Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1995-06-07
2001-04-24
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S006120, C435S091310, C435S252300, C435S320100, C435S325000, C435S375000, C435S410000, C435S455000, C435S468000, C435S476000, C536S023100, C536S023200, C536S024500
Reexamination Certificate
active
06221661
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an RNA catalyst which cleaves specific RNA sequences into a fragment having a 5′ hydroxyl and a fragment having a 2′,3′ cyclic phosphate. The products of the reaction described herein resemble those resulting from the natural hydrolysis of RNA.
BACKGROUND OF THE INVENTION
Certain naturally occurring satellite, virusoid and viroid RNAs possess the property of self-catalyzed cleavage. Self-cleavage has been demonstrated in vitro for avocado sunblotch viroid (ASBV) (Hutchins, C. J., Rathjen, P. D., Forster, A. C. and Symons, R. H. (1986)
Nucleic Acids Res.,
14: 3627-3640), satellite RNA from tobacco ringspot virus (sTRSV) (Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R. and Bruening, G. (1986)
Science,
231: 1577-1580; Buzayan, J. M., Gerlach, W. L. and Bruening, G. B. (1986)
Proc. Natl. Acad. Sci. U.S.A.
83: 8859-8862) and lucerne transient streak virus (vLTSV) (Forster, A. C. and Symons, R. H. (1987)
Cell,
49: 211-220). These self-catalyzed RNA cleavage reactions share a requirement for divalent metal ions and neutral or higher pH and cleave target RNA sequences to give 5′ hydroxyl and 2′,3′-cyclic phosphate termini (Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R. and Bruening, G. (1986)
Science,
231: 1577-1580; Forster, A. C. and Symons, R. H. (1987)
Cell,
49: 211-220; Epstein, L. M. and Gall, J. G. (1987)
Cell,
48: 535-543; Buzayan, J. M. Gerlach, W. L., Bruening, G. B., Keese, P. and Gould, A. R. (1986)
Virology,
151: 186-199).
A “hammerhead” model has been proposed and accurately describes the catalytic center of (+)sTRSV RNA, the (+) and (−) strands of ASBV and the (+) and (−) strands of VLTSV (Forster, A. C. and Symons, R. H. (1987)
Cell,
49: 211-220). The single exception is (−)sTRSV RNA which does not fit the “hammerhead” model (Forster, A. C. and Symons, R. H. (1987)
Cell,
49: 211-220; Buzayan, J. M., Gerlach, W. L. and Bruening, G. (1986)
Nature,
323: 349-352; Buzayan, J. M., Hampel, A. and Bruening, G. B. (1986)
Nucleic Acids Res.,
14: 9729-9743), and the structure of whose catalytic center was unknown prior to the present invention. It is therefore understandable that the primary scientific focus has been on studying the “hammerhead” consensus structure and, as regards sTRSV, on studying the (+) strand.
Intermolecular cleavage of an RNA substrate by an RNA catalyst that fits the “hammerhead” model was first shown in 1987 (Uhlenbeck, O. C. (1987)
Nature,
328: 596-600). The RNA catalyst was recovered and reacted with multiple RNA molecules, demonstrating that it was truly catalytic.
Catalytic RNAs designed based on the “hammerhead” motif have been used to cleave specific target sequences by making appropriate base changes in the catalytic RNA to maintain necessary base pairing with the target sequences (Haseloff and Gerlach,
Nature,
334, 585 (1988); Walbot and Bruening,
Nature,
334, 196 (1988); Uhlenbeck, O. C. (1987)
Nature,
328: 596-600; Koizumi, M., Iwai, S. and Ohtsuka, E. (1988)
FEBS Lett.,
228: 228-230). This has allowed use of the catalytic RNA to cleave specific target sequences and indicates that catalytic RNAs designed according to the “hammerhead” model may possibly cleave specific substrate RNAs in vivo. (see Haseloff and Gerlach,
Nature,
334, 585 (1988); Walbot and Bruening,
Nature,
334, 196 (1988); Uhlenbeck, O. C. (1987)
Nature,
328: 596-600).
However, catalytic RNAs such as those that were designed based on the “hammerhead” model have several limitations which restrict their use in vitro and may forestall their use in vivo. For example, the temperature optimum for the reaction is 50-55° C., which is well above physiological, and the kcat (turnover number) is only 0.5/min even at 55° C. (Uhlenbeck, O. C. (1987)
Nature,
328:596-600; Haseloff and Gerlach,
Nature,
334, 585 (1988)). In addition, the Km is 0.6 uM (Uhlenbeck, O. C. (1987)
Nature,
328:596-600), meaning that the reaction requires high concentrations of substrate which makes it difficult, if not impossible, for the catalytic RNA to cleave low levels of target RNA substrate such as would be encountered in vivo.
Cech et al. published application WO 88/04300 and U.S. Pat. No. 4,987,071 also report the preparation and use of certain synthetic ribozymes that have several activities, including endoribonuclease activity. The design of these ribozymes is based on the properties of the Tetrahymena ribosomal RNA self-splicing reaction. A temperature optimum of 50° C. is reported (page 39 of WO 88/04300; col. 20, lines 4-5, of U.S. Pat. No. 4,987,071) for the endoribonuclease activity, and the Km and kcat reported for this activity are 0.8 uM and 0.13/minute, respectively (Example VI, last paragraph).
In view of the above, there is a need for an RNA catalyst having a lower temperature optimum, preferably near physiological temperatures, a higher turnover number and a smaller Km and which can be engineered to cut specific target RNA substrates. Accordingly, based on the discovery of a totally different structure disclosed hereinafter, it is an object of the present invention to provide such an RNA catalyst. Other objects and features of the invention will be in part apparent and in part pointed out. The invention, accordingly, comprises the products and methods hereinafter described and their equivalents, the scope of the invention being indicated in the appended claims.
SUMMARY OF THE INVENTION
The invention comprises a synthetic RNA catalyst capable of cleaving an RNA substrate which contains the sequence:
5′—F
1
—CS—F
2
—3′,
wherein,
CS is a cleavage sequence; and
F
1
and F
2
each is a sequence of bases flanking the cleavage sequence.
The catalyst comprises a substrate binding portion and a “hairpin” portion. The substrate binding portion of the catalyst has the sequence:
3′—F
4
—L
1
—F
3
—5′
wherein,
F
3
is a sequence of bases selected so that F
3
is substantially base paired with F
2
when the catalyst is bound to the substrate;
F
4
is a sequence of bases selected so that F
4
is substantially base paired with F
1
when the catalyst is bound to the substrate;
the sequences of F
3
and F
4
being selected so that each contains an adequate number of bases to achieve sufficient binding of the RNA substrate to the RNA catalyst so that cleavage of the substrate can take place; and
L
1
is a sequence of bases selected so that L
1
does not base pair with CS when the catalyst is bound to the substrate.
The “hairpin” portion is a portion of the catalyst that assumes a hairpin-like configuration when the substrate-catalyst complex is modeled in two dimensions for minimum energy folding. The “hairpin” portion of the catalyst preferably has the sequence:
wherein,
P
1
and P
4
each is a sequence of bases, the sequences of P
1
and P
4
being selected so that P
1
and P
4
are substantially base paired;
P
1
is covalently attached to F
4
;
S
1
and S
2
each is a sequence of bases, the sequences of S
1
and S
2
being selected so that S
1
and S
2
are substantially unpaired;
P
2
and P
3
each is a sequence of bases, the sequences of P
2
and P
3
being selected so that P
2
and P
3
are substantially base paired; and
L
2
is a sequence of unpaired bases.
RNA catalysts according to the invention can cleave substrates of any length or type as long as they contain an appropriate cleavage sequence. In particular, the catalysts can be used to cleave a specific sequence in naturally-occurring RNA having a cleavage sequence, as well as RNAs which have been engineered to contain a cleavage sequence.
The invention further comprises an engineered DNA molecule and a vector, each of which comprises a DNA sequence that codes for an RNA catalyst according to the invention. The invention also comprises a host transformed with the vector, the host being capable of expressing the RNA catalyst. In particular, hosts can be transformed with vectors that, when transcribed, will produce RNA catalysts whic
Hampel Arnold E.
Hicks Margaret F.
Tritz Richard H.
Cooper & Dunham LLP
LeGuyader John L.
The of Regents for Northern Illinois University of DeKalb
White John P.
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