This work is supported by the US National Institutes of Health (R01GM111452, Y.W., S.X.). Y. W. acknowledges support from the Welch Foundation (E-1721).

1. Preparation of the ribosome complexes
<ol>
	<li>Make 1,000 mL of TAM10 buffer, which consists of 20 mM tris-HCl (pH 7.5), 10 mM Mg (OAc)2, 30 mM NH4Cl, 70 mM KCl, 5 mM EDTA, 7 mM BME (2-mercaptoethanol), and 0.05% Tween20.</li>
	<li>Prepare the five mixtures listed in Table 1. 
	NOTE: The ribosome was from the MRE600 strain11. EF-Tu: elongation factor thermo unstable. EF-Ts: elongation factor thermo stable. GTP: guanosine triphosphate. PEP: phospho(...

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In our dual ruler assay, the magnetic beads play two essential roles. First, they serve as the force transducers because the centrifugal force is proportional to their buoyant mass. Second, the beads are signal carriers detected by an atomic magnetometer, which is currently the most accurate magnetic sensor. Combining mechanical manipulation and magnetic detection, the FIRMS technique is able to resolve a large number of molecular interactions based on their critical dissociation forces, which is the basis of the DNA rul...

Biomolecular displacement is a fundamental parameter in studying the mechanism of the related biological functions. One particular example is the ribosome translocation1,2, during which the ribosome moves by exactly three nucleotides (nt) on the messenger RNA (mRNA) normally, and by one, two, or other numbers of nt except three in the case of frameshifting. Therefore, a molecular ruler system single-nt resolution is required to distinguish the different step sizes. A greater challenge is to probe the ribosome movement on both the entrance and exit sides. In other words, only with a dual ruler system will we be able to reveal whether the mRNA is smoothly threaded through the ribosome, or there are intermediate steps in which the two sides have different step sizes leading to a kinked or looped mRNA conformation inside the ribosome.
Several methods have been developed to address the first challenge of resolving different steps on the exit side of the ribosome (the 3&#39;&#160;end of the mRNA). The dual luciferase assay resolves the different reading frames by measuring the ratios of the resulting different proteins3,4. It is only applicable for the 3&#39;&#160;end of the mRNA and thus insufficient to provide a complete picture of translocation. Mass spectrometry can analyze the different peptide fragments as the consequence of the corresponding code rearrangements5. But it cannot pinpoint to how many nt the ribosome moves on the mRNA. The toe-printing assay is another common method that uses a reverse transcriptase primed at the 3&#39;-distal end to transcribe the mRNA toward the ribosome6. However, it is not applicable for the 5&#39;&#160;end of mRNA that is entering the ribosome. Other techniques, including single molecule approaches and fluorescence methods7, are difficult to achieve single-nt resolution.
We have developed the dual DNA ruler assay that can uniquely determine both the entrance and exit positions of the uncovered mRNA in ribosome-mRNA complexes. &#160;The ruler DNAs are DNA oligomers that form duplexes of certain numbers of basepairs (bp) with the mRNA uncovered by the ribosome, regardless of which end of the mRNA. The bp numbers then precisely reveal the ribosome position on the mRNA during translocation. The bp numbers of the duplexes are determined by their critical dissociation forces obtained from force-induced remnant magnetization spectroscopy (FIRMS)8. With 2-3 pN force uncertainty, the critical forces are sufficient to offer single-nt resolution. By implementing a linker molecule on the DNA rulers, the sterically hindered side of the mRNA by the ribosome can be probed. Different ribosomal displacements can thus be accurately resolved. We have successfully revealed a unique looped conformation of mRNA trapped by antibiotics during translocation9, and resolved different reading frames that coexisted on a slippery mRNA sequence10. This article describes the details of the dual ruler assay, which include preparation of the ribosome complexes, surface functionalization of the glass slides, immobilization of the ribosome complexes and their hybridization with magnetically labeled DNA ruler molecules, magnetic detection, and force spectrum analysis by FIRMS.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:1412px;" width="1411">
	<colgroup>
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			<td height="21" style="height:21px;width:217px;">Styrene Strip</td>
			<td style="width:239px;">City of Industry</td>
			<td style="width:161px;">MS-861</td>
			<td style="width:795px;"></td>
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			<td style="width:239px;">Evaporated Coatings</td>
			<td style="width:161px;"></td>
			<td>60.0 &#215; 4.0 &#215; 0.3 mm3</td>
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			<td height="21" style="height:21px;width:217px;">Acetic acid</td>
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			<td style="width:161px;">A6283-500ML</td>
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			<td height="21" style="height:21px;width:217px;">3-Aminopropyltriethoxysilane</td>
			<td style="width:239px;">UCT specialties</td>
			<td style="width:161px;">21400088</td>
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			<td height="21" style="height:21px;width:217px;">mPEG-SVA</td>
			<td style="width:239px;">Laysan Bio</td>
			<td style="width:161px;">154-82</td>
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			<td height="21" style="height:21px;width:217px;">Biotin-PEG-SVA</td>
			<td style="width:239px;">Laysan Bio</td>
			<td style="width:161px;">152-84</td>
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			<td height="21" style="height:21px;width:217px;">Sodium bicarbonate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">S5761-500G</td>
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			<td height="21" style="height:21px;width:217px;">Epoxy glue</td>
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			<td style="width:161px;">31345</td>
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			<td style="width:239px;">ThermoFisher</td>
			<td style="width:161px;">434301</td>
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			<td height="21" style="height:21px;width:217px;">Fusidic Acid</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">F0756-1G</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Neomycin Sulfate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">1458009</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Viomycin Sulfate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">1715000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Hygromycin</td>
			<td style="width:239px;">invitrogen</td>
			<td style="width:161px;">10687-010</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Tris-HCl</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">T5941-100G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Magnesium acetate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">M5661-50G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Ammonium chloride</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">A9434-500G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Potassium chloride</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P9333-500G</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">EDTA</td>
			<td style="width:239px;">GIBCO</td>
			<td style="width:161px;">774750</td>
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			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">M6250-500ML</td>
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			<td height="21" style="height:21px;width:217px;">Tween20</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P1379-250ML</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">GTP</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">G8877-100MG</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">PEP</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P7127-100MG</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Pyruvate Kinase</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P1506-5KU</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Sucrose&#160;</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">S7903-5KG</td>
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			<td height="21" style="height:21px;width:217px;">Dynabeads M-280 Streptavidin</td>
			<td style="width:239px;">ThermoFisher</td>
			<td style="width:161px;">11205D</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">mRNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">133899727</td>
			<td>5&#8242;-Bio- CAA CUG UUA AUU AAA UUA AAU UAA AAA GGA AAU AAAA AUG UUU AAU UUU UUA GGG CGC AAU CUA CUG CUG AAC UC-3&#8242;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">157468630</td>
			<td>3&#697;- TAA TTT AAT TTA ATT TTT CGA AAU AT50/TEGBio/-5&#697;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">164845370</td>
			<td>3&#697;-AAT TTA ATT TTT CCT TTA AAA AT50/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">157468628</td>
			<td>3&#697;-AAA ATC CCG CGT TAG AAC UGG GG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">163472705</td>
			<td>3&#697;-CCG CGT TAG ATG ACG AGA ACG GG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678130</td>
			<td>3&#697;-AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678131</td>
			<td>3&#697;-T AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678132</td>
			<td>3&#697;-TT AGA TGA CGA CTT CTC GGG/TEGBio/-5&#697;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678133</td>
			<td>3&#697;-GTT AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;&#160;</td>
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			<td height="21" style="height:21px;width:217px;">Centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:161px;">5427R</td>
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			<td height="21" style="height:21px;width:217px;">Micro Ultracentrifuge</td>
			<td style="width:239px;">Hitachi</td>
			<td style="width:161px;">CS150FNX</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Vortex mixer</td>
			<td style="width:239px;">VWR</td>
			<td style="width:161px;">VM-3000</td>
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			<td height="21" style="height:21px;width:217px;">Lock-in Amplifier</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR530</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Lock-in Amplifier</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR830</td>
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			<td height="21" style="height:21px;width:217px;">Laser</td>
			<td style="width:239px;">Newport</td>
			<td style="width:161px;">TLB-6918-D</td>
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			<td height="21" style="height:21px;width:217px;">Function generator</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">DS345</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Photo detectors</td>
			<td style="width:239px;">Thorlabs</td>
			<td style="width:161px;">DET36A</td>
			<td></td>
		</tr>
	</tbody>
</table>

dual dna rulers to study the mechanism of ribosome translocation with single-nucleotide resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Figure 1 shows the detection scheme and photographs of the major components. Magnetic detection is achieved by an atomic magnetometer using the scanning scheme (Figure 1A)13. The sample is placed on a rod mounted on a linear motor. The motor transports the sample to the atomic sensor inside a magnetic shield, then back to the original site for unloading. The atomic magnetometer detects the magnetic signal during the sampl...

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Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

The dual DNA ruler assay can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements. Our dual DNA ruler assay is currently the only method that can objectively probe both the entrance and exit sides of mRNA in the ribosome complexes with single-nucleotide resolution. Similar methods are also applied to measure DNA binding strength and selectivity of chemotherapeutical drugs.Visual demonstration of the method is a good chance for us to intuitively show the advantages of our technique and help other researchers to further develop it into more applications. To begin, make a plastic sample well with a styrene strip by drilling a cube in the center with dimensions of four millimeters by three millimeters by two millimeters. Then, using epoxy, glue a piece of biotin-coated glass, of approximately five millimeters long, to the bottom surface.Add 20 microliters of 0.25 milligram per milliliter streptavidin aqueous solution into the sample well, and incubate at room temperature for 40 minutes. Then rinse the sample well twice with TAM10 buffer. To immobilize the ribosome complexes without antibiotics, first remove the buffer from the sample well, and add 20 microliters of 0.1 micromolar MF-Pre or MF-Post ribosome complex into the sample well.Incubate at 37 degrees Celsius for one hour, and then rinse once with TAM10 buffer. The ribosome complex now binds with the streptavidin on the surface via the five-prime end biotin on the mRNA. For the experiment using antibiotics, incubate nine microliters of the MF-Pre complex with one microliter of four millimolar neomycin, 37 degrees Celsius for 10 minutes.Then combine in a tube 0.6 microliters of 60 micromolar EFG, 0.8 microliters of 100 micromolar GTP, 0.8 microliters of 100 millimolar PEP, 0.3 microliters of 1.3 milligram per milliliter pyruvate kinase, 6.43 microliters TAM10 buffer, and one microliter of five millimolar fusidic acid. Incubate at 37 degrees Celsius for 20 minutes. Then combine the two mixtures, and incubate at 37 degrees Celsius for an additional five minutes.Carry out the other antibiotics experiments similarly, with concentrations of 0.2 millimolar viomycin, 0.4 millimolar hygromycin B, and 0.25 millimolar fusidic acid. For frame shifting study, repeat the incubation for the MFNF-Pre complexes involving the slippery motif, U6A. Include antibiotics of fusidic acid plus neomycin.Sufficient time is necessary to ensure the completion of the hybridization process. It is also advised to use the TAM10 buffer with actual one mole sodium chloride to maintain a homosteady system. After incubation, remove buffer from the sample well.Add 20 microliters of one micromolar biotinylated probing DNA strand, and incubate at room temperature overnight. In the morning, rinse the formed DNA/mRNA duplex once with TAM10 buffer. Remove the buffer from the sample well.Then add 20 microliters of 0.5 milligrams per milliliter streptavidin-coated magnetic beads into the sample well, and incubate at room temperature for two hours. After that, carefully insert the sample well into a holder, and place it in a centrifuge to remove the free magnetic particles from the surface at 84 times g for five minutes. Turn on the laser.Then press the power button. Adjust the sensitivity to 500 millivolts, and wait for about two hours to warm up and stabilize the atomic magnetometer. To set up the atomic magnetometer, run the instrument control software, and set up the Motor moving mode to Noise and default position to zero.Press Lock on the front panel. Adjust the sensitivity back to 200 millivolts. Adjust the current and voltage of the laser, and find the proper resonance peak and signal-to-noise level for the best signal resolution.Press Sweep on the front panel. Make sure the amplitude-to-width ratio is above 0.5 and the phase value is smaller than five degrees with width less than 170 hertz. Plug in the output of another SR530 lock-in amplifier to the feedback of the laser to lock its frequency.Plug in function generator ATF20B to input a square wave of 500 millivolts peak to peak, 100 millihertz as the reference signal for converting current to magnetic signal. Then unplug the function generator. Set up the Motor moving mode to Two-way and default position to 260 millimeters.Check the status of the temperature controller, to ensure the temperature is at approximately 37 degrees Celsius, of the atomic sensor. Check the stability of the whole system twice by directly running the program without loading samples to measure the signals of the empty sample holder. To magnetize the samples, gently use tweezers to take the sample out of the holder, and place on the magnetization stage for two minutes.Then put the sample back into the sample holder, and centrifuge at 335.4 times g for 20 minutes. Remove the nonspecifically-bound magnetic particles. Next load the sample onto the motor with tweezers.Click Lock on the front panel to run the program. Meanwhile use tweezers to load another sample into the holder, and place it into the centrifuge. Make the coated glass side face the center of the centrifuge, and start the centrifugation at 335.4 times g for four minutes.When the motor comes back to zero, after approximately five minutes, click Save on the front panel. Carefully use tweezers to transfer one sample from the motor to the centrifuge holder. Apply a stronger force by increasing the centrifugal speed by 100 rpm or a similar step size.Exchange the sample for the centrifuge. Process alternately to gradually increase the force each time. A complete force spectrum is obtained after 10 to 12 data points.When all planned experiments are finished, turn off the equipment, proceeding in the opposite order as it was turned on. Remove the samples from the holder, and immerse them in ethanol for cleaning and future use. Clean up the sample holder with acetone in case of magnetic bead contamination.In this protocol, the ribosome complex was immobilized on the surface, via the five-prime end of the mRNA, and made both the five-prime and three-prime side easily accessible for the probing DNA rulers. When the linker for the five-prime side was longer than 50 thymine, a strong magnetic signal was detected, indicating successful formation of DNA/mRNA duplexes. By varying the number of nucleotides on the DNA, the correlation of dissociation force to duplex length was achieved for the three-prime and five-prime sides, respectively.The results of normal translocation from MF-Pre to MF-Post, in the absence of antibiotics, show that the ribosome moved by three nucleotides toward the three-prime end on both sides. MF-Pre carried a vagrant fMet-tRNA and phenylalanine-tRNA at the P and A sites, respectively, while MF-Post carried fMet-tRNA and phenylalanine-tRNA at the E and P sites, respectively, with a vacant A site. However, when both fusidic acid and adiomycin antibiotics were present, the ribosome moved by only one nucleotide at the five-prime side but two nucleotides at the three-prime side.After washing away the antibiotics, normal translocation occurred, as evidenced by three nucleotide movements from both the five-prime and three-prime sides. When magnetizing the sample, the coated surface should approach and leave the magnet vertically. And removing the nonspecifically-bound magnetic particles is very important for data accuracy and quality.Our technique provides a high resolution and a generally applicable way to investigate molecular displacement of nucleic acid which is widely encountered in molecular biology.

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Biochemistry

6.2K visualizações.  University of Houston. O ensaio da régua de DNA duplo pode fornecer insights mecanicistas para translocação ribossosome e sondar outros deslocamentos de ácido nucleico. Nosso ensaio de régua de DNA duplo é atualmente o único método que pode sondar objetivamente os lados de entrada e saída do mRNA nos complexos ribossomos com resolução de nucleotídeos únicos. Métodos semelhantes também são aplicados para medir a força de ligação de DNA e a seletividade de drogas quimioterápicas.A demonstra...