Analysis of Multidimensional G‐Quadruplex Melting Curves

Gray, Robert D.; Chaires, Jonathan B.

doi:10.1002/0471142700.nc1704s45

Cited by 60 publications

(63 citation statements)

References 15 publications

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“…The most stable sequence was LTR-III (T m 62.1 ± 0.1 °C), followed by LTR-IV and LTR-III + IV (T m 59.1 ± 0.1 °C and 55.2 ± 0.1 °C, respectively); LTR-II + III + IV was the least stable sequence (T m 49.1 ± 0.1), confirming that the observed unfolding scale depended on the oligonucleotide stability in the FRET system. To note that in the unlabelled oligonucleotides measured by circular dichroism (CD), LTR-IV was the least stable sequence (Table 2), bias which likely derives by the absence of fluorophores at the oligonucleotides’ 5′- and 3′-end, which affect T m values, as previously noted32.…”

Section: Resultsmentioning

confidence: 88%

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

Scalabrin

Frasson

Ruggiero

et al. 2017

Sci Rep

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G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

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Section: Resultsmentioning

confidence: 88%

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

Scalabrin

Frasson

Ruggiero

et al. 2017

Sci Rep

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“…To enumerate the number of significant spectral species involved in the melting processes, we performed singular value decomposition analysis (SVD) on the data matrices obtained by monitoring CD spectra as a function of temperature 51,62 . The magnitudes of the singular values provide the first indication of the number of significant spectral species.…”

Section: Results and Analysismentioning

confidence: 99%

Structure and Stability of Higher-Order Human Telomeric Quadruplexes

et al. 2011

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G-quadruplex formation in the sequences 5′-(TTAGGG)n and 5′(TTAGGG)nTT (n=4,8,12) was studied using circular dichroism, sedimentation velocity, differential scanning calorimetry and molecular dynamics simulations. Sequences containing 8 and 12 repeats formed higher-order structures with two and three contiguous quadruplexes, respectively. Plausible structures for these sequences were determined by molecular dynamics simulations followed by experimental testing of predicted hydrodynamic properties by sedimentation velocity. These structures featured folding of the strand into contiguous quadruplexes with mixed hybrid conformations. Thermodynamic studies showed the strands folded spontaneous to contain the maximum number contiguous quadruplexes. For the sequence 5′(TTAGGG)12TT, more than 90% of the strands contained completely folded structures with three quadruplexes. Statistical mechanical-based deconvolution of thermograms for three quadruplex structures showed that each quadruplex melted independently with unique thermodynamic parmameters. Thermodynamic analysis revealed further that quadruplexes in higher-ordered structures were destabilized relative to their monomeric counterparts, with unfavorable coupling free energies. Quadruplex stability thus depends critically on the sequence and structural context.

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“…39,43,48,49 Simultaneously collected wavelength data show non-linear correlations (Supporting Information Figure 2), while derivative curves (δΔε/δT) demonstrated the presence of multiple unique minimum and maximum values (Figure 2). These two analysis techniques indicate that ht-quadruplex unfolding cannot be described by a two-state mechanism.…”

Section: Discussionmentioning

confidence: 99%

“…39 The three-dimensional data matrix contains elements D i,j , where each row i corresponds to a melting profile at λ i and each column j represents a CD spectra collected at temperature T j . The matrix D was analyzed by SVD, generating three additional matrices such that D = USV T .…”

Section: Methodsmentioning

confidence: 99%

Thermodynamic characterization of human telomere quadruplex unfolding

2013

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The 3'-terminal extension of eukaryotic chromosomes are a unique example of functional single-stranded DNA. Human telomeres are constructed of the repeated DNA sequence 5'-d(TTAGGG). Four-repeats of human telomeric DNA have been characterized by high-resolution techniques to be capable of forming at least five distinct monomeric conformations. The predominant solution topology is influenced by solution conditions and the presence of 3'- or 5'-flanking residues. This study extends recently described sequential unfolding mechanisms for human telomeric quadruplexes to eight telomeric DNA sequences that form three unique topologies. Thermal unfolding monitored by circular dichroism is subjected to singular value decomposition to enumarate the number of significant spectral species required to model the unfolding process. Thermal denaturations are found to be best modeled by a four-state sequential mechanism with ΔGave = −8.8 kcal mol−1. The thermal unfolding was also investigated in 50% (v/v) acetonitrile. Under dehydrated conditions, quadruplex thermal denaturations are best modeled by a three-state sequential unfolding mechanism. Dehydrated quadruplexes demonstrate increased thermal stability with ΔGave = −12.7 kcal mol−1. A common spectral intermediate is shared amongst human telomere quadruplex thermal unfolding regardless of starting topology and hydration state. This unfolding intermediate was recently characterized as an ensemble of triple-helical structures for a 22-nt human telomeric quadruplex.

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Analysis of Multidimensional G‐Quadruplex Melting Curves

Cited by 60 publications

References 15 publications

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

Structure and Stability of Higher-Order Human Telomeric Quadruplexes

Thermodynamic characterization of human telomere quadruplex unfolding

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