Topoisomerase-based ligation and cloning methods

Details Topoisomerase-based ligation and cloning methods Topoisomerase-based ligation and cloning methods

1998-06-12

2003-11-25

Marschel, Ardin H. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091200, C435S091410, C435S091510, C435S091500, C435S091520

Reexamination Certificate

active

06653106

ABSTRACT:

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, preceding the sequence listing and claims.
BACKGROUND OF THE INVENTION
Vaccinia topoisomerase binds duplex DNA and forms a covalent DNA-(3′-phosphotyrosyl)-protein adduct at the sequence 5′-CCCTT
1
. The enzyme reacts readily with a 36-mer CCCTT strand (DNA-p-RNA) composed of DNA 5′ and RNA 3′ of the scissile bond. However, a 36-mer composed of RNA 5′ and DNA 3′ of the scissile phosphate (RNA-p-DNA) is a poor substrate for covalent adduct formation. Vaccinia topoisomerase efficiently transfers covalently held CCCTT-containing DNA to 5′—OH terminated RNA acceptors; the topoisomerase can therefore be used to tag the 5′ end of RNA in vitro.
Religation of the covalently bound CCCTT-containing DNA strand to a 5′—OH terminated DNA acceptor is efficient and rapid (k
rel
>0.5 sec
−1
), provided that the acceptor DNA is capable of base-pairing to the noncleaved DNA strand of the topoisomerase-DNA donor complex. The rate of strand transfer to DNA is not detectably affected by base mismatches at the 5′ nucleotide of the acceptor strand. Nucleotide deletions and insertions at the 5′ end of the acceptor slow the rate of religation; the observed hierarchy of reaction rates is: +1 insertion >−1 deletion >+2 insertion >>−2 deletion. These findings underscore the importance of a properly positioned 5′ OH terminus in transesterification reaction chemistry, but also raise the possibility that topoisomerase may generate mutations by sealing DNA molecules with mispaired or unpaired ends.
Vaccinia topoisomerase, a 314-amino acid eukaryotic type I enzyme, binds and cleaves duplex DNA at a specific target sequence 5′-(T/C)CCTT
1
(1-3). Cleavage is a transesterification reaction in which the Tp
1
N phosphodiester is attacked by Tyr-274 of the enzyme, resulting in the formation of a DNA-(3′-phosphotyrosyl) protein adduct (4). The covalently bound topoisomerase catalyzes a variety of DNA strand transfer reactions. It can religate the CCCTT-containing strand across the same bond originally cleaved (as occurs during the relaxation of supercoiled DNA) or it can ligate the strand to a heterologous acceptor DNA 5′ end, thereby creating a recombinant molecule (5-7).
Duplex DNA substrates containing a single CCCTT target site have been used to dissect the cleavage and strand transfer steps. A cleavage-religation equilibrium is established when topoisomerase transesterifies to DNA ligands containing ≧18-bp of duplex DNA 3′ of the cleavage site (8-11). The reaction is in equilibrium because the 5′—OH terminated distal segment of the scissile strand remains poised near the active site by virtue of the fact that it is stably base-paired with the nonscissile strand. About 20% of the CCCTT-containing strand is covalently bound at equilibrium (11). “Suicide” cleavage occurs when the CCCTT-containing substrate contains no more than fifteen base pairs 3′ of the scissile bond, because the short leaving strand dissociates from the protein-DNA complex. In enzyme excess, >90% of the suicide substrate is cleaved (11).
The suicide intermediate can transfer the incised CCCTT strand to a DNA acceptor. Intramolecular strand transfer occurs when the 5′ —OH end of the noncleaved strand of the suicide intermediate attacks the 3′ phosphotyrosyl bond and expels Tyr-274 as the leaving group. This results in formation of a hairpin DNA loop (5). Intermolecular religation occurs when the suicide intermediate is provided with an exogenous 5′—OH terminated acceptor strand, the sequence of which is complementary to the single strand tail of the noncleaved strand in the immediate vicinity of the scissile phosphate (5). In the absence of an acceptor strand, the topoisomerase can transfer the CCCTT strand to water, releasing a 3′-phosphate-terminated hydrolysis product, or to glycerol, releasing a 3′-phosphoglycerol derivative (12). Although the hydrolysis and glycerololysis reactions are much slower than religation to a DNA acceptor strand, the extent of strand transfer to non-DNA nucleophiles can be as high as 15-40%.
The specificity of vaccinia topoisomerase in DNA cleavage and its versatility in strand transfer have inspired topoisomerase-based strategies for polynucleotide synthesis in which DNA oligonucleotides containing CCCTT cleavage sites serve as activated linkers for the joining of other DNA molecules with compatible termini (13). The present study examines the ability of the vaccinia topoisomerase to cleave and rejoin RNA-containing polynucleotides. It was shown previously that the enzyme did not bind covalently to CCCTT-containing molecules in which either the scissile strand or the complementary strand was composed entirely of RNA (9). To further explore the pentose sugar specificity of the enzyme, we have prepared synthetic CCCTT-containing substrates in which the scissile strand is composed of DNA-and RNA-containing halves. In this way, we show that the enzyme is indifferent to RNA downstream of the scissile phosphate, but is does not form the covalent complex when the region 5′ of the scissile phosphate is in RNA form. Also assessed is the contribution of base-pairing by the 5′ end of the acceptor strand to the rate of the DNA strand transfer reaction.
SUMMARY OF THE INVENTION
The present invention provides a method of covalently joining a DNA strand to an RNA strand comprising (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5′ single-strand tails; and (b) adding to the topoisomerase-DNA intermediate an acceptor RNA strand complementary to the 5′ single-strand tail under conditions permitting a ligation of the 5′ single-strand tail of the topoisomerase-DNA intermediate to the RNA acceptor strand and dissociation of the topoisomerase, thereby covalently joining the DNA strand to the RNA strand. The DNA cleavage substrate may be created by hybridizing a DNA strand having a topoisomerase cleavage site to one or more complementary DNA strands, thereby forming a DNA cleavage substrate having a topoisomerase cleavage site and a oligonucleotide leaving group located 3′ of a scissile bond or may be a plasmid vector comprising a topoisomerase cleavage site.
The present invention also provides a covalent topoisomerase-DNA intermediate having a 5′ single-strand tail.
Another aspect of the present invention provides a DNA-RNA molecule covalently joined by topoisomerase catalysis.
The present invention provides a covalently joined DNA-RNA molecule having a labeled 5′ end.
The present invention further provides a method of tagging a 5′ end of an RNA molecule comprising: (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5′ single-strand tails; and (b) adding to the topoisomerase-DNA intermediate a 5′-hydroxyl terminated RNA molecule complementary to the 5′ single-strand tail under conditions permitting a ligation of the covalently bound DNA strand of the topoisomerase-DNA intermediate to the RNA molecule and dissociation of the topoisomerase, thereby forming a 5′ end tagged DNA-RNA ligation product. The DNA cleavage substrate can be created, for example, by hybridizing a DNA strand having a topoisomerase cleavage site to a complementary DNA strand, thereby formi

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