Sequence-dependent theory of oligonucleotide hybridization kinetics

Marimuthu, Karthikeyan; Chakrabarti, Raj

doi:10.1063/1.4873585

Cited by 13 publications

(15 citation statements)

References 31 publications

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“…The correlation of the association rate with extent of base pairing could be due to RNAs with greater complementarity also having a greater probability of nucleating duplex formation due to the increased number of possible toeholds or short stretches of pairing interactions. This hypothesis is consistent with previous single molecule fluorescence resonance energy transfer studies and ensemble measurements of DNA and RNA oligo hybridization that show nucleation of nucleic acid duplex formation by base pairing interactions of only 2-4 nt in length (Cisse et al, 2012;Craig et al, 1971;Marimuthu and Chakrabarti, 2014;Wetmur, 1991;Wetmur and Davidson, 1968). Additionally, we observed that oligos capable of pairing towards the 3' end of the SSRS formed observable complexes more quickly than those where the pairing was shifted towards the 5' end (Fig.…”

Section: Base Pairing Potential Accelerates U1/rna Complex Formationsupporting

confidence: 93%

Multi-Factor Authentication of Potential 5’ Splice Sites by the Saccharomyces cerevisiae U1 snRNP

Hansen

Scalf²,

et al. 2021

Preprint

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In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.

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Section: Base Pairing Potential Accelerates U1/rna Complex Formationsupporting

confidence: 93%

Multi-Factor Authentication of Potential 5’ Splice Sites by the Saccharomyces cerevisiae U1 snRNP

Hansen

Scalf²,

et al. 2021

Preprint

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“…Single-molecule experiments, including fluorescence-based methods such as resonance energy transfer123 and force-based methods4 such as atomic force microscopy (AFM)5 and optical tweezers6, can be used to investigate the kinetics of association (hybridization) and dissociation (melting) of two single-stranded oligonucleotides at equilibrium7891011. While ensemble versions of such experiments are possible, single-molecule versions are much more informative121314.…”

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confidence: 99%

Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification

et al. 2017

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The study of biomolecular interactions at the single-molecule level holds great potential for both basic science and biotechnology applications. Single-molecule studies often rely on fluorescence-based reporting, with signal levels limited by photon emission from single optical reporters. The point-functionalized carbon nanotube transistor, known as the single-molecule field-effect transistor, is a bioelectronics alternative based on intrinsic molecular charge that offers significantly higher signal levels for detection. Such devices are effective for characterizing DNA hybridization kinetics and thermodynamics and enabling emerging applications in genomic identification. In this work, we show that hybridization kinetics can be directly controlled by electrostatic bias applied between the device and the surrounding electrolyte. We perform the first single-molecule experiments demonstrating the use of electrostatics to control molecular binding. Using bias as a proxy for temperature, we demonstrate the feasibility of detecting various concentrations of 20-nt target sequences from the Ebolavirus nucleoprotein gene in a constant-temperature environment.

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“…The resulting solutions were denatured at 95°C in a thermostat for 15 minutes followed by slow (overnight) cooling to room temperature (around 20°C). This temperature will allow complete hybridization [126]. The denaturation step was necessary to eliminate secondary structures to ensure ultimate probe-target binding.…”

Section: Sample Preparationmentioning

confidence: 99%

Quantitation without Calibration: Response Profile as an Indicator of Target Amount

et al. 2018

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Quantitative assessment of biomarkers is essential in numerous contexts from decision-making in clinical situations to food quality monitoring to interpretation of life-science research findings. However, appropriate quantitation techniques are not as widely addressed as detection methods. One of the major challenges in biomarker's quantitation is the need to have a calibration for correlating a measured signal to a target amount. The step complicates the methodologies and makes them less sustainable. In this work we address the issue via a new strategy: relying on position of response profile rather than on an absolute signal value for assessment of a target's amount. In order to enable the capability we develop a target-probe binding mechanism based on a negative cooperativity effect. A proof-of-concept example demonstrates that the model is suitable for quantitative analysis of nucleic acids over a wide concentration range. The general principles of the platform will be applicable toward a variety of biomarkers such as nucleic acids, proteins, peptides, and others.

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Sequence-dependent theory of oligonucleotide hybridization kinetics

Cited by 13 publications

References 31 publications

Multi-Factor Authentication of Potential 5’ Splice Sites by the Saccharomyces cerevisiae U1 snRNP

Multi-Factor Authentication of Potential 5’ Splice Sites by the Saccharomyces cerevisiae U1 snRNP

Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification

Quantitation without Calibration: Response Profile as an Indicator of Target Amount

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