Microsecond Equilibrium Dynamics of Hairpin-Forming Oligonucleotides Quantified by Two-Color Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Abstract

RNA and DNA play distinct roles in biological systems. However, the underlying physicochemical difference has been poorly understood, in particular, that in dynamical aspects. In this paper, we report on a comparative study of the formation−dissociation dynamics of a hairpin structure of RNA and DNA with development of two-color two-dimensional fluorescence lifetime correlation spectroscopy (two-color 2D FLCS). In this extension of 2D FLCS, we newly introduce the two-color detection scheme to analyze not only donor fluorescence photons but also acceptor fluorescence photons from a doubly labeled Forster resonance energy transfer (FRET) pair. This new 2D FLCS is utilized to resolve multiple species present in an equilibrated condition with a microsecond time resolution and enhanced sensitivity, and the combined use with the filtered fluorescence correlation spectroscopy (FCS) method enables a quantitative discussion on microsecond structural dynamics occurring in the equilibrium. This integrated approach is applied to FRET-labeled RNA/DNA oligonucleotides having analogous hairpin-forming sequences, and it was revealed that the hairpin dissociation rate of RNA is an order of magnitude slower than that of DNA while their hairpin-forming rates are comparable. This marked difference is attributable to the distinct duplex structure of RNA and DNA. The present study demonstrates that the integrated approach combining two-color 2D FLCS and filtered FCS has a high potential for quantifying microsecond kinetics at the single-molecule level, which allows us to experimentally construct a free energy landscape.

Microsecond Folding of preQ1 Riboswitch and Its Biological Significance Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

“…We performed the filtered FCS analysis , using the independent fluorescence compoments determined by 2D FLCS to obtain species-specific auto- and cross-correlations . Note that we performed “restricted” filtered FCS analysis in which the D-only component is restricted to show zero cross-correlations with the O and the F forms, consistent with the restricted global 2D MEM analysis used for determining the independent fluorescence components in 2D FLCS (see Supporting Information for details).…”

“…Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy (2D FLCS). To clarify the structural dynamics of the Bsu preQ 1 aptamer on the time scale from microsecond to millisecond, we performed two-color 2D FLCS measurements 32 on the FRET-pair-labeled aptamer in the apo state, in the presence of 5 μM preQ 1 , and in the presence of 2 mM Mg 2+ . The details of two-color 2D FLCS and the analysis of the obtained data are described in the Supporting Information.…”

“…We utilized the independent fluorescence components obtained with two-color 2D FLCS for the filtered FCS analysis as reported in ref . In this analysis, for separating the “unreactive” component (D-only) from others, we “corrected” the obtained species-specific correlations by setting cross-correlations of D-only to zero.…”

“…In this study, we investigate the microsecond to millisecond dynamics of the Bsu preQ 1 aptamer by two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). , Two-dimensional FLCS is a powerful single-molecule spectroscopy method that can identify individual conformers of a biopolymer based on the fluorescence lifetime and can clarify their interconversion dynamics with a microsecond time resolution. − The 2D FLCS data obtained indicates that the Bsu preQ 1 aptamer actually undergoes a three-state folding/unfolding process that proceeds in a wide time range from 1 μs to more than 10 ms. Furthermore, it is indicated that the binding of preQ 1 significantly accelerates the microsecond folding process with an induced-fit mechanism, while Mg 2+ binding only changes the dynamics on the slower time scale with a conformational selection mechanism.…”

Targeting G-quadruplexes in the rhinovirus genome by Pyridostatin inhibits uncoating and highlights a critical role for sodium ions.

“…Presumably, during positive-strand RNA synthesis in the infected cells, sequences of the nascent (+) strand comprising these QGRS fold much faster into alternative, metastable conformations such as RNA hairpins than into the more stable GQ (tens of microseconds vs hundreds of milliseconds 110,111 ) as soon as they emerge from the active centre of the viral RNA replicase. If separated from each other by an appreciable activation energy barrier, as indicated by the ThT analysis, these metastable structures, following their rapid encapsidation (which is tightly coupled with replication 112 ), will persist in the progeny virions.…”