Chemically Modified Hammerhead Ribozymes with Improved Catalytic Rates

Burgin, Alex B.; Gonzalez, C.; Matulić‐Adamić, Jasenka; Karpeisky, Alexander; Usman, Nassim; McSwiggen, James A.; Beigelman, Leonid

doi:10.1021/bi961264u

Cited by 72 publications

(46 citation statements)

References 36 publications

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“…All analogs were incorporated into RNA using standard solid phase RNA synthesis protocols. The fidelity of incorporation of the modified nucleosides into RNA was confirmed by base compositional analysis as described previously (12). All oligonucleotides were gel-purified prior to use (1).…”

Section: Methodsmentioning

confidence: 99%

Specific Recognition of an rU2-N15-rU Motif by VP55, the Vaccinia Virus Poly(A) Polymerase Catalytic Subunit

Deng

Beigelman²,

Matulić‐Adamić³

et al. 1997

Journal of Biological Chemistry

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VP55, the vaccinia poly(A) polymerase catalytic subunit, interacts with oligonucleotide primers via two uridylate recognition sites (Deng, L., and Gershon, P. D. (1997) EMBO J. 16, 1103-1113). Here, we show that the cognate RNA sequence comprises a 5-rU 2 -N 15 -rU-3 motif (where N ‫؍‬ any deoxyribo or ribonucleotide), embedded within oligonucleotide primers 29 -30 nucleotides (nt), or greater, in length. Nine residues separate the 3-most ribouridylate of the optimally positioned motif from the primer 3-OH. A ribose sugar at the extreme 3-terminal nucleotide of the primer is absolutely required for VP55's adenylyltransferase activity, but not for stable VP55-RNA interaction. A ribose at position ؊3 markedly stimulates both adenylyltransferase activity and stable binding. The use of uridine analogs indicated (i) those functional groups of the uracil base which contribute to stable VP55-primer interaction, and (ii) that VP55's ability to discriminate uracil from cytosine stems largely from the requirement for a protonated N3 nitrogen within the pyrimidine ring. The rU 2 -N 15 -rU motif was identified within the uridylate-rich 3 end of a naturally occurring vaccinia mRNA. However, oligonucleotides whose only internal uridylates comprised the motif supported only a 3-5-nt processive burst of oligo(A) tail addition, as opposed to the ϳ30 -35-nt burst observed with the naturally occurring 3 end.One unusual feature of the poly(A) polymerase (PAP) 1 encoded by vaccinia virus is its heterodimeric structure (2-4). However, roles for the individual subunits within the heterodimer have been established by examination of the in vitro properties of the individual subunits. Thus, the isolated larger (VP55) subunit possesses PAP catalytic activity, and is able to add ϳ30 -35-nt oligo(A) tails to RNA 3Ј ends in a rapid and processive burst of polyadenylylation, before switching, abruptly, to a very slow and non-processive mode of adenylate addition (2, 5). The isolated smaller (VP39) subunit has no PAP catalytic activity, but its addition to VP55 permits tails that are greater than ϳ35 nt in length to be processively elongated to an overall length of several hundred nucleotides, in vitro (6). In addition to its activity as a PAP processivity factor, VP39 has an entirely unrelated function, at the mRNA 5Ј end. Thus, as an mRNA cap-specific 2Ј-O-methyltransferase, VP39 modifies the ribose sugar of the penultimate nucleotide of the mRNA type 0 cap structure to a 2Ј-O-methylated nucleotide (7).Since VP55 extends oligo(A) primers greater than 30 -35 nt in length in only a slow, non-processive mode of adenylate addition, the ability of this subunit to catalyze the processive polyadenylylation burst is apparently triggered by some signal other than oligo(A) or poly(A) (5). This signal was shown to comprise, in some manner, a high content of uridylate residues within the 3Ј-terminal 30 -40 nt of the initial RNA primer, in no obvious arrangement or pattern (8). Consistent with this, VP55 interacts stably with uridylate-rich RNA segments ...

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Section: Methodsmentioning

confidence: 99%

Specific Recognition of an rU2-N15-rU Motif by VP55, the Vaccinia Virus Poly(A) Polymerase Catalytic Subunit

Deng

Beigelman²,

Matulić‐Adamić³

et al. 1997

Journal of Biological Chemistry

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“…Such modifications would be expected to enhance the cleavage rate of the minimal hammerhead compared to the wild-type sequence. An example of such a modification that was found accidentally in the minimal hammerhead involved introducing a pyridin-4-one at position 7 that appears to enhance cleavage by destabilizing the A14-U7 pair in the minimal hammerhead structure and not affecting the structure of the extended hammerhead (Burgin et al 1996;Nelson and Uhlenbeck 2006). It will be interesting to see whether a pyridin-4-one at position 7 or any other activating modification would also enhance the rate of cleavage when introduced into an extended hammerhead.…”

Section: Introductionmentioning

confidence: 99%

Hammerhead redux: Does the new structure fit the old biochemical data?

Nelson¹,

Uhlenbeck

2008

RNA

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The cleavage rates of 78 hammerhead ribozymes containing structurally conservative chemical modifications were collected from the literature and compared to the recently determined crystal structure of the Schistosoma mansoni hammerhead. With only a few exceptions, the biochemical data were consistent with the structure, indicating that the new structure closely resembles the transition state of the reaction. Since all the biochemical data were collected on minimal hammerheads that have a very different structure, the minimal hammerhead must be dynamic and occasionally adopt the quite different extended structure in order to cleave.

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“…Because the first exponential decay accounted for 70% of total degradation, this portion was taken for the calculation of the rate constant k obs . As other authors have suggested, the second, slower segment of substrate cleavage presumably reflects ribozyme-substrate complexes in alternative (inactive or less active) conformations (23).…”

Section: Kinetic Analysismentioning

confidence: 75%

“…1. The sequences of ribozyme and substrate are based on literature reports of catalytically active hammerhead structures (21)(22)(23). The central core motif contains 17 nucleotides with only 5 purine ribose residues, namely G5, A6, G8, G12, and A15.1.…”

Section: Ribozyme-substrate Complexmentioning

confidence: 99%