Background: When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. Methods: I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and > 20 complementary oligonucleotides ('antisenses'). Results: Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. Conclusions: The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. General significance: While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process.

Dissecting the hybridization of oligonucleotides to structured complementary sequences / Peracchi, Alessio. - In: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS. - ISSN 0304-4165. - 1860:6(2016), pp. 1107-1117. [10.1016/j.bbagen.2016.02.006]

Dissecting the hybridization of oligonucleotides to structured complementary sequences

PERACCHI, Alessio
2016-01-01

Abstract

Background: When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. Methods: I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and > 20 complementary oligonucleotides ('antisenses'). Results: Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. Conclusions: The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. General significance: While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process.
Dissecting the hybridization of oligonucleotides to structured complementary sequences / Peracchi, Alessio. - In: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS. - ISSN 0304-4165. - 1860:6(2016), pp. 1107-1117. [10.1016/j.bbagen.2016.02.006]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2823091
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