Mimicking the first step of RNA splicing: An artificial DNA enzyme can synthesize branched RNA using an oligonucleotide leaving group as a 5′-exon analogue

Rebecca L. Coppins, Scott K Silverman

Research output: Contribution to journalArticle

Abstract

The 7S11 deoxyribozyme synthesizes 2′,5′-branched RNA by mediating the nucleophilic attack of an internal 2′-hydroxyl group of one RNA substrate into the 5′-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5′-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5′-diphosphate or 5′-monophosphate, the same 2′,5′-branched product is formed as when pyrophosphate is the leaving group, but with an ∼50- or ∼1000-fold lower rate (Brønsted βLG = -0.40). When the 5′-end of the RNA substrate that bears the leaving group is longer by one or more nucleotides, either the new 5′-terminal α-phosphate or the original α-phosphate can be attacked by the branch-site 2′-hydroxyl group; in the latter case, the leaving group is an oligonucleotide. The choice between these α-phosphate reaction sites is determined by the subtle balance between the length of the single-stranded 5′-extension and the stability of the leaving group. Because the branch-site adenosine is a bulged nucleotide flanked by Watson-Crick duplex regions, we earlier concluded that 7S11 structurally mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5′-exon in the first step. These findings reinforce the notion that splicing-related catalysis can be achieved by artificial nucleic acid enzymes that are much smaller than the spliceosome and group II introns.

Original languageEnglish (US)
Pages (from-to)13439-13446
Number of pages8
JournalBiochemistry
Volume44
Issue number41
DOIs
StatePublished - Oct 18 2005

Fingerprint

RNA Splicing
Oligonucleotides
Exons
RNA
Phosphates
DNA
Enzymes
Hydroxyl Radical
Nucleotides
Substrates
Catalytic DNA
Spliceosomes
Diphosphates
Catalysis
Adenosine
Introns
Nucleic Acids
Anions
Phosphorylation
Observation

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{78f0c137394b4c74be6a7c0728c11922,
title = "Mimicking the first step of RNA splicing: An artificial DNA enzyme can synthesize branched RNA using an oligonucleotide leaving group as a 5′-exon analogue",
abstract = "The 7S11 deoxyribozyme synthesizes 2′,5′-branched RNA by mediating the nucleophilic attack of an internal 2′-hydroxyl group of one RNA substrate into the 5′-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5′-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5′-diphosphate or 5′-monophosphate, the same 2′,5′-branched product is formed as when pyrophosphate is the leaving group, but with an ∼50- or ∼1000-fold lower rate (Br{\o}nsted βLG = -0.40). When the 5′-end of the RNA substrate that bears the leaving group is longer by one or more nucleotides, either the new 5′-terminal α-phosphate or the original α-phosphate can be attacked by the branch-site 2′-hydroxyl group; in the latter case, the leaving group is an oligonucleotide. The choice between these α-phosphate reaction sites is determined by the subtle balance between the length of the single-stranded 5′-extension and the stability of the leaving group. Because the branch-site adenosine is a bulged nucleotide flanked by Watson-Crick duplex regions, we earlier concluded that 7S11 structurally mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5′-exon in the first step. These findings reinforce the notion that splicing-related catalysis can be achieved by artificial nucleic acid enzymes that are much smaller than the spliceosome and group II introns.",
author = "Coppins, {Rebecca L.} and Silverman, {Scott K}",
year = "2005",
month = "10",
day = "18",
doi = "10.1021/bi0507229",
language = "English (US)",
volume = "44",
pages = "13439--13446",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "41",

}

TY - JOUR

T1 - Mimicking the first step of RNA splicing

T2 - An artificial DNA enzyme can synthesize branched RNA using an oligonucleotide leaving group as a 5′-exon analogue

AU - Coppins, Rebecca L.

AU - Silverman, Scott K

PY - 2005/10/18

Y1 - 2005/10/18

N2 - The 7S11 deoxyribozyme synthesizes 2′,5′-branched RNA by mediating the nucleophilic attack of an internal 2′-hydroxyl group of one RNA substrate into the 5′-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5′-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5′-diphosphate or 5′-monophosphate, the same 2′,5′-branched product is formed as when pyrophosphate is the leaving group, but with an ∼50- or ∼1000-fold lower rate (Brønsted βLG = -0.40). When the 5′-end of the RNA substrate that bears the leaving group is longer by one or more nucleotides, either the new 5′-terminal α-phosphate or the original α-phosphate can be attacked by the branch-site 2′-hydroxyl group; in the latter case, the leaving group is an oligonucleotide. The choice between these α-phosphate reaction sites is determined by the subtle balance between the length of the single-stranded 5′-extension and the stability of the leaving group. Because the branch-site adenosine is a bulged nucleotide flanked by Watson-Crick duplex regions, we earlier concluded that 7S11 structurally mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5′-exon in the first step. These findings reinforce the notion that splicing-related catalysis can be achieved by artificial nucleic acid enzymes that are much smaller than the spliceosome and group II introns.

AB - The 7S11 deoxyribozyme synthesizes 2′,5′-branched RNA by mediating the nucleophilic attack of an internal 2′-hydroxyl group of one RNA substrate into the 5′-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5′-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5′-diphosphate or 5′-monophosphate, the same 2′,5′-branched product is formed as when pyrophosphate is the leaving group, but with an ∼50- or ∼1000-fold lower rate (Brønsted βLG = -0.40). When the 5′-end of the RNA substrate that bears the leaving group is longer by one or more nucleotides, either the new 5′-terminal α-phosphate or the original α-phosphate can be attacked by the branch-site 2′-hydroxyl group; in the latter case, the leaving group is an oligonucleotide. The choice between these α-phosphate reaction sites is determined by the subtle balance between the length of the single-stranded 5′-extension and the stability of the leaving group. Because the branch-site adenosine is a bulged nucleotide flanked by Watson-Crick duplex regions, we earlier concluded that 7S11 structurally mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5′-exon in the first step. These findings reinforce the notion that splicing-related catalysis can be achieved by artificial nucleic acid enzymes that are much smaller than the spliceosome and group II introns.

UR - http://www.scopus.com/inward/record.url?scp=26644465737&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=26644465737&partnerID=8YFLogxK

U2 - 10.1021/bi0507229

DO - 10.1021/bi0507229

M3 - Article

C2 - 16216067

AN - SCOPUS:26644465737

VL - 44

SP - 13439

EP - 13446

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 41

ER -