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`28 JANUARY 2000
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`ELSEVIER
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`1
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`MTX1023
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`2
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`

`

`ELSEVIER
`
`Journal of Chromatography A, 868 (2000) 109-114
`
`JOURNAL OF
`CHROMATOGRAPHY A
`
`www.elsevier. com/ locate/ clu·oma
`
`Short communication
`Use of high-performance liquid chromatographic fractionation of
`large RNA molecules in the assay of group I intron
`ribozyme activity
`D.E. Georgopoulos, M.J. Leibowitz*
`University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School and Cancer Institute of Ne1v Jersey,
`675 Hoes Lane, Piscataway, NJ 08854-5635, USA
`
`Received 15 September 1999; accepted 4 November 1999
`
`Abstract
`
`Ion-pair .reversed-phase high-performance liquid clu·omatography (HPLC), which has previously been used to fractionate
`double-stranded DNA molecules, can be applied to single-stranded RNA molecules in the size range of 200- 1000
`nucleotides. This procedure permits RNA molecules to be separated and recovered rapidly in liquid medium, thereby
`facilitating recovery. We have used this system to separate an in vitro transcription product containing a group I intron
`J·ibozyme from the intermediates and products of the splicing reaction, permitting rapid assay of ribozyme activity without
`ti1e use of radioactivity. © 2000 Elsevier Science B.V. All rights reserved.
`~~
`Keywords: RNA; Ribozymes
`
`1. Introduction
`
`Self-splicing introns, including the group I and
`group II ribozymes [1 ,2], catalyze their own excision
`from precursor RNA, with ligation of the flanking
`exons. Specifically, in the splicing reaction of group
`I intron ribozymes, precursor RNA (denoted El-I(cid:173)
`E2, where E 1 indicates the 5' -ex on, I indicates the
`intron, and E2 indicates the 3' -ex on) reacts with a
`guanosine nucleotide (G) to yield the El and GI-E2
`intermediates. These further react to result in the
`ligated exons (El-E2) and the excised guanylylated
`intron (GI) reaction products. Self-splicing reactions
`are
`typically assayed, using
`internally
`labeled
`
`*Con·esponding author.
`
`radioactive precursor RNA, by polyacrylamide gel
`electrophoresis, followed by detection of radioactivi(cid:173)
`ty, to monitor the conversion of precursor RNA into
`the intermediates and products of the splicing re(cid:173)
`action.
`The reaction can also be assayed, by using non(cid:173)
`radioactive precursor RNA, and following the in(cid:173)
`corporation of [ex- 32P]guanosine nucleotide into the
`GI-E2 intermediate and excised GI product by gel
`electrophoresis. Alternatively, this reaction can be
`monitored by incorporation of radioactivity from the
`nucleotide into trichloroacetic acid (TCA)-insoluble
`material, although this rapid and convenient assay
`method, as previously reported, suffers from prob(cid:173)
`lems in quantitative reproducibility and high blanks
`[3] . A chromatographic method to separate RNA
`
`0021-9673/ 00 /$- see front matter © 2000 Elsevier Science BV. All rights reserved.
`PII: S00 2 1- 9673(99 )01178-4
`
`3
`
`

`

`110
`
`D.E. Georgopoulos, M.J. Leibowitz I J. Chromatogr. A 868 (2000) 109-114
`
`molecules based on size has the potential of being
`much faster than gel electrophoresis . A preparative
`liquid phase system would also eliminate the losses
`incuiTed by gel purification and recovery of the RNA
`from a solid gel matrix.
`Anion-exchange high-performance liquid chroma(cid:173)
`tography (HPLC) has been used to separate small
`RNA molecules and purify them for crystallography
`and other physical studies [ 4]. This method has been
`applied to the assay of hairpin and hammerhead
`ribozyme-catalyzed RNA cleavage [5,6]. However, it
`has only been used for ribozymes and RNA sub(cid:173)
`strates (for trans-acting ribozymes) of less than 50
`nucleotides in length, but not for larger ribozymes,
`such as
`the self-splicing introns. Reversed-phase
`chromatography on non-porous alkylated poly(cid:173)
`(styrene-divinylbenzene) particles has been shown to
`resolve DNA oligonucleotides of less
`than 60
`nucleotides in length [7]. Ion-pair reversed-phase
`HPLC on these same beads extended the range of
`sizes of DNA molecules resolved to make this
`method useful for restriction fragments or polymer(cid:173)
`ase chain reaction (PCR) products [8,9], with res(cid:173)
`olution documented for fragments up to 1000 base
`pairs in length. Similar resolution of DNA fragments
`has also been attained using anion-exchange HPLC
`[10].
`This paper describes the application of ion-pair
`reversed-phase HPLC to RNA molecules comparable
`in size to the DNA molecules separable by this
`method. This method makes possible a rapid ana(cid:173)
`lytical and preparative method for size fractionation
`of RNA molecules, which we have applied to the
`assay of the self-splicing reaction catalyzed by the
`Pcl.LSU group I intron ribozyme from Pneumocystis
`carinii sp. f. carinii [11].
`
`2. Experimental
`
`2.1. RNA samples
`
`RNA standards included a 0.24-9.5 kilobase pair
`(kb) RNA ladder, a 0.16-1.77 kb RNA ladder (both
`from GIBCO-BRL, Rockville, MD, USA) and a
`0.3-1.6 kb RNA
`ladder (Boehringer-Mannheim,
`Indianapolis, IN, USA). Preparation of the 660-
`nucleotide RNA molecule derived from the rRNA
`
`gene of P. carinii sp. f. carinii contammg the
`Pc l .LSU group I intron was performed as described
`[12]. The DNA containing this intron and fl anking
`exon fragments was amplified by PCR from a cloned
`DNA template, using DNA primers which added the
`seventeen nucleotide bacteriophage SP6 promoter
`[13]. The 660-nucleotide precursor RNA was tran(cid:173)
`scribed from the amplified DNA using bacteriophage
`SP6 RNA polymerase (Promega, Madison, WI,
`USA), and the crude transcript RNA was separated
`from other
`reaction components by QIAquick
`Nucleotide Clean-Up spin column (Qiagen, Valencia,
`CA, USA). Splicing of crude precursor RNA was
`performed in the presence of 50 mM Tris-HCl (pH
`7.5), 5 mM magnesium chloride, 0.4 mM spermidine
`and 10 f.LM GTP for 30 min at 50°C. The reaction
`was stopped by addition of EDTA (tetrasodium salt,
`BioChernica MicroSelect Grade, Fluka, Milwaukee,
`WI, USA) to a final concentration of 2.5 mM and
`chilling on ice. In the splicing reaction, the precursor
`RNA (denoted El-I-E2) is spliced in a two-ste~
`process to yield the splicing intermediates, El +GI;:(cid:173)
`E2, and final products, GI+El-E2.
`
`'
`
`2.2. HPLC analysis
`
`HPLC was performed using a Model HP1090M
`HPLC System (Hewlett-Packard Instruments, Wil(cid:173)
`mington, DE, USA) which had been modified to
`eliminate most metallic components corning in con(cid:173)
`tact with the mobile phase, as recommended by the
`manufacturer of the column. Chromatography was
`performed on a DNASep column (Sarasep, San Jose,
`CA, USA), a 50 X4.6 mrn deactivated stainless steel
`column with titanium frits, with a stationary phase
`consisting of C 18 alkylated non-porous poly(styrene(cid:173)
`divinylbenzene) copolymer, 2.2 f.Lm particle size [8].
`Samples of 5 f.Ll (for RNA standards) or 90 f.Ll
`(ribozyme crude precursors or reaction products)
`were injected. RNA separation was performed as for
`DNA [8] with the following modifications. Eluent A
`was 0.1 M triethylamrnonium acetate (TEAA), pH
`7.0, containing 0.1 mM EDTA; eluent B was eluent
`A containing 25 % acetonitrile, prepared as described
`[9]. All eluent reagents used were HPLC grade from
`Fluka. HPLC-grade acetonitrile was from Fisher
`(Springfield, NJ, USA). Chromatography was per(cid:173)
`formed at 55°C at a flow-rate of 0.75 ml/rnin, with
`
`4
`
`

`

`D.E. Georgopoulos, M.J. LeiboiVitz I J. Ch romatogr. A 868 (2000) 109-114
`
`Ill
`
`A.
`
`24
`
`U)
`.~
`1:
`;:::) 18
`<t
`
`E -(I) 12
`
`(,)
`1:
`Ill
`..0
`....
`g 6
`..0
`<t
`
`0 ..,.
`
`N
`
`0
`
`0 ..,.
`..,.
`
`0
`LO
`M
`
`2
`
`3
`
`6
`5
`4
`Time (min)
`
`7
`
`8
`
`9
`
`10
`
`;· :
`"
`
`B.
`
`44
`
`0
`0
`M
`
`(I) 22
`(,)
`1:
`Ill
`..0
`....
`g 11
`<t
`
`0
`0
`CD
`
`0
`
`0 ..,.
`
`..0 o.I..J,....,....~~Uu~~:;;;;:;;..g~:;:;;;;;:;;
`
`2
`
`3
`
`6
`5
`4
`Time (min)
`
`7
`
`8
`
`9
`
`10
`
`c.
`
`12
`
`0 c:o
`N
`
`(I) 6
`(,)
`1:
`Ill
`..0
`
`.... g 3
`..0
`<t
`
`0
`0 M
`..,.
`0
`LO
`
`0 c:o
`1'-
`
`2
`
`3
`
`6
`5
`4
`Time (min)
`
`7
`
`8
`
`9
`
`10
`
`Fig. I. Chromatographic resolution of RNA standards. Three sets of single-stranded RNA molecular mass markers were separated by HPLC
`on a DNASep column using the elution gradient profile described in the Experimental section. (A) 0.24-9.5 kb RNA ladder (GLBCO-BRL),
`(B) 0.3-1.6 kb RNA molecular mass marker III (Boehringer-Mannheim), (C) 0.16-1.77 kb RNA ladder (GLBCO-BRL).
`
`5
`
`

`

`112
`
`D.E. Georgopoulos, M.J. Leibowitz I J. Chromatogr. A 868 (2000) 109-114
`
`the elution peiformed with the linear gradient pro(cid:173)
`files: 35 to 45% B (0 to 2 min), 45 to 55 % B (2 to 12
`min), 55 % B (12 to 14 min), 55 to 100% B (14 to 15
`min) followed by a 5 min wash in 100% B, and
`re-equilibration by a gradient from 100% to 35% B
`over 4 min.
`
`3. Results and discussion
`
`3.1. Fractionation of RNA molecules by HPLC
`
`Fig. 1 shows the chromatographic resolution of
`various single-stranded RNA standards by HPLC. It
`can be seen that in the size range of 240 to 1000
`nucleotides, different RNA species are well-resolved.
`Elution order is according to RNA chain length in
`the size range from 400 to 1000 nucleotides. As
`shown in Fig. 2, retention time can be used to
`
`10
`
`9
`
`t:
`
`6 E
`i=
`t:
`
`5
`
`8 -
`E 7 -CJ.)
`0 - 4
`CJ.) -
`
`t:
`
`CJ.)
`0:
`
`3
`
`•
`
`•
`
`•
`
`2
`
`RNA Size (nucleotides)
`
`Fig. 2. Molecular mass vs. retention time. Molecular masses of
`separated single-stranded RNA markers shown in Fig. I were
`plotted vs. elution time.
`
`this size range with
`estimate RNA size within
`accuracy comparable to that achieved by gel electro(cid:173)
`phoresis. It should be noted that chromatographic
`mobility of single-stranded RNA molecules may be
`determined by other factors in addition to size, as
`indicated by the apparently aben·ant retention times
`of some of the RNA molecules with sizes outside the
`apparent linear portion of the plot in Fig. 2. Electro(cid:173)
`phoretic gel mobility of native single-stranded RNA
`molecules can also be affected by conformation and/
`or sequence (for example, [14]) . Separation of RNA
`molecules in this size range suggested that this
`HPLC method could also be used to assay group I
`intron splicing.
`
`3.2. HPLC assay of ribozyme activity
`
`When the 660-nucleotide precursor RNA contain(cid:173)
`ing the Pcl.LSU group I intron is produced by in
`vitro
`transcription of a PCR product with th~~
`sequence downstream from a bacteriophage SRp
`promoter, the crude transcription reaction contains ·
`the precursor RNA (E1-I-E2) along with the inter(cid:173)
`mediates and products of the splicing reaction, which
`result from splicing occurring under transcription
`conditions [12]. For gel electrophoretic analysis of
`the splicing of in vitro radioactive transcript RNA,
`the precursor RNA is generally purified by prepara(cid:173)
`tive gel electrophoresis, to produce electrophoretical(cid:173)
`ly pure precursor RNA, which serves as the enzyme
`and substrate for the self-splicing reaction [12]. The
`use of radioactive RNA for ribozyme assays by gel
`analysis requires two electrophoresis steps: one to
`purify the precursor and another to assay the conver(cid:173)
`sion of precursor
`to reaction
`intermediates and
`products. Not only is
`this time consuming, but
`internally labeled radioactive precursor tends to show
`significant radiochemical breakdown over the course
`of several days, limiting the usefulness of each
`preparation. For this reason, we assayed ribozyme
`activity of a crude non-radioactive precursor using
`the HPLC method described here.
`As can be seen in Fig. 3A, the crude transcription
`reaction contains the predicted mix of RNA species,
`including the 660-nucleotide precursor RNA and the
`other species in descending size order: GI-E2 (382
`nucleotides), GI (355 nucleotides), E1-E2 (305
`
`6
`
`

`

`D.E. Georgopoulos, M.J. Leibowitz I J. Chromatogr. A 868 (2000) 109-114
`
`11 3
`
`A.
`
`2.0
`~ 1.8
`s:::
`~ 1.6
`<(
`_§_ 1.4
`1.2
`
`Q)
`0
`s:::
`ro
`..c ...
`0
`Ill
`..c
`<(
`
`1.0
`
`0.8
`
`0.6 °
`
`0.4
`
`3
`
`j
`
`"' ~
`w
`
`}
`
`8
`
`~
`r) . ) ,
`
`~~ ~~~'"'~
`
`4
`
`5
`
`6
`
`Time (min)
`
`B.
`
`(i) 1.2 -·c:
`
`1.4
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`-0.2
`
`3
`
`~
`<(
`_§_
`
`Q)
`0
`s:::
`ro
`...
`..c
`0
`Ill
`..c
`<(
`
`"'
`~
`
`"'
`
`);Jl~H~~~
`
`4
`
`5
`
`Time (min)
`
`6
`
`Fig. 3. HPLC chromatographic analys is of splicing of 660-nucleotide precursor RNA containing Pcl.LSU group I intron. (A) Unreacted
`660-nucleotide RNA transcript chromatogram, indicating positions of precursor (El - I- E2), intermediate (GI-E2 and El ), and final product
`(GI and EIE2) peaks. (B) Reacted RNA transcript chromatogram showing progress of group I splicing reaction, indicating di sappearance of
`El-I-E2 precursor peak and increase of GI- E2 intermediate.
`
`nucleotides) and E1 (279 nucleotides) [12]. Peak
`collection and analysis by agarose gel electrophoresis
`confirmed the identification of the splicing product
`species (data not shown) . In this chromatogram, the
`level of GI-E2 is barely detectable, while the
`precursor is a major peak. When the same sample
`was analyzed after allowing the splicing reaction to
`occur (Fig. 3B ), the precursor peak was markedly
`reduced while the GI-E2 intermediate became a
`major species. Thus, HPLC can be used instead of
`gel electrophoresis to fractionate RNA species in the
`
`group I intron ribozyme assay, which can now be .
`performed without the use of radioactivity.
`The HPLC method described here should also be
`applicable to the analysis and purification of other
`RNA molecules of up to 1 kb in length.
`
`4. Conclusion
`
`The present method allows HPLC to be used for
`
`7
`
`

`

`11 4
`
`DE. Georgopoulos, M.J. Leibo111itz I J. Ch romatog r. A 868 (2000) 109- 114
`
`the rapid fractionation of RNA molecules of up to 1
`kb in length .
`
`Acknowledgements
`
`This work was partially supported by grant num(cid:173)
`ber GM53815 from the National Institutes of Health.
`
`References
`
`[I] T.R. Cech, Annu. Rev. Biochem. 59 ( 1990) 543-568.
`[2] F. Michel, J.L. Ferat, Annu. Rev. Biochem. 64 (1995)
`435- 46 1.
`[3] H.Y. Mei, M . Cui, S.T. Sutton, H.N. Truong, F.Z. Chung,
`A.W. Czarnik, Nucleic Acids Res. 24 ( 1996) 5051-5053.
`[4] A.C. Anderson, S.A. Scaringe, B.E. Earp, C.A. Frederick,
`RNA 2 (1996) 110-117.
`
`[5] R. Vinayak, A. Andrus, N .D. Sinha, A. Hampel, Anal.
`Biochem. 232 ( 1995) 204-209.
`[6] L. Citti, L. Boldrini , S. Nevischi, L. Mariani, G . Rai naldi ,
`BioTechniques 23 ( 1997) 898-903.
`[7] C.G. Huber, P.J. Oefner, G.K. Bonn, Anal. Biochem. 212
`(1993) 35 1-358 .
`[8] C.G. Huber, P.J. Oefner, E. Preuss, G.K. Bonn, Nucleic
`Acids Res. 21 (1 993) 1061-1066.
`[9] P.J. Oefner, C .G. Huber, E. Puchhammer-Stockl, F. Umlauft,
`K. Grunewald, G.K. Bonn, C. Kunz, BioTechniques 16
`( 1994) 898-908.
`[ l 0] E. D. Katz, M.W. Dong, BioTechniques 8 ( 1990) 546-555.
`[ll] Y. Liu, M.J. Leibowitz, Nucleic Acids Res. 21 (1993) 2415-
`2421.
`[1 2] Y. Liu, R.R. Tidwell, M.J. Le ibow itz, J. Euk. Microbiol. 41
`( 1994) 31-38.
`[1 3] S.C. Nam, C. Kang, J. Bioi. Chem. 263 (1988) 18123-
`18 127.
`[14] D.J. Thiele, M.J. Leibowitz, Nucleic Acids Res. 10 (1982)
`6903-6918.
`
`8
`
`