Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin

Joanne M. Bevilacqua, Philip C. Bevilacqua

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The T(MS) of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson- Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in ΔG°37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.

Original languageEnglish (US)
Pages (from-to)15877-15884
Number of pages8
JournalBiochemistry
Volume37
Issue number45
DOIs
StatePublished - Nov 10 1998

Fingerprint

Denaturing Gradient Gel Electrophoresis
Thermodynamics
Gels
RNA
Electrophoresis
Thermal gradients
Base Pairing
Thermodynamic stability
Nucleic Acids
Magnesium Chloride
Potassium Chloride
Guanosine
Random Allocation
Sodium Chloride
Small Interfering RNA
Freezing
Libraries
Urea
Salts
Binding Sites

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{8d6ac62668aa416e8af70ebdf1b08296,
title = "Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin",
abstract = "Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The T(MS) of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson- Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in ΔG°37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.",
author = "Bevilacqua, {Joanne M.} and Bevilacqua, {Philip C.}",
year = "1998",
month = "11",
day = "10",
doi = "10.1021/bi981732v",
language = "English (US)",
volume = "37",
pages = "15877--15884",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "45",

}

Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin. / Bevilacqua, Joanne M.; Bevilacqua, Philip C.

In: Biochemistry, Vol. 37, No. 45, 10.11.1998, p. 15877-15884.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Thermodynamic analysis of an RNA combinatorial library contained in a short hairpin

AU - Bevilacqua, Joanne M.

AU - Bevilacqua, Philip C.

PY - 1998/11/10

Y1 - 1998/11/10

N2 - Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The T(MS) of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson- Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in ΔG°37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.

AB - Prediction of nucleic acid structure from sequence requires thermodynamic parameters for a variety of motifs, many of which are complex and consist of a large number of possible sequence combinations. Here we report an experimental approach for identifying the stable and unstable members of an RNA combinatorial library. Short model RNA hairpins consisting of 13 base pairs (bp) flanked by primer binding sites are constructed and separated according to their relative thermodynamic stabilities using temperature gradient gel electrophoresis (TGGE). Partially denaturing TGGE is carried out with potassium chloride, sodium chloride, or magnesium chloride salts in the gel. The T(MS) of model hairpins can be tuned by adjusting the concentration of urea in the gel while maintaining the correct order of stabilities for the hairpins. Mixtures of RNAs differing by a single Watson- Crick base pair are resolved according to their relative thermodynamic stabilities, as are mixtures of GC or AU base pair transversions differing in ΔG°37 by only 0.3-0.5 kcal/mol. In addition, a simple combinatorial library with one position of randomization opposite a guanosine is prepared and separated into its four members by parallel and perpendicular TGGE. The order of thermodynamic stabilities for the library determined by TGGE is shown to be the same when assayed by UV-melting experiments. Analysis of the thermodynamics of folding of combinatorial libraries is general and may be applied to a wide variety of complex nucleic acid secondary and tertiary motifs in order to identify the stable and unstable members.

UR - http://www.scopus.com/inward/record.url?scp=0032506045&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032506045&partnerID=8YFLogxK

U2 - 10.1021/bi981732v

DO - 10.1021/bi981732v

M3 - Article

C2 - 9843393

AN - SCOPUS:0032506045

VL - 37

SP - 15877

EP - 15884

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 45

ER -