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Remote Sensing

Immobilization and stretching of DNA molecules in a microchannel

A new technique can immobilize and stretch a large number of DNA molecules for single-molecule DNA analysis applications.
15 March 2007, SPIE Newsroom. DOI: 10.1117/2.1200702.0622

DNA-protein interactions drive the cellular machinery for maintaining and transcribing DNA. To study the motion and kinetics of proteins along a DNA strand at the single-molecule level, it is critical that the DNA molecules be stretched and immobilized. However, existing stretching and immobilization techniques, such as optical tweezers1 and molecular combing,2 either produce very few stretched molecules or molecules that do not readily allow normal DNA-protein interactions. Hence the interest in improving these techniques. Advances in the ability to stretch and immobilize large numbers of DNA molecules in micro- and nanofluidic channels will provide a powerful tool for single-molecule DNA sequencing and DNA-protein interaction analysis, gene mutation detection, and for the manufacture of DNA-based electronic devices.

We recently developed a DNA immobilization technique—protein-assisted DNA immobilization (PADI)—that can deposit a large number of stretched and immobilized DNA molecules in a microchannel, as shown in Figure 1. The PADI technique works as follows. DNA-interacting proteins, such as restriction enzymes and RNA polymerases (RNAPs), are first allowed to bind to DNA in bulk solution at nonspecific segments. This solution is then introduced into a microchannel whose hydrodynamic flow stretches the DNA-protein complex. When the complex diffuses to the channel surface, its protein moiety adsorbs on the surface, resulting in immobilization of stretched DNA molecules inside the microchannel. Figure 1 shows a large number of λ-DNA molecules stretched and immobilized in a 100µm-wide and 1µm-deep microchannel. PADI thus allows immobilization of thousands of stretched DNA molecules from solutions of very low concentration (pM and fM). In principle, the PADI technique can be used to immobilize double-stranded DNA of any sequence and size. We expect that its application to single-molecule detection in microchannels will prove inexpensive and more sensitive than DNA microarrays and nanowire-based detection methods.

Figure 1. Image of λ-DNA molecules stretched and immobilized in the presence of T7 RNA polymerase (10nM) in a microfluidic device. Reproduced by kind permission of the American Chemical Society from Nanoletters 6, 2499–3504, 2006.3

PADI has several attractive features. It avoids overstretching of DNA molecules. The degree of attachment of DNA to the substrate can be controlled by changing the protein concentration without changing the substrate material. The number of DNA molecules immobilized onto the substrate is time- and concentration-dependent and can be controlled simply by varying the pumping time as well as the concentration. And stretching and immobilization are achieved at physiological pH.

Using total internal reflection fluorescence microscopy (TIRFM), single-protein molecules bound to DNA are imaged in Figure 2. Combining TIRFM with PADI clearly could allow large-scale DNA mapping with fluorescent tags such as peptide nucleic acids for single-molecule DNA sequencing and gene mutation detection.

Figure 2. T7 DNA molecules (green) stretched and immobilized with the assistance of T7 RNA polymerase (red). Arrows indicate positions of bound T7 RNA polymerases. Scale bar=2.5µm. Reproduced by kind permission of the American Chemical Society from Nanoletters 6, 2499–3504, 2006.3

Controlling the degree of attachment of the DNA backbone to the surface is critical for such applications. This can be achieved by modifying protein concentrations. At high concentrations, the DNA is firmly attached to the surface as shown in Figure 3(a). Lowering the concentration leads to fewer proteins bound to DNA, resulting in loose DNA attachment to the surface as shown in Figure 3(b).

Figure 3. T7 DNA molecules are immobilized at (a) 5nM and (b) 0.5nM T7 RNA polymerase concentrations followed by DNA photo-cleavage upon exposure to illumination. Reproduced by kind permission of the American Chemical Society from Nanoletters 6, 2499–3504, 2006.3

Optical mapping is a single-molecule DNA sequencing method that involves digesting the stretched DNA with restriction enzymes, followed measuring the length of the resulting DNA fragments. Figure 4 shows fragments of λ-DNA molecules after digestion with the restriction enzyme Sma I.

Figure 4. DNA fragment after digestion with the Sma I restriction enzyme. Scale bar=2.5µm. Reproduced by kind permission of the American Chemical Society from Nanoletters 6, 2499–3504, 2006.3

We have also carried out single-molecule transcription. The RNA transcripts were detected by incorporating fluorescently labeled uridine triphosphate into the growing RNA chain. Figure 5 shows the transcripts as bright red dots along the stretched DNA molecules stained with a YOYO nucleic acid dye.

Figure 5. RNA transcripts labeled with Alexa Fluor 546 uridine triphosphate (red) formed along YOYO-stained T7 DNA (green). Scale bar=5µm. Reproduced by kind permission of the American Chemical Society from Nanoletters 6, 2499–3504, 2006.3

In summary, we have developed a novel DNA immobilization method to stretch and immobilize DNA molecules onto a substrate. Our PADI technique can also be applied to single-molecule DNA sequencing and DNA-protein optical mapping.

Venkat Dukkipati, Ji Hoon Kim, Stella Pang, and Ronald Larson
University of Michigan
Ann Arbor, MI