Improved deoxyribozymes for synthesis of covalently branched DNA and RNA

Christine S. Lee, Timothy P. Mui, Scott K. Silverman

Research output: Contribution to journalArticle

Abstract

A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn2+ as a cofactor, rather than Mg2+ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has kobs on the order of 0.1min-1, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.

Original languageEnglish (US)
Pages (from-to)269-279
Number of pages11
JournalNucleic acids research
Volume39
Issue number1
DOIs
StatePublished - Jan 17 2011

Fingerprint

Catalytic DNA
RNA
DNA
Nucleic Acids
Ribonucleotides
Biotechnology
Oligonucleotides
Hydroxyl Radical
Biochemistry
Phosphorus
Nucleotides

ASJC Scopus subject areas

  • Genetics

Cite this

Improved deoxyribozymes for synthesis of covalently branched DNA and RNA. / Lee, Christine S.; Mui, Timothy P.; Silverman, Scott K.

In: Nucleic acids research, Vol. 39, No. 1, 17.01.2011, p. 269-279.

Research output: Contribution to journalArticle

Lee, Christine S. ; Mui, Timothy P. ; Silverman, Scott K. / Improved deoxyribozymes for synthesis of covalently branched DNA and RNA. In: Nucleic acids research. 2011 ; Vol. 39, No. 1. pp. 269-279.
@article{48dc504bf32240949380a1bd908dbe2b,
title = "Improved deoxyribozymes for synthesis of covalently branched DNA and RNA",
abstract = "A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn2+ as a cofactor, rather than Mg2+ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has kobs on the order of 0.1min-1, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.",
author = "Lee, {Christine S.} and Mui, {Timothy P.} and Silverman, {Scott K.}",
year = "2011",
month = "1",
day = "17",
doi = "10.1093/nar/gkq753",
language = "English (US)",
volume = "39",
pages = "269--279",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "1",

}

TY - JOUR

T1 - Improved deoxyribozymes for synthesis of covalently branched DNA and RNA

AU - Lee, Christine S.

AU - Mui, Timothy P.

AU - Silverman, Scott K.

PY - 2011/1/17

Y1 - 2011/1/17

N2 - A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn2+ as a cofactor, rather than Mg2+ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has kobs on the order of 0.1min-1, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.

AB - A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn2+ as a cofactor, rather than Mg2+ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has kobs on the order of 0.1min-1, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA.

UR - http://www.scopus.com/inward/record.url?scp=78651267394&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78651267394&partnerID=8YFLogxK

U2 - 10.1093/nar/gkq753

DO - 10.1093/nar/gkq753

M3 - Article

C2 - 20739352

AN - SCOPUS:78651267394

VL - 39

SP - 269

EP - 279

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 1

ER -