NDLI: Single-molecule mechanics of protein-labelled DNA handles

Content Provider	PubMed Central
Author	Jadhav, Vivek S. Brüggemann, Dorothea Wruck, Florian Hegner, Martin
Editor	Ruitenbeek, Jan M. Van
Copyright Year	2016
Abstract	DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.
Related Links	http://dx.doi.org/10.3762/bjnano.7.16
Ending Page	148
Page Count	11
Starting Page	138
File Format	PDF
ISSN	21904286
e-ISSN	21904286
Journal	Beilstein Journal of Nanotechnology
Volume Number	7
Language	English
Publisher	Beilstein-Institut
Publisher Date	2016-01-29
Access Restriction	Open
Rights Holder	Beilstein-Institut
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Nanoscience and Nanotechnology Physics and Astronomy Electrical and Electronic Engineering

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

Charge transport in a zinc–porphyrin single-molecule junction

Electrical properties and mechanical stability of anchoring groups for single-molecule electronics

Interaction of spin and vibrations in transport through single-molecule magnets

Single-molecule magnet behavior in 2,2’-bipyrimidine-bridged dilanthanide complexes

Modification of a single-molecule AFM probe with highly defined surface functionality

Charge transfer through single molecule contacts: How reliable are rate descriptions?

Manipulating Protein Conformations by Single-Molecule AFM-FRET Nanoscopy

Controlled switching of single-molecule junctions by mechanical motion of a phenyl ring

Single-molecule mechanics of protein-labelled DNA handles

Similar Documents

Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

Charge transport in a zinc–porphyrin single-molecule junction

Electrical properties and mechanical stability of anchoring groups for single-molecule electronics

Interaction of spin and vibrations in transport through single-molecule magnets

Single-molecule magnet behavior in 2,2’-bipyrimidine-bridged dilanthanide complexes

Modification of a single-molecule AFM probe with highly defined surface functionality

Charge transfer through single molecule contacts: How reliable are rate descriptions?

Manipulating Protein Conformations by Single-Molecule AFM-FRET Nanoscopy

Controlled switching of single-molecule junctions by mechanical motion of a phenyl ring

Single-molecule mechanics of protein-labelled DNA handles