mM Tris–HCl, pH 8.0 with 1 mM EDTA and 0.01% Tween-20).
SVP cleavage
The isolated full-length transcripts were incubated with 0.01 U SVP of Crotalus adamenteus (Amersham Biosciences, Freiburg, Germany) in a total reaction volume of 10 μl with Tris–HCl, pH 9.2, 1 mM MgCl2 at room temperature for 45 min. Occasionally, the cleavage products were further purified by a reversed phase purification using ZipTips. The additional purification resulted in minor improvements in spectral quality.
MALDI-MS
Spectra were acquired in positive ion mode with a Bruker ReflexIII (Bruker Daltonik, Bremen, Germany) mass spectrometer using a ternary matrix mixture consisting of 0.2 M 2,3,4-trihydroxyacetophenone (THAP) and 0.2 M 2,4,6-trihydroxyacetophenone and 0.3 M diammonium citrate (v/v/v = 1/2/1) (26). A nitrogen laser (337 nm) was used for desorption and ions were accelerated to 25 kV by delayed ion extraction. The recorded spectra (accumulation of 15–20 single shot spectra) were smoothed using a Golay–Savitzky filter and baseline corrected using the software package XMASS 5.0 (provided by the manufacturer). The same set of mass calibration constants was used for all analyses (external calibration).
RESULTS AND DISCUSSION
In vitro transcription
Figure 1 shows a MALDI-MS analysis of RNA transcripts generated under standard conditions (expected RNA 60mer, plasmid pGEM linearized by HindIII and expected RNA 23mer plasmid pGEM linearized by Sma).
Figure 1. MALDI mass spectra of the RNA in vitro transcription performed with pGEM 3Zf linearized with (A) HindIII (sequence of 60mer: 5'-PPP-GGGCGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCU-3') or with (B) SmaI (sequence of 23mer: 5'-PPP-GGGCGAAUUCGAGCUCGGUACCC-3') and transcribed with T7 RNA polymerase utilizing unmodified NTP after EtOH-precipitation and ZipTip purification. The byproducts, abortive products (2–12 nt) and slippage products (G4–G7), are labeled according to their length.
After ethanol/ZipTip purification several signals were observed by MALDI-MS, which were characterized as byproducts of RNA in vitro transcriptions. The byproducts consisted of abortive products, Poly-G transcripts, the so-called slippage products and template-independent, non-specific elongation. In the lower mass range the MALDI spectrum is dominated by abortive products, which are labeled according to their length. In addition to abortive products G-ladders are detected, ranging from 4 to 7 nt. In contrast to previous studies (21), our studies reveal the presence of these slippage products even under regular transcription conditions with non-limiting NTP concentrations. Template-independent, non-specific elongations are manifested in the mass spectrum by signals. In addition to the singly charged ions, doubly charged ions are also observed. All these byproducts were not detected under standard conditions by PAGE and staining with ethidium bromide or methylene blue (data not shown). However, these byproducts generate undesirable signals in MALDI-MS and thereby limit the sensitivity for the detection of the product of interest. Therefore a purification method was developed, which allows the specific isolation and purification of the desired full-length transcript to increase the validity of subsequent analysis.
Purification results
In general, the generation of pronounced byproducts such as abortive and slippage products as a characteristic for RNA transcriptions is an intrinsic problem of in vitro transcription, but the amount even increases when modified NTPs are applied. In mass spectra, these byproducts are detected despite an EtOH-precipitation and a reversed-phase purification using ZipTip. Figure 2 shows representative mass spectra of purified and isolated transcripts.
Figure 2. MALDI mass spectra of RNA transcripts after solid-phase purification by hybridization using magnetic beads No. 2 (A) or magnetic beads No. 1 (B). In vitro transcription was performed with pGEM 3Zf linearized (A) with HindIII (sequence of 60mer: 5'-GGGCGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCU-3'). Signals marked with open circle: leakage bond of the universal complementary sequence coated to magnetic beads; (B) with SmaI (sequence of 23mer: 5'-PPP-GGGCGAAUUCGAGCUCGGUACCC-3') and transcribed with T7 RNA polymerase utilizing unmodified NTP.
A selective enrichment of the full-length transcript was achieved by the specific immobilization via hybridization of the transcription products to magnetic beads, coated with complementary universal sequences, which are defined by the sequence of the cloning site in the transcription vector. The complementary sequences (underlined and bold) are given within the RNA transcript sequence in the caption of Figure 2. Transcriptional byproducts such as abortive (from 3 to 13 nt) and slippage products (G-ladders ranging from 4 to 9 nt) and contamination, originating from transcription buffer such as spermidine and DDT are not bound on the solid phase. After washing, the unmodified and modified full-length transcripts were released by heating in water. The magnetic beads can be used several times (up to 10 times), but continued use of the magnetic beads leads to bond cleavage of the universal sequences coated to magnetic beads, which can be observed as several signals in the lower mass range by MALDI-MS (corresponding signals are marked with an open circle in Figure 2A).
Figure 3A represents an overlay of two mass spectra of transcripts isolated with magnetic beads No. 1, one transcribed with all four native NTPs and one where the native ATP was replaced by 100% of -S-ATP.
Figure 3. MALDI mass spectra of unmodified and modified full-length transcripts (23mer) isolated by solid phase purification. (A) RNA in vitro transcriptions performed with T7 RNA polymerase and with 100% ATP (black) and 100% -S-ATP (green); (B) containing 25% -S-ATP, which results in a heterogeneity of the products.
The hybridization conditions with magnetic beads No. 1 were optimized to prevent immobilization of abortive products from 3 to 13 nt, which have the correct sequence but are too short to be efficiently hybridized. In the transcription using 100% of -S-ATP the incorporation of four modified rATPs results in the expected mass shift by 64 u. As judged by the comparable signal intensities natural and Sp--S-NTP transcriptions have about equal yield, as also reported by Eckstein (27). Template-independent, non-specific elongations cannot be eliminated by the capture purification and are, therefore, still detected in the mass spectrum by + and +, respectively (28). The small mass difference of 1 u does not allow differentiation between UTP and CTP, however.
A mixture of modified and native NTPs (one-third) of one nucleotide species in the in vitro transcription results in a statistical incorporation in all positions. The average number of incorporated -S-NTPs in the transcripts is determined by the concentration ratio r = cn(-S-NTP)/cn(NTP). This ratio is critical since the cleavage reaction of the resulting transcripts by SVP renders a sequence ladder terminated by the thiophosphate-containing nucleotide nearest to the 3' terminus. The probability p(m) for the generation of products terminated by an -S-NTP in position m of the possible positions counting from the 3' terminus of the transcript is given by
(1)
with i equal to the total number of potential incorporation sites. In this nomenclature the product length increases with increasing m. This equation assumes equal incorporation probability for the modified and unmodified NTP by the polymerase discussed above. The influence of the concentration ratio r = cn(-S-NTP)/cn(NTP) on the yield of products of different length is demonstrated for an RNA with five potential incorporation sites of -S-NTP (e.g. -S-ATP): using only 25% of -S-ATP in the transcription reaction, 25% of the longest and 7.9% of the shortest product are generated. The total product yield amounts to 76.3%; the rest is lost to total cleavage reaction because of no -S-ATP incorporation. This concentration ratio was found to be optimal for transcripts up to 25 bases. Figure 3B shows the mass spectra of the isolated 23 nt transcription product with 25% of -S-ATP and four incorporation sites. Incorporation of more than one -S-ATP per transcript generates products of the same length but with different masses, which depend on the number x of thiophosphate-containing nucleotides. Therefore the full-length transcript displays five signals, corresponding to the incorporation of zero to four -S-ATP. The total peak area of the signal is comparable to that of the full-length transcript in the reaction using unmodified NTPs or 100% -S-NTPs, but the mass signal for the modified transcript is split into several species.
Cleavage
The SVP belongs to the class of exonucleases, hydrolyzing phosphodiester groups from the 3' terminus of DNA and RNA, and releasing successively, nucleotides carrying a 5'-phosphate group. Reverse Sanger sequencing was hitherto not considered feasible for RNA, since it was reported that RNA polymerases incorporate specifically the Sp-isomer of -S-NTP with inversion to the Rp-isomer, and the Rp-isomer was reported to be efficiently cleaved by SVP (27,29). To investigate the cleavage rate of SVP an isolated and purified transcript containing 100% of -S-ATP was cleaved and analyzed by MALDI-MS (Figure 4).
Figure 4. MALDI mass fragmentation spectrum of RNA in vitro transcript (23mer) obtained by SVP-cleavage after solid-phase purification. Transcription was performed with T7 RNA polymerase utilizing 100% -S-ATP. ?: Sequence-independent signals in the lower mass range.
The spectrum is dominated by the signal of the longest fragment with an -S-ATP in position 20 at its 3' terminus. Some less intense signals are also detected in products with -S-ATP in positions 12 and 7. This demonstrates that the SVP cleavage is significantly slowed down at -S-ATP-sites, but not fully inhibited. Surprisingly, no signal of the product with -S-ATP in position 6 corresponding to the sequence is observed using 100% of -S-ATP. This result might indicate that the cleavage activity of the exonuclease is completely stopped by two neighboring, modified NTPs. SVP shows a somewhat lower hydrolysis rate for RNA compared with DNA and is even less active in cleaving RNA in vitro transcripts, especially when they contain thiophosphates as required in the reverse Sanger sequencing approach (data not shown). However, a clearly readable complete sequence ladder can be obtained under appropriate conditions. In separate in vitro transcriptions all four -S-NTPs were used, all at a ratio of 25% -S-NTP. The solid-phase purified transcripts were cleaved by SVP and then analyzed. Figure 5 represents an overlay of the four base-specific mass spectra.
Figure 5. Overlay of four base-specific fragmentation mass spectra (23mer) obtained by SVP-cleavage after solid-phase purification. Transcription was performed with T7 RNA polymerase utilizing 25% -S-rNTP (25 -S-rATP, 25 -S-rCTP, 25 -S-rGTP, 25 -S-rUTP).
The masses for the sequence ladders are given in Table 1.
Table 1. Calculated SVP fragments of unmodified and modified RNA
Signals corresponding to all of the expected products of the reaction could be observed using 25% -S-NTP except the first five nucleotides starting at the 5' terminus. In addition to the singly charged ions doubly charged ions are also observed. Using 25% -S-NTP the signals corresponding of two neighboring NTPs were also detected. The resolution in the mass spectra is considerably decreased due to the fact that for each peak there is a distribution of peaks containing different numbers of -S-NTPs. In addition, the intensity of the signals varies depending on the base-specific reactions. The longest products are more abundant in the G/C bases cleavage reaction as compared with modified A/U bases. This result might indicate that the cleavage rate of SVP is somewhat sequence dependent using modified RNA. However, the mass spectra were obtained in four separate reactions. To verify this result the four transcriptions and subsequent cleavage reaction would have to be performed and analyzed jointly in only one reaction. This observation is in contrast to previous studies by Faulstich et al. (30), who observed higher signal intensities for C-terminated products of unmodified RNA only for calf spleen phosphodiesterase, which hydrolyzes from the 5' terminus, but not for SVP cleavage reactions.
CONCLUSION
Reverse Sanger sequencing was hitherto not considered feasible for RNA. Here, however, it was shown that the cleavage reaction of the modified transcript by SVP can obviously give sequence ladders terminated by the thiophosphate-containing nucleotide at the 3' terminus. The cleavage reaction of SVP is not stopped completely at such linkage, but despite this lack of full suppression sequence ladders are obtained for a MALDI-MS analysis. Also the ratio of -S-NTP to NTP must be chosen properly for the SVP cleavage reaction to produce a sequence ladder amenable to mass spectrometric analysis. A high concentration of -S-NTP results in the occupation of many positions by the modified nucleotide and subsequent cleavage reaction with SVP stops mostly close to the 3' terminus. On the other hand, low concentrations of -S-NTP lead to a dominance of shorter fragments and a complete cleavage of non-modified transcripts since fewer positions are occupied by thiophosphate-containing nucleotides. It was found that 25% -S-NTP is the optimal ratio for the subsequent cleavage reaction of transcripts of 25 nt in length. Incorporation of more than one -S-NTP per transcript generates products of the same length but with a different number of -S-NTP, the mass of which differs by 16 u. This leads to a heterogeneity of the products in terms of mass and lowers the amplitude of each one of them in the spectrum, if they are mass resolved, or leads to peak broadening with the associated loss in resolution and sensitivity. The synthesis of modified NTPs, which allow cleavage by SVP but are isobaric to the corresponding -S-NTP, would improve the mass spectra quality. The sequence analysis of RNA in one single reaction is very limited by the small mass difference of 1 u between uridine and cytidine, but the generation of three or four separate sequence ladders for each different base solves this problem. Also the problem concerning the byproducts characteristic for RNA transcription, is resolved using a solid-phase purification. The immobilization of the full-length transcripts via hybridization eliminates the byproducts, additives and metal cations and improves the mass spectra interpretation. The reverse Sanger reaction has great potential for various attractive applications, e.g. signature sequencing for characterizing libraries of clones or for tags used as forensic markers. The maximum length of templates that can be fully sequenced by this method is not yet known, but should be somewhere between 25 and 50 bases. Particularly for longer sequences the availability of isobaric nucleotides with and without the -S-modification would be necessary to retain the sensitivity and specificity of the assay.
ACKNOWLEDGEMENTS
We would like to thank J. Reiss for helpful discussions. This work was done in partial fulfillment of the requirement for the Ph.D. (Dr rer. nat.) of B.S. and J.G. at the University of Münster. Financial support by the Bundesministerium für Bildung und Forschung (BMBF, grant No. BD 081535) is gratefully acknowledged.
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* To whom correspondence should be addressed. Tel: +49 251 83 55103; Fax: +49 251 83 55121; Email: hillenk@uni-muenster.de
ABSTRACT
Several DNA/RNA sequencing strategies have been developed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In the reverse Sanger sequencing approach -thiophosphate-containing NTPs are employed. Sequencing ladders are produced by the subsequent exonuclease cleavage, which is inhibited by the -S-NTP at the 3' terminus. Here the reverse Sanger sequencing of RNA is described. The stability of RNA during the UV-MALDI process is higher relative to DNA, and RNA can be easily synthesized by transcription using bacteriophage RNA polymerase. -S-rNTP was added to the reaction in a ratio of 1:3 to the native rNTPs and was incorporated statistically by the RNA polymerase. Four separate sequence ladders were produced, to avoid the problem of the only 1u mass difference between uridine and cytidine. However, it was shown that RNA transcription does not produce homogeneous transcripts. Therefore isolation of the full-length transcript is required to attain a non-ambiguous interpretation of cleavage spectra. This is achieved by the exclusive immobilization of the full-length transcript on a solid phase. The full-length transcripts were hybridized to magnetic beads, coated with short universal sequences, complementary to the in vitro RNA. After purification and isolation the RNA full-length transcript is cleaved by snake venom phosphodiesterase (SVP) and the obtained sequence ladder is analyzed by MALDI-MS.
INTRODUCTION
With the draft sequence of the human genome in place (1), new genomic applications are expected in clinical diagnostics and forensics which require high-throughput analysis of short nucleic acids sequences. Mass spectrometry (MS) offers advantages over gel electrophoretic separation and various forms of hybridization, particularly for automated, rapid and large-scale DNA/RNA screening. The use of electrophoretic mobility or fluorescence of a label are only indirect measures, but mass is an intrinsic property of the molecule. As a result, mass spectrometric assays are intrinsically more accurate. The development of the ‘soft’ ionization technique, matrix-assisted laser desorption/ionization (MALDI) (2), in the late 1980s enabled the analysis of large biomolecules (3) and has been applied to the analysis of DNA and RNA sequencing (4,5). However, base losses and metastable backbone fragmentation (6) as well as a dramatic decrease of ion yield with increasing fragment length limit the accessible mass range for DNA analysis to essentially less than 15 kDa (7,8) This problem is especially marked for Sanger sequencing ladders because of the intrinsic bias of ladder production to yield higher concentrations of shorter termination products. This adds to the suppression of longer termination products by MALDI-MS (9). Nonetheless, application of MALDI-MS for the sequencing by chain termination principle for RNA was demonstrated by Kwon et al. (10). Reverse Sanger sequencing as an alternative approach also uses four separate assays for the four different nucleotides but employs -thiophosphate containing dNTPs (-S-dNTPs) instead of using ddNTPS (11). Exonuclease activity is almost completely inhibited by the -S-dNTPs and therefore a sequence ladder is produced by cleavage reactions of full-length products. This approach is especially suitable for MALDI-TOF-MS analysis since the longer species are more abundant in the mixture. Additionally it is not necessary to acquire mass spectra at different time points of cleavage reaction to achieve a complete sequence coverage. So far, the combination of reverse Sanger sequencing and MALDI-MS has only been reported for DNA (12,13). RNA, however, shows higher sensitivity and is far more stable than DNA under UV-MALDI conditions due to the stabilizing effect of the electronegative 2'-hydroxyl group. Therefore, several strategies have been devised for comparative sequence analysis using MALDI-MS after base-specific endonucleolytic or exonucleolytic cleavage reaction (14–17). Moreover, RNA can be rapidly generated by in vitro transcription using bacteriophage RNA polymerases (18,19) with an intrinsic amplification factor. However, it is well known that RNA transcription does not produce homogeneous transcripts (20–22). Here it is shown that this interferes strongly with mass spectrometric assays of such transcripts. Therefore a procedure for the purification and isolation of the full-length transcripts has been developed.
MATERIALS AND METHODS
In vitro transcription
RNA was synthesized by runoff transcription from a linearized plasmid DNA vector pGEM 3Zf (Promega, Mannheim, Germany) carrying two promotors directed at each other from opposite ends of a multiple cloning site. The total reaction volume of 20 μl containing T7 transcription buffer , 1.5 μg DNA template, three unmodified NTPs (5 mM each) and 25 or 100% -S—NTP, respectively, of the fourth nucleotide (5 mM total) as Sp-stereoisomer as well as 25 U T7 RNA polymerase (New England Biolabs, Frankfurt, Germany). The transcription reaction was incubated at 37°C for 1 h, followed by addition of another 25 U polymerase and incubation for 1 h.
Isolation and purification
Method 1. RNA transcripts were purified by an Ethanol precipitation (23). The precipitate was dissolved in 200 mM TEAA, pH 7, for reversed phase purification using ZipTips-C18 (Millipore, Bedford, MA) (9,24). ZipTips were washed twice with 10 μl 200 mM TEAA, pH 7.0 and twice with 10 μl 20 mM TEAA, pH 7.0 followed by MALDI-MS.
Method 2. RNA in vitro transcripts were immobilized to 100–200 μl suspension of magnetic beads, coated with the vectors (+1–16) universal complementary sequence No. 1 to capture RNA transcripts at 5' end or with the vectors (+24–42) universal complementary sequence No. 2 to capture at 3' end. The magnetic beads were supplied by Dynal at a concentration of 10 mg/ml and a functionality of 150–200 pmol oligonucleotide load per milligram. The immobilization of the transcripts with magnetic beads No. 1 or No. 2 was obtained by adding 20 μl 1 M TEAA, pH 7.0 to the transcription reaction volume and keeping it at 40–50°C for 5 min. Using magnetic beads No. 1 the hybridization temperature is critical in order to discriminate against early quittings which are only 12 nt in length or less. The temperature was calculated by a home-made software using nearest-neighbor parameters as described by Sugimoto (25). After washing twice with 200 mM TEAA, pH 7.0 followed by cold 10 mM Tris–HCl, pH 7.5 the full-length transcripts were released by heating to 80°C for 5 min in 8 μl H2Obidest. The magnetic beads can be regenerated several times by washing twice with 0.1 N NaOH and storage buffer (10 mM Tris–HCl, pH 8.0 with 1 mM EDTA and 0.01% Tween-20).
SVP cleavage
The isolated full-length transcripts were incubated with 0.01 U SVP of Crotalus adamenteus (Amersham Biosciences, Freiburg, Germany) in a total reaction volume of 10 μl with Tris–HCl, pH 9.2, 1 mM MgCl2 at room temperature for 45 min. Occasionally, the cleavage products were further purified by a reversed phase purification using ZipTips. The additional purification resulted in minor improvements in spectral quality.
MALDI-MS
Spectra were acquired in positive ion mode with a Bruker ReflexIII (Bruker Daltonik, Bremen, Germany) mass spectrometer using a ternary matrix mixture consisting of 0.2 M 2,3,4-trihydroxyacetophenone (THAP) and 0.2 M 2,4,6-trihydroxyacetophenone and 0.3 M diammonium citrate (v/v/v = 1/2/1) (26). A nitrogen laser (337 nm) was used for desorption and ions were accelerated to 25 kV by delayed ion extraction. The recorded spectra (accumulation of 15–20 single shot spectra) were smoothed using a Golay–Savitzky filter and baseline corrected using the software package XMASS 5.0 (provided by the manufacturer). The same set of mass calibration constants was used for all analyses (external calibration).
RESULTS AND DISCUSSION
In vitro transcription
Figure 1 shows a MALDI-MS analysis of RNA transcripts generated under standard conditions (expected RNA 60mer, plasmid pGEM linearized by HindIII and expected RNA 23mer plasmid pGEM linearized by Sma).
Figure 1. MALDI mass spectra of the RNA in vitro transcription performed with pGEM 3Zf linearized with (A) HindIII (sequence of 60mer: 5'-PPP-GGGCGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCU-3') or with (B) SmaI (sequence of 23mer: 5'-PPP-GGGCGAAUUCGAGCUCGGUACCC-3') and transcribed with T7 RNA polymerase utilizing unmodified NTP after EtOH-precipitation and ZipTip purification. The byproducts, abortive products (2–12 nt) and slippage products (G4–G7), are labeled according to their length.
After ethanol/ZipTip purification several signals were observed by MALDI-MS, which were characterized as byproducts of RNA in vitro transcriptions. The byproducts consisted of abortive products, Poly-G transcripts, the so-called slippage products and template-independent, non-specific elongation. In the lower mass range the MALDI spectrum is dominated by abortive products, which are labeled according to their length. In addition to abortive products G-ladders are detected, ranging from 4 to 7 nt. In contrast to previous studies (21), our studies reveal the presence of these slippage products even under regular transcription conditions with non-limiting NTP concentrations. Template-independent, non-specific elongations are manifested in the mass spectrum by signals. In addition to the singly charged ions, doubly charged ions are also observed. All these byproducts were not detected under standard conditions by PAGE and staining with ethidium bromide or methylene blue (data not shown). However, these byproducts generate undesirable signals in MALDI-MS and thereby limit the sensitivity for the detection of the product of interest. Therefore a purification method was developed, which allows the specific isolation and purification of the desired full-length transcript to increase the validity of subsequent analysis.
Purification results
In general, the generation of pronounced byproducts such as abortive and slippage products as a characteristic for RNA transcriptions is an intrinsic problem of in vitro transcription, but the amount even increases when modified NTPs are applied. In mass spectra, these byproducts are detected despite an EtOH-precipitation and a reversed-phase purification using ZipTip. Figure 2 shows representative mass spectra of purified and isolated transcripts.
Figure 2. MALDI mass spectra of RNA transcripts after solid-phase purification by hybridization using magnetic beads No. 2 (A) or magnetic beads No. 1 (B). In vitro transcription was performed with pGEM 3Zf linearized (A) with HindIII (sequence of 60mer: 5'-GGGCGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCU-3'). Signals marked with open circle: leakage bond of the universal complementary sequence coated to magnetic beads; (B) with SmaI (sequence of 23mer: 5'-PPP-GGGCGAAUUCGAGCUCGGUACCC-3') and transcribed with T7 RNA polymerase utilizing unmodified NTP.
A selective enrichment of the full-length transcript was achieved by the specific immobilization via hybridization of the transcription products to magnetic beads, coated with complementary universal sequences, which are defined by the sequence of the cloning site in the transcription vector. The complementary sequences (underlined and bold) are given within the RNA transcript sequence in the caption of Figure 2. Transcriptional byproducts such as abortive (from 3 to 13 nt) and slippage products (G-ladders ranging from 4 to 9 nt) and contamination, originating from transcription buffer such as spermidine and DDT are not bound on the solid phase. After washing, the unmodified and modified full-length transcripts were released by heating in water. The magnetic beads can be used several times (up to 10 times), but continued use of the magnetic beads leads to bond cleavage of the universal sequences coated to magnetic beads, which can be observed as several signals in the lower mass range by MALDI-MS (corresponding signals are marked with an open circle in Figure 2A).
Figure 3A represents an overlay of two mass spectra of transcripts isolated with magnetic beads No. 1, one transcribed with all four native NTPs and one where the native ATP was replaced by 100% of -S-ATP.
Figure 3. MALDI mass spectra of unmodified and modified full-length transcripts (23mer) isolated by solid phase purification. (A) RNA in vitro transcriptions performed with T7 RNA polymerase and with 100% ATP (black) and 100% -S-ATP (green); (B) containing 25% -S-ATP, which results in a heterogeneity of the products.
The hybridization conditions with magnetic beads No. 1 were optimized to prevent immobilization of abortive products from 3 to 13 nt, which have the correct sequence but are too short to be efficiently hybridized. In the transcription using 100% of -S-ATP the incorporation of four modified rATPs results in the expected mass shift by 64 u. As judged by the comparable signal intensities natural and Sp--S-NTP transcriptions have about equal yield, as also reported by Eckstein (27). Template-independent, non-specific elongations cannot be eliminated by the capture purification and are, therefore, still detected in the mass spectrum by + and +, respectively (28). The small mass difference of 1 u does not allow differentiation between UTP and CTP, however.
A mixture of modified and native NTPs (one-third) of one nucleotide species in the in vitro transcription results in a statistical incorporation in all positions. The average number of incorporated -S-NTPs in the transcripts is determined by the concentration ratio r = cn(-S-NTP)/cn(NTP). This ratio is critical since the cleavage reaction of the resulting transcripts by SVP renders a sequence ladder terminated by the thiophosphate-containing nucleotide nearest to the 3' terminus. The probability p(m) for the generation of products terminated by an -S-NTP in position m of the possible positions counting from the 3' terminus of the transcript is given by
(1)
with i equal to the total number of potential incorporation sites. In this nomenclature the product length increases with increasing m. This equation assumes equal incorporation probability for the modified and unmodified NTP by the polymerase discussed above. The influence of the concentration ratio r = cn(-S-NTP)/cn(NTP) on the yield of products of different length is demonstrated for an RNA with five potential incorporation sites of -S-NTP (e.g. -S-ATP): using only 25% of -S-ATP in the transcription reaction, 25% of the longest and 7.9% of the shortest product are generated. The total product yield amounts to 76.3%; the rest is lost to total cleavage reaction because of no -S-ATP incorporation. This concentration ratio was found to be optimal for transcripts up to 25 bases. Figure 3B shows the mass spectra of the isolated 23 nt transcription product with 25% of -S-ATP and four incorporation sites. Incorporation of more than one -S-ATP per transcript generates products of the same length but with different masses, which depend on the number x of thiophosphate-containing nucleotides. Therefore the full-length transcript displays five signals, corresponding to the incorporation of zero to four -S-ATP. The total peak area of the signal is comparable to that of the full-length transcript in the reaction using unmodified NTPs or 100% -S-NTPs, but the mass signal for the modified transcript is split into several species.
Cleavage
The SVP belongs to the class of exonucleases, hydrolyzing phosphodiester groups from the 3' terminus of DNA and RNA, and releasing successively, nucleotides carrying a 5'-phosphate group. Reverse Sanger sequencing was hitherto not considered feasible for RNA, since it was reported that RNA polymerases incorporate specifically the Sp-isomer of -S-NTP with inversion to the Rp-isomer, and the Rp-isomer was reported to be efficiently cleaved by SVP (27,29). To investigate the cleavage rate of SVP an isolated and purified transcript containing 100% of -S-ATP was cleaved and analyzed by MALDI-MS (Figure 4).
Figure 4. MALDI mass fragmentation spectrum of RNA in vitro transcript (23mer) obtained by SVP-cleavage after solid-phase purification. Transcription was performed with T7 RNA polymerase utilizing 100% -S-ATP. ?: Sequence-independent signals in the lower mass range.
The spectrum is dominated by the signal of the longest fragment with an -S-ATP in position 20 at its 3' terminus. Some less intense signals are also detected in products with -S-ATP in positions 12 and 7. This demonstrates that the SVP cleavage is significantly slowed down at -S-ATP-sites, but not fully inhibited. Surprisingly, no signal of the product with -S-ATP in position 6 corresponding to the sequence is observed using 100% of -S-ATP. This result might indicate that the cleavage activity of the exonuclease is completely stopped by two neighboring, modified NTPs. SVP shows a somewhat lower hydrolysis rate for RNA compared with DNA and is even less active in cleaving RNA in vitro transcripts, especially when they contain thiophosphates as required in the reverse Sanger sequencing approach (data not shown). However, a clearly readable complete sequence ladder can be obtained under appropriate conditions. In separate in vitro transcriptions all four -S-NTPs were used, all at a ratio of 25% -S-NTP. The solid-phase purified transcripts were cleaved by SVP and then analyzed. Figure 5 represents an overlay of the four base-specific mass spectra.
Figure 5. Overlay of four base-specific fragmentation mass spectra (23mer) obtained by SVP-cleavage after solid-phase purification. Transcription was performed with T7 RNA polymerase utilizing 25% -S-rNTP (25 -S-rATP, 25 -S-rCTP, 25 -S-rGTP, 25 -S-rUTP).
The masses for the sequence ladders are given in Table 1.
Table 1. Calculated SVP fragments of unmodified and modified RNA
Signals corresponding to all of the expected products of the reaction could be observed using 25% -S-NTP except the first five nucleotides starting at the 5' terminus. In addition to the singly charged ions doubly charged ions are also observed. Using 25% -S-NTP the signals corresponding of two neighboring NTPs were also detected. The resolution in the mass spectra is considerably decreased due to the fact that for each peak there is a distribution of peaks containing different numbers of -S-NTPs. In addition, the intensity of the signals varies depending on the base-specific reactions. The longest products are more abundant in the G/C bases cleavage reaction as compared with modified A/U bases. This result might indicate that the cleavage rate of SVP is somewhat sequence dependent using modified RNA. However, the mass spectra were obtained in four separate reactions. To verify this result the four transcriptions and subsequent cleavage reaction would have to be performed and analyzed jointly in only one reaction. This observation is in contrast to previous studies by Faulstich et al. (30), who observed higher signal intensities for C-terminated products of unmodified RNA only for calf spleen phosphodiesterase, which hydrolyzes from the 5' terminus, but not for SVP cleavage reactions.
CONCLUSION
Reverse Sanger sequencing was hitherto not considered feasible for RNA. Here, however, it was shown that the cleavage reaction of the modified transcript by SVP can obviously give sequence ladders terminated by the thiophosphate-containing nucleotide at the 3' terminus. The cleavage reaction of SVP is not stopped completely at such linkage, but despite this lack of full suppression sequence ladders are obtained for a MALDI-MS analysis. Also the ratio of -S-NTP to NTP must be chosen properly for the SVP cleavage reaction to produce a sequence ladder amenable to mass spectrometric analysis. A high concentration of -S-NTP results in the occupation of many positions by the modified nucleotide and subsequent cleavage reaction with SVP stops mostly close to the 3' terminus. On the other hand, low concentrations of -S-NTP lead to a dominance of shorter fragments and a complete cleavage of non-modified transcripts since fewer positions are occupied by thiophosphate-containing nucleotides. It was found that 25% -S-NTP is the optimal ratio for the subsequent cleavage reaction of transcripts of 25 nt in length. Incorporation of more than one -S-NTP per transcript generates products of the same length but with a different number of -S-NTP, the mass of which differs by 16 u. This leads to a heterogeneity of the products in terms of mass and lowers the amplitude of each one of them in the spectrum, if they are mass resolved, or leads to peak broadening with the associated loss in resolution and sensitivity. The synthesis of modified NTPs, which allow cleavage by SVP but are isobaric to the corresponding -S-NTP, would improve the mass spectra quality. The sequence analysis of RNA in one single reaction is very limited by the small mass difference of 1 u between uridine and cytidine, but the generation of three or four separate sequence ladders for each different base solves this problem. Also the problem concerning the byproducts characteristic for RNA transcription, is resolved using a solid-phase purification. The immobilization of the full-length transcripts via hybridization eliminates the byproducts, additives and metal cations and improves the mass spectra interpretation. The reverse Sanger reaction has great potential for various attractive applications, e.g. signature sequencing for characterizing libraries of clones or for tags used as forensic markers. The maximum length of templates that can be fully sequenced by this method is not yet known, but should be somewhere between 25 and 50 bases. Particularly for longer sequences the availability of isobaric nucleotides with and without the -S-modification would be necessary to retain the sensitivity and specificity of the assay.
ACKNOWLEDGEMENTS
We would like to thank J. Reiss for helpful discussions. This work was done in partial fulfillment of the requirement for the Ph.D. (Dr rer. nat.) of B.S. and J.G. at the University of Münster. Financial support by the Bundesministerium für Bildung und Forschung (BMBF, grant No. BD 081535) is gratefully acknowledged.
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