Name: single-molecule manipulation of g-quadruplexes by magnetic tweezers
Uploaded: 2017-09-19T14:00:00.000+00:00
Duration: 8 min 28 s
Description: Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

Gli autori ringraziano Meng Pan per la correzione del manoscritto. Questo lavoro è supportato da Singapore Ministero di formazione accademica ricerca fondo Tier 3 (MOE2012-T3-1-001) per J.Y.; la Fondazione di ricerca nazionali attraverso l'Istituto Mechanobiology da Singapore a J.Y.; il National Research Foundation, ufficio, Singapore del primo ministro, nell'ambito del programma Investigatorship NRF (NRF Investigatorship premio No. NRF-NRFI2016-03 al J.Y.; il fondo di ricerca fondamentale per le Università centrale (2017KFYXJJ153) di H. Y.

Ho ></ol></li> <li>esempio di impostazione e identificare singole dsDNA tether <ol><li>formazione Tether <ol><li>delicatamente il flusso in 200 X diluito M-280, paramagnetici perle nella soluzione di saggio (100 mM KCl, 2 mM MgCl 2, 10 mM Tris-pH 7,4 ) in un canale. Incubare per 10 minuti consentire le perline da associare alla biotina di molecole di DNA immobilizzati sulla superficie rivestita con SMCC fino alla fine del tiolo-etichettati. Delicatamente lavare via un-tethered perline utilizzando 200 µ l di soluzione di reazione standard. 
			Nota: Le perle di M-280 mostrano più basso legame non specifico alla superficie rispetto alle altre biglie magnetiche commerciali come M-270.</li>
		</ol></li> <li>Montare il canale sul palco microscopio. Ricerca per le perle sulla superficie inferiore usando 100 X obiettivo a immersione in olio.</li>
		<li>Selezionare una perlina di riferimento sulla superficie e una perlina tethered commovente. Compilare le librerie di immagine iniziale del branello di riferimento e perlina legato alle diverse defocus aerei.</li>
		<li>Calibrazione dell'immagine di perline <ol><li>prima di esperimenti, utilizzare un attuatore piezoelettrico oggettivi per ottenere immagini di riferimento e di perle legati al defocus diversi piani distanziati di 20-50 nm, che vengono archiviati come due immagine separata della perla librerie e sono rispettivamente indicizzati con la distanza di sfocatura ( Figura 2D).</li>
		</ol></li> <li>x, y, z posizione determinazione <ol><li>durante gli esperimenti, per determinare la posizione del tallone nel piano x-y il centroide della perla. Determinare la modifica dell'altezza del tallone tethered confrontando l'immagine corrente della perla con quelli memorizzati nella libreria. Utilizzare l'analisi della funzione di auto-correlazione dello spettro di potenza della trasformata di Fourier del branello immagini 16.</li>
		</ol></li> <li>Anti-drift feedback utilizzando perlina riferimento <ol><li>durante gli esperimenti, utilizzare il piezo a &#34; blocco &#34; la distanza tra l'obiettivo e un'immagine di perlina di riferimento specifici memorizzati nella libreria tramite una bassa frequenza feedback di controllo, affinché l'immagine di tallone bloccato ha la migliore correlazione con una specifica immagine memorizzata nella libreria ( Figura 2D).</li>
		</ol></li> <li>Determinare se il tether è una molecola singola dsDNA applicando ~ 65 pN forza determinare una molecola singola dsDNA se il tether subisce il DNA caratteristico overstretching transizione 17 , 18 , 19. Ripetere la procedura finché non viene trovato un singolo dsDNA tether. (Fare riferimento alla sezione successiva per forza calibrazione e DNA overstretching transizione).</li>
	</ol></li></ol> 4. Calibrazione di forza magnetica pinzette <ol><li>determinare direttamente la forza (fino a 100 pN) per lunghe molecole di DNA (48.502 bp λ-DNA) utilizzando fluttuazione tallone attraverso: <img alt="Equation 1" src="/files/ftp_upload/56328/56328eq1.jpg" /> , dove k B è la costante di Boltzmann, T è la temperatura in Kelvin scala, δ 2 y è la varianza della fluttuazione della perla nella direzione perpendicolare al campo magnetico e la forza direzione. In questa direzione, il movimento del tallone può essere descritto come una fluttuazione di pendolo con una lunghezza di l + r 0, dove l è la distanza di end-to-end lungo la direzione della forza (estensione) del DNA, e r 0 è il raggio del branello 10 ( Figura 3A).</li>
	<li>Determinare la curva di taratura per forza F in funzione dei magneti a distanza tallone d e F (d) per differenti perline usando il lungo λ-DNA. Di solito, la distanza tra il magnete e la lamella viene utilizzata come la lunghezza del legare di DNA può essere ignorata. Appoggiare la F-curva d dalla funzione decadimento doppio-esponenziale: <img alt="Equation 2" src="/files/ftp_upload/56328/56328eq2.jpg" /> , dove il raccordo parametri α1, α2, γ1, γ2 sono determinati dalla pinzetta magnetica e il parametro C è determinato dalla proprietà eterogenea di biglie magnetiche. Spostando la C, la F-curva d ottenuto da differenti perline può essere sovrapposta 10 ( Figura 3B).</li>
	<li>Esperimenti di calibrazione del parametro C per biglie magnetiche individuali per DNA breve.
	<ol><li>Per determinare la forza basata sulla fluttuazione richiede la posizione del tallone ad una frequenza superiore alla frequenza di angolo di registrazione: <img alt="Equation 3" src="/files/ftp_upload/56328/56328eq3.jpg" /> , dove γ è il trascinamento coefficiente del tallone. Per breve DNA a forza elevata, richiede più veloce di 100 Hz frequenza di registrazione, pertanto, la forza non può essere misurata direttamente basata sulla fluttuazione con la fotocamera. Tuttavia, il parametro C può essere determinato misurando la forza a bassa forza gamma (&#60; 15 pN) utilizzando fluttuazione e montaggio i dati con un calibrato F-curva d per ottenere il parametro C 20.</li>
		<li>In alternativa, per esperimenti con dsDNA, determinare il parametro C usando il DNA overstretching transizione una forza di ~ 65 pN ( Figura 3) 21 , 22 , 23 registrando la posizione d al salto di estensione ha mostrato quale dsDNA (0,6 X aumento di lunghezza di lunghezza di contorno). Dopo aver determinato il parametro C, calcolare la forza per le perline tethered.</li>
	</ol></li> <li>Controllare la velocità di caricamento di programmazione il movimento dei magneti attraverso la funzione inversa di F (d). Il magnete dovrebbe affrontare il campione doppio esponenzialmente. 
	Nota: L'errore relativo della forza determinata da un metodo di estrapolazione è ~ 10% ed è principalmente causata dalla perlina raggio eterogeneità 20.</li>
</ol> 5. Manipolazione di singola molecola del G4 in presenza ed assenza di Binding Proteins <ol><li>Force-rampa esperimenti <ol><li>dopo aver identificato la lombata di dsDNA, eseguire una scansione di forza-aumento ad un tasso di carico di 0,2 pN/s seguita da un scansione di forza-decesso a -0,2 pN/s. Dopo ogni ciclo di allungamento, tenere la molecola di DNA a 1 pN per 30 s per consentire il ssDNA a ripiegare a G4.</li>
	</ol></li> <li>Force-salto esperimenti <ol><li>ciclo la forza tra 54 pN per 30 s in base al quale un G4 piegato potrebbe essere spiegato e 1 pN per 60s, sotto il quale potrebbe ripiegare un piegato G4-15T.</li>
	</ol></li> <li>Soluzione proteica Flowing <ol><li>fluire la soluzione della proteina quando al ~ 20 pN forza per evitare allegato delle perline per il vetrino coprioggetti. Il helicase RHAU DEAH-box, che ha mostrato elevata specificità la struttura G4, è stato usato 24. Il ricombinante dell'elicasi RHAU (DmRHAU) di Drosophila melanogaster è stata espressa in Escherichia coli e purificata come descritto in precedenza 25. Il G4 svolgimento attività di DmRHAU è stata analizzata su un G4 DNA substrato 13 tetramolecolari.</li>
	</ol></li> <li>Analizzare la forza di spiegamento utilizzando un in-House scritti Matlab programma 14.</li></ol>

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Come descritto sopra, una piattaforma per lo studio della stabilità meccanica del G4 DNA e le interazioni della proteina a G4 utilizzando una pinzetta magnetica di singola molecola è segnalata. Accompagnando la piattaforma, sono sviluppati protocolli altamente efficienti di trovare G4 DNA tether e misura della chiusura-apertura dinamica e la stabilità della struttura G4 con risoluzione speciale nanometri. Il bloccaggio del piano focale consente controllo antislittamento altamente stabile, che è importante per la rile...

Quattro-stranded DNA o RNA G4 strutture svolgono un ruolo critico in molti processi biologici importanti1. Molte proteine sono coinvolte nel legame G4 e regolamento, comprese le proteine obbligatorie del telomero (telomerase, POT1, RPA, TEBPs, TRF2)1,2, fattori di trascrizione (nucleolina, PARP1)3, RNA, proteine (hnRNP A1, di elaborazione hnRNP A2)4, elicasi (BLM, Pif1, FANCJ, RHAU, WRN, Dna2)5e replicazione del DNA relative proteine (Rif1, REV1, PrimPolymerase)6. Legame con le proteine può stabilizzare o destabilizzare G4 strutture; regolando le successive funzioni biologiche. La stabilità del G4 è stata misurata da thermal fusione utilizzando raggi ultravioletti (UV) o di metodi di dicroismo circolare (CD)7. Tuttavia, tali condizioni non sono fisiologiche rilevanti e sono difficili da applicare per lo studio degli effetti di binding proteine7.
Il rapido sviluppo delle tecnologie di manipolazione di singola molecola ha permesso studi di chiusura e apertura di una biomolecola, ad esempio un DNA o una proteina, a livello di singola molecola con risoluzione nanometrica in tempo reale8. Microscopia a forza atomica (AFM), pinzette ottiche e magnetiche pinzette sono i più comunemente usati metodi di manipolazione di singola molecola. Rispetto a AFM e pinzette ottiche9, magnetiche pinzette permettono misurazioni stabili di chiusura-apertura dinamica di una singola molecola nei giorni utilizzando una tecnica anti-drift10,11.
Qui, una piattaforma di singola molecola manipolazione usando pinzetta magnetica per studiare la regolazione della stabilità di G4 da proteine leganti è segnalato12,13. Questo lavoro delinea gli approcci di base, tra cui campione e flusso preparazione di canale, il programma di installazione di pinzette magnetiche e la calibrazione di forza. Il controllo di forza e i protocolli di antislittamento come descritto nel passaggio 3 consentono misure di tempo lungo sotto diversi controlli di forza, ad esempio costante forza (forza di bloccaggio) e carico costanti votare (forza-rampa) e forza-salto misura. Il protocollo di calibrazione forza descritto nel passaggio 4 consente forza calibrazione di &#60; 1 µm cavezze breve sopra una vasta forza gamma pN fino a 100, con un errore relativo all'interno di 10%. Un esempio di regolazione della stabilità del RNA Helicase associato AU-rich element (RHAU) elicasi (alias DHX36, G4R1) che gioca un ruolo essenziale nella risoluzione di che RNA G4 viene utilizzato per illustrare le applicazioni di questa piattaforma13.

<table><tbody><tr><td>DNA PCR primers</td><td>IDT</td><td></td><td>DNA preparations</td></tr><tr><td>DNA PCR chemicals</td><td>NEB</td><td></td><td>DNA preparations</td></tr><tr><td>restriction enzyme BstXI</td><td>NEB</td><td>R0113S</td><td>DNA preparations</td></tr><tr><td>coverslips (#1.5, 22*32 mm, and 20*20 mm)</td><td>BMH.BIOMEDIA</td><td>72204</td><td>flow channel preparation</td></tr><tr><td>Decon90</td><td>Decon Laboratories Limited</td><td></td><td>flow channel preparation</td></tr><tr><td>APTES</td><td>Sigma</td><td>440140-500ML</td><td>flow channel preparation</td></tr><tr><td>Sulfo-SMCC</td><td>ThermoFisher Scientific</td><td>22322</td><td>flow channel preparation</td></tr><tr><td>M-280, paramganetic beads,streptavidin</td><td>ThermoFisher Scientific</td><td>11205D</td><td>flow channel preparation</td></tr><tr><td>Polybead Amino Microspheres 3.00 &amp;mu;m</td><td>Polysciences, Inc</td><td>17145-5</td><td>flow channel preparation</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M6250-250ML</td><td>flow channel preparation</td></tr><tr><td>Olympus Microscopes IX71</td><td>Olympus</td><td>IX71</td><td>Magnetic tweezers setup</td></tr><tr><td>Piezo-Z Stages P-721</td><td>Physik Instrumente</td><td>P-721</td><td>Magnetic tweezers setup</td></tr><tr><td>Olympus Objective lense MPLAPON-Oil 100X</td><td>Olympus</td><td>MPLAPON-Oil 100X</td><td>Magnetic tweezers setup</td></tr><tr><td>CCD/CMOS camera</td><td>AVT</td><td>Pike F-032B</td><td>Magnetic tweezers setup</td></tr><tr><td>Translation linear stage</td><td>Physik Instrumente</td><td>MoCo DC</td><td>Magnetic tweezers setup</td></tr><tr><td>LED</td><td>Thorlabs</td><td>MCWHL</td><td>Magnetic tweezers setup</td></tr><tr><td>Cubic Magnets</td><td>Supermagnete</td><td></td><td>Magnetic tweezers setup</td></tr><tr><td>Labview</td><td>National Instruments</td><td></td><td>Magnetic tweezers setup</td></tr><tr><td>OriginPro/Matlab</td><td>OriginLab/MathWorks</td><td></td><td>Data analysis</td></tr></tbody></table>

single-molecule manipulation of g-quadruplexes by magnetic tweezers

Non-canonico dell'acido nucleico struttura secondaria che g-quadruplex (G4) sono coinvolti in diversi processi cellulari, come ad esempio la replicazione del DNA, trascrizione, elaborazione del RNA e l'allungamento dei telomeri. Durante questi processi, varie proteine legano e risolvere strutture G4 per svolgere la loro funzione. Come la funzione di G4 spesso dipende dalla stabilità della sua struttura piegata, è importante studiare come G4 proteine regolano la stabilità di G4. Questo lavoro presenta un metodo per modificare singole molecole di G4 usando la pinzetta magnetica, che permette studi del regolamento delle proteine leganti G4 su una singola molecola di G4 in tempo reale. In generale, questo metodo è adatto per un ampio campo di applicazioni negli studi per interazioni proteine/ligandi e regolamenti su varie strutture secondarie del DNA o RNA.

qui per visualizzare la versione ingrandita di questa figura.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56328/56328fig5.jpg"> 
Figura 5: Misurazioni di forza-salto del G4 svolgimento. (A) traccia di tempo rappresentativo del cambiamento di altezza tallone durante forza saltare cicli tra 1 pN e pN 54 misurata senza DmRHAU, con 10 nM DmRHAU ma senza ATP e con 10 nM DmRHAU e 1 mM ATP, come indicato nel pannello di figura. (B...

Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Manipolazione di singola molecola di G-quadruplex di pinzette magnetiche

Una piattaforma di singola molecola magnetico pinzette per manipolare G-quadruplex è segnalata, che permette per lo studio della stabilità di G4 e regolamento di varie proteine.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

single-molecule-manipulation-of-g-quadruplexes-by-magnetic-tweezers

Research

JoVE Journal

Biochemistry

7.9K Views.  Huazhong University of Science and Technology. Una piattaforma di singola molecola magnetico pinzette per manipolare G-quadruplex è segnalata, che permette per lo studio della stabilità di G4 e regolamento di varie proteine.

Video: Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Un approccio G-quadruplex del DNA-affinità per purificazione di enzimaticamente attiva G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Ricerca

Biochimica

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

FRET di alta precisione a singolo livello molecolare per la determinazione della struttura biomolecola

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplex singola molecola Forza Misure proteolisi con delle pinzette magnetiche

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Bioingegneria

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

Pinzetta magnetici per la misurazione di Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Nanomanipolazione di Single RNA Molecole da pinzette ottiche

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

Identificazione dei G-quadruplex in un modello di DNA utilizzando la mappatura chimica

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

Autore in primo piano: Caratterizzazione del DNA G-quadruplex mediante mappatura chimica basata su bis-3-cloropiperidina 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

In vitro Mappatura chimica delle strutture del DNA G-quadruplex mediante bis-3-cloroperidine

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Pinzette magnetiche ad alta velocità per misure nanomeccaniche su elementi sensibili alla forza

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Manipolazione di singola molecola di G-quadruplex di pinzette magnetiche

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un approccio G-quadruplex del DNA-affinità per purificazione di enzimaticamente attiva G4 Resolvase1

FRET di alta precisione a singolo livello molecolare per la determinazione della struttura biomolecola

Multiplex singola molecola Forza Misure proteolisi con delle pinzette magnetiche

Pinzetta magnetici per la misurazione di Twist and Torque

Nanomanipolazione di Single RNA Molecole da pinzette ottiche

Magnetic Tweezers in a Microplate Format

In vitro Mappatura chimica delle strutture del DNA G-quadruplex mediante bis-3-cloroperidine

In vitro Mappatura chimica delle strutture del DNA G-quadruplex mediante bis-3-cloroperidine

In vitro Mappatura chimica delle strutture del DNA G-quadruplex mediante bis-3-cloroperidine

Pinzette magnetiche ad alta velocità per misure nanomeccaniche su elementi sensibili alla forza