7 research outputs found
Recognition Tunneling of Canonical and Modified RNA Nucleotides for Their Identification with the Aid of Machine Learning
In
the present study, we demonstrate a tunneling nanogap technique
to identify individual RNA nucleotides, which can be used as a mechanism
to read the nucleobases for direct sequencing of RNA in a solid-state
nanopore. The tunneling nanogap is composed of two electrodes separated
by a distance of <3 nm and functionalized with a recognition molecule.
When a chemical entity is captured in the gap, it generates electron
tunneling currents, a process we call recognition tunneling (RT).
Using RT nanogaps created in a scanning tunneling microscope (STM),
we acquired the electron tunneling signals for the canonical and two
modified RNA nucleotides. To call the individual RNA nucleotides from
the RT data, we adopted a machine learning algorithm, support vector
machine (SVM), for the data analysis. Through the SVM, we were able
to identify the individual RNA nucleotides and distinguish them from
their DNA counterparts with reasonably high accuracy. Since each RNA
nucleoside contains a hydroxyl group at the 2′-position of
its sugar ring in an RNA strand, it allows for the formation of a
tunneling junction at a larger nanogap compared to the DNA nucleoside
in a DNA strand, which lacks the 2′ hydroxyl group. It also
proves advantageous for the manufacture of RT devices. This study
is a proof-of-principle demonstration for the development of an RT
nanopore device for directly sequencing single RNA molecules, including
those bearing modifications
A Three-Arm Scaffold Carrying Affinity Molecules for Multiplex Recognition Imaging by Atomic Force Microscopy: The Synthesis, Attachment to Silicon Tips, and Detection of Proteins
We have developed a multiplex imaging
method for detection of proteins using atomic force microscopy (AFM),
which we call multiplex recognition imaging (mRI). AFM has been harnessed
to identify protein using a tip functionalized with an affinity molecule
at a single molecule level. However, many events in biochemistry require
identification of colocated factors simultaneously, and this is not
possible with only one type of affinity molecule on an AFM tip. To
enable AFM detection of multiple analytes, we designed a recognition
head made from conjugating two different affinity molecules to a three-arm
linker. When it is attached to an AFM tip, the recognition head would
allow the affinity molecules to function in concert. In the present
study, we synthesized two recognition heads: one was composed of two
nucleic acid aptamers, and the other one composed of an aptamer and
a cyclic peptide. They were attached to AFM tips through a catalyst-free
click reaction. Our imaging results show that each affinity unit in
the recognition head can recognize its respective cognate in an AFM
scanning process independently and specifically. The AFM method was
sensitive, only requiring 2 to 3 μL of protein solution with
a concentration of ∼2 ng/mL for the detection with our current
setup. When a mixed sample was deposited on a surface, the ratio of
proteins could be determined by counting numbers of the analytes.
Thus, this mRI approach has the potential to be used as a label-free
system for detection of low-abundance protein biomarkers
Application of Catalyst-Free Click Reactions in Attaching Affinity Molecules to Tips of Atomic Force Microscopy for Detection of Protein Biomarkers
Atomic force microscopy (AFM) has
been extensively used in studies
of biological interactions. Particularly, AFM based force spectroscopy
and recognition imaging can sense biomolecules on a single molecule
level, having great potential to become a tool for molecular diagnostics
in clinics. These techniques, however, require affinity molecules
to be attached to AFM tips in order to specifically detect their targets.
The attachment chemistry currently used on silicon tips involves multiple
steps of reactions and moisture sensitive chemicals, such as (3-aminopropyl)Âtriethoxysilane
(APTES) and <i>N</i>-hydroxysuccinimide (NHS) ester, making
the process difficult to operate in aqueous solutions. In the present
study, we have developed a user-friendly protocol to functionalize
the AFM tips with affinity molecules. A key feature of it is that
all reactions are carried out in aqueous solutions. In summary, we
first synthesized a molecular anchor composed of cyclooctyne and silatrane
for introduction of a chemically reactive function to AFM tips and
a bifunctional polyethylene glycol linker that harnesses two orthogonal
click reactions, copper free alkyne–azide cycloaddition and
thiol-vinylsulfone Michael addition, for attaching affinity molecules
to AFM tips. The attachment chemistry was then validated by attaching
antithrombin DNA aptamers and cyclo-RGD peptides to silicon nitride
(SiN) tips, respectively, and measuring forces of unbinding these
affinity molecules from their protein cognates human α-thrombin
and human α<sub>5</sub>β<sub>1</sub>-integrin immobilized
on mica surfaces. In turn, we used the same attachment chemistry to
functionalize silicon tips with the same affinity molecules for AFM
based recognition imaging, showing that the disease-relevant biomarkers
such as α-thrombin and α<sub>5</sub>β<sub>1</sub>-integrin can be detected with high sensitivity and specificity by
the single molecule technique. These studies demonstrate the feasibility
of our attachment chemistry for the use in functionalization of AFM
tips with affinity molecules
Optical and Electrical Detection of Single-Molecule Translocation through Carbon Nanotubes
Ion current through a single-walled carbon nanotube (SWCNT) was monitored at the same time as fluorescence was recorded from charged dye molecules translocating through the SWCNT. Fluorescence bursts generally follow ion current peaks with a delay time consistent with diffusion from the end of the SWCNT to the fluorescence collection point. The fluorescence amplitude distribution of the bursts is consistent with single-molecule signals. Thus each peak in the ion current flowing through the SWCNT is associated with the translocation of a single molecule. Ion current peaks (as opposed to blockades) were produced by both positively (Rhodamine 6G) and negatively (Alexa 546) charged molecules, showing that the charge filtering responsible for the current bursts is caused by the molecules themselves
Click Addition of a DNA Thread to the N‑Termini of Peptides for Their Translocation through Solid-State Nanopores
Foremost among the challenges facing single molecule sequencing of proteins by nanopores is the lack of a universal method for driving proteins or peptides into nanopores. In contrast to nucleic acids, the backbones of which are uniformly negatively charged nucleotides, proteins carry positive, negative and neutral side chains that are randomly distributed. Recombinant proteins carrying a negatively charged oligonucleotide or polypeptide at the C-termini can be translocated through a α-hemolysin (α-HL) nanopore, but the required genetic engineering limits the generality of these approaches. In this present study, we have developed a chemical approach for addition of a charged oligomer to peptides so that they can be translocated through nanopores. As an example, an oligonucleotide PolyT<sub>20</sub> was tethered to peptides through first selectively functionalizing their N-termini with azide followed by a click reaction. The data show that the peptide-PolyT<sub>20</sub> conjugates translocated through nanopores, whereas the unmodified peptides did not. Surprisingly, the conjugates with their peptides tethered at the 5′-end of PolyT<sub>20</sub> passed the nanopores more rapidly than the PolyT<sub>20</sub> alone. The PolyT<sub>20</sub> also yielded a wider distribution of blockade currents. The same broad distribution was found for a conjugate with its peptide tethered at the 3′-end of PolyT<sub>20</sub>, suggesting that the larger blockades (and longer translocation times) are associated with events in which the 5′-end of the PolyT<sub>20</sub> enters the pore first
Electronic Signatures of all Four DNA Nucleosides in a Tunneling Gap
Nucleosides diffusing through a 2 nm electron-tunneling junction generate current spikes of sub-millisecond duration with a broad distribution of peak currents. This distribution narrows 10-fold when one of the electrodes is functionalized with a reagent that traps nucleosides in a specific orientation with hydrogen bonds. Functionalizing the second electrode reduces contact resistance to the nucleosides, allowing them to be identified via their peak currents according to deoxyadenosine > deoxycytidine > deoxyguanosine > thymidine, in agreement with the order predicted by a density functional calculation
Fixed-Gap Tunnel Junction for Reading DNA Nucleotides
Previous measurements of the electronic conductance of DNA nucleotides or amino acids have used tunnel junctions in which the gap is mechanically adjusted, such as scanning tunneling microscopes or mechanically controllable break junctions. Fixed-junction devices have, at best, detected the passage of whole DNA molecules without yielding chemical information. Here, we report on a layered tunnel junction in which the tunnel gap is defined by a dielectric layer, deposited by atomic layer deposition. Reactive ion etching is used to drill a hole through the layers so that the tunnel junction can be exposed to molecules in solution. When the metal electrodes are functionalized with recognition molecules that capture DNA nucleotides <i>via</i> hydrogen bonds, the identities of the individual nucleotides are revealed by characteristic features of the fluctuating tunnel current associated with single-molecule binding events