Nanomanipulation of Single RNA Molecules by Optical Tweezers

Stephenson, William; Wan, Gorby; Tenenbaum, Scott A.; Li, Pan T.X.

doi:10.3791/51542-v

Cited by 4 publications

(1 citation statement)

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“…Purified f HTT mRNA transcripts were combined with the dsDNA handles at 25 nM equimolar ratio, and annealed and refolded by denaturation at 85°C for 10 min, 65°C for 1.5 h, 55°C for 1.5 h and slow cooling to 4°C (0.1 °C/s) in 200 mM NaCl, 20 mM PIPES, pH 6.5, 60% formamide 44 . Formation of the desired DNA-RNA complexes were verified by agarose gel electrophoresis ( Supplementary Figure S1 ), purified using a PCR clean up kit (NEB, Cat# T1030L) and eluted in water.…”

Section: Methodsmentioning

confidence: 99%

Stick-slip unfolding favors self-association of expandedHTTmRNA

O’Brien,

Moulick,

Jiménez-Avalos

et al. 2024

Preprint

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In Huntington’s Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expandedhuntingtin(expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTTmRNA using single-molecule force spectroscopy. Whereas normalHTTmRNAs unfold reversibly and cooperatively, expHTTmRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTTmRNA.

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Section: Methodsmentioning

confidence: 99%

Stick-slip unfolding favors self-association of expandedHTTmRNA

O’Brien,

Moulick,

Jiménez-Avalos

et al. 2024

Preprint

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show abstract

The Physics of micro- and nano-manipulation: Fundamentals and applications

Zhuang,

Lin,

et al. 2024

Physics Reports

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Stick-slip unfolding favors self-association of expanded HTT mRNA

O’Brien,

Moulick,

Jiménez-Avalos

et al. 2024

Nat Commun

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In Huntington’s Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (exp HTT ) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of exp HTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, exp HTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of exp HTT mRNA.

show abstract

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Cited by 4 publications

References 0 publications

Stick-slip unfolding favors self-association of expandedHTTmRNA

Stick-slip unfolding favors self-association of expandedHTTmRNA

The Physics of micro- and nano-manipulation: Fundamentals and applications

Stick-slip unfolding favors self-association of expanded HTT mRNA

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