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(enol)pyruvate.</li>
	<li>Incubate the five mixes separately at 37 &#176;C for 25 min before making the ribosome complexes. Prepare the five ribosome complexes as per Table 2. 
	NOTE: Post: post-translocation; Pre: pre-translocation.</li>
	<li>Add each of the five ribosome complexes onto a 1.1 M sucrose cushion separately, with volume ratio 1:1. Purify each with 450,000 &#215; g for 2 h in an ultra-centrifuge. Use a pipet to remove the supernatant and restore the ribosome complexes at -80 &#176;C after resuspension of the pellet with TAM10 buffer.</li>
</ol>
2. Preparation of biotin-coated glass slides
<ol>
	<li>Preliminary cleaning of the glass slides
	<ol>
		<li>Place 12 glass slides with dimensions of 60.0 &#215; 4.0 &#215; 0.3 mm3 (L &#215; W &#215; T) in a short and wide glass dish.</li>
		<li>Fill the glass dish with acetone and sonicate for 5 min. Then wash the slides with ultrapure water 5 times and fill &#190; of the dish with water.</li>
		<li>Add 10 M KOH to fill the dish and sonicate the glass slides for 20 min. Wash the slides with water 5 times.</li>
		<li>Add ethanol and sonicate for 5 min, pour out the ethanol and then dry them separately at 300 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Aminosilane coating
	<ol>
		<li>Place the 12 cleaned slides back into the glass dish containing methanol. Clean a PEGylation flask with methanol by sonicating for 5 min, then fill it with 25 mL methanol, 1.25 mL water, 0.125 mL HAc, 0.25 mL 3-aminopropyltriethoxysilane (AMEO).</li>
		<li>Immediately replace the methanol in the glass dish with the prepared AMEO solution. Incubate at room temperature for 30 min.</li>
		<li>Rinse the slides with water several times, then dry them by nitrogen purge. Place the dried slides in clean glass dishes.</li>
	</ol>
	</li>
	<li>PEGlation
	<ol>
		<li>Prepare NaHCO3 solution (8.4 mg/mL) and PEGylation buffer (37.5 mg PEG, 6 mg biotinylated PEG, 150 &#956;L NaHCO3 solution). Mix them well by spinning at 6,000 rpm for 1 min.</li>
		<li>Place 25 &#956;L of PEG solution onto each slide. Cover it with the other slide on top. Make sure that there are no bubbles in between the two slides.&#160; Place the slides in an empty pipet tip box. Be sure that the box is leveled and place it in a dark drawer for about 3 h.</li>
		<li>Rinse the slides with water and dry again. Store the dried slides at room temperature under vacuum for up to 2 weeks.</li>
	</ol>
	</li>
</ol>
3. Sample preparation prior to magnetic and force measurements
<ol>
	<li>Machine a plastic sample well with dimensions 4 &#215; 3 &#215; 2 mm3 (L &#215; W &#215; D). Glue a piece of biotin-coated glass (approximately 5 mm long, cut from the 60 mm long slides prepared in section 2) on the bottom surface using epoxy.</li>
	<li>Add 20 &#956;L of 0.25 mg/mL streptavidin aqueous solution into the sample well and incubate at room temperature for 40 min. Then rinse the sample well twice with TAM10 buffer.</li>
	<li>Immobilize the ribosome complexes.
	<ol>
		<li>Without antibiotics: Use a pipette to remove buffer from the sample well, then add 20 &#956;L of 0.1 &#956;M ribosome complex (MF-Pre or MF-Post) into the sample well. The ribosome complex will bind with the streptavidin on the surface via the 5&#39;-end biotin on the mRNA. Incubate at 37 &#176;C for 1 h and then rinse once with TAM10 buffer.</li>
		<li>For the experiment using both neomycin and fusidic acid: incubate the MF-Pre complex with neomycin at 37 &#176;C for 10 min; incubate EF-G with fusidic acid at 37 &#176;C for 20 min. The concentrations are as follows: 0.1 &#181;M ribosome complex, 2 &#181;M EF-G, 4 mM GTP, 4 mM PEP, 0.02 mg/mL pyruvate kinase, 0.2 mM neomycin, and 0.25 mM fusidic acid.</li>
		<li>Carry out the other antibiotics experiments similarly. The concentrations are as follows: 0.2 mM viomycin, 0.4 mM hygromycin B, and 0.25 mM fusidic acid.</li>
		<li>For frameshifting study, repeat the above steps for the MFNF-Pre and MFNF-Post complexes involving the slippery motif U6A. Use antibiotics fusidic acid plus neomycin, and fusidic acid alone, respectively.</li>
	</ol>
	</li>
	<li>Remove buffer from the sample well, then add 20 &#956;L of 1 &#956;M biotinylated probing DNA strand and incubate at room temperature overnight. Rinse the formed DNA-mRNA duplex once with TAM10 buffer.</li>
	<li>Remove the buffer from the sample well. Then add 20 &#956;L of 0.5 mg/mL streptavidin-coated magnetic beads into the sample well and incubate at room temperature for 2 h.</li>
	<li>Carefully insert the sample well into a holder and place it in a centrifuge. Remove the free magnetic particles from the surface by centrifuging at 84 x g for 5 min.</li>
</ol>
4. Magnetic and force measurements
<ol>
	<li>Turning on the laser
	<ol>
		<li>Turn on the laser using the key. Then press the power button.</li>
		<li>Adjust the sensitivity of lock-in amplifier 1 (LIA1) to 500 mV and wait for about 2 h to warm up and stabilize the atomic magnetometer.</li>
	</ol>
	</li>
	<li>Setting up the atomic magnetometer
	<ol>
		<li>Run the instrument control software and set up the measurement parameters. Some parameters may slightly vary in each measurement. 
		NOTE: The atomic magnetometer comprises a laser (described above), an SR830 lock-in amplifier (referred to as LIA1), an SR530 lock-in amplifier (referred to as LIA2), DS345 (referred to as FG1) and ATF20B (referred to as FG2) function generators, a high-resolution motor and a computer. All of these should be turned on at this step of the protocol.</li>
		<li>Set up motor moving mode to Noise and default position to 0. Press Lock on front panel, adjust the sensitivity of LIA1 back to 200 mV.</li>
		<li>Adjust the current and voltage of the laser and find the proper resonance peak and signal-to-noise level. Press Sweep on the front panel. Note the amplitude/width ratio should be above 0.5 and phase value should be less than 5 degree. If not, re-do the sweep step.</li>
		<li>Plug-in the output of LIA2 to the feedback of the laser to lock its frequency. This amplifier measures the optical rotation of an auxiliary cesium cell to maintain the laser frequency on resonance12. The state remains until the end of the measurement.</li>
		<li>Plug-in function generator FG2 to input a square wave (500 mVpp, 100 mHz) as the reference signal. The square wave corresponds to 100 pT and is used to convert the current output of the amplifier to magnetic signal. Unplug the function generator.</li>
		<li>Set up motor moving mode to Two-way and default position to 260 mm. Check the status of the temperature controller to ensure the proper temperature (~37 &#176;C) of the atomic sensor.</li>
		<li>Check the stability of the whole system by measuring the signals of the empty sample holder twice. Evaluate stability and noise level after subtracting the two traces. Typical noise level and fluctuation should be &#177;2 pT at 30 ms integration time.</li>
	</ol>
	</li>
	<li>Magnetizing the sample
	<ol>
		<li>Gently place the sample on the magnetization station and let it stay for 2 min. 
		NOTE: The magnetization station consists of a permanent magnet (~0.5 T) and a plastic spacer.</li>
		<li>Put the sample back into the sample holder. Use 335.4 &#215; g centrifugal force to remove the nonspecifically bound magnetic particles.</li>
	</ol>
	</li>
	<li>Magnetic measurements after applying forces
	<ol>
		<li>Use tweezers to load the sample onto the motor. Click Lock on the front panel to run the program. Meanwhile, use tweezers to load the other sample in the holder and place it in the centrifuge. Note that the coated glass side should face the center of the centrifuge. 
		NOTE: The technique of force-induced remnant magnetization spectroscopy (FIRMS) is used, which uses an atomic magnetometer to measure the magnetic signal of the sample after applying mechanical force on the molecular interactions in the sample. Here, the molecular interactions are between the DNA ruler molecules and the mRNA in the ribosome complex. The force is increased stepwise by increasing the centrifugal speed. After applying each force, the motor translates the sample to the atomic sensor and then moves back. Hence, two magnetic field profiles are obtained, one during the forward scan and the other during backward scan13. We only use the latter to extract the peak height due to its better signal-to-noise ratio. The peak height in current (nA) is converted to magnetic signal amplitude (pT) based on the calibration square wave.</li>
		<li>Every measurement lasts approximately 5 min. When motor comes back to 0, click Save on the front panel. Carefully use tweezers to take samples from motor and centrifuge. Use initial speed corresponding to 335.4 &#215; g (2000 rpm, revolution per minute for the centrifuge listed in the Table of Materials).</li>
		<li>Exchange the two samples and apply a stronger force by increasing the centrifugal speed by 100 rpm or similar step size. Process alternately to gradually increase the force; a complete force spectrum is obtained after 10-12 data points.</li>
	</ol>
	</li>
	<li>Finishing the experiment
	<ol>
		<li>When all planned experiments are finished, turn off the equipment, proceeding in the opposite order as it was turned on.</li>
		<li>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 beads contamination.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Open the Python analysis script and input all the experimental data. Click Load square to input the square wave as the reference. Then click Load baseline to input residual magnetic signal as background.</li>
		<li>Define the overall magnetic signal decrease as B0. Normalize each magnetic signal decrease B to B0 and express it as a percentage. Plot the percentage (B/B0) versus centrifugal force to obtain the FIRMS spectrum. 
		NOTE: The centrifugal force F is calculated from the buoyant mass of the magnetic beads m (4.6 &#215; 10&#8722;15 kg), centrifugal speed w, and radius of the centrifuge r (7.5 cm here) via equation F = mw2r. The typical force resolution is 2-4 pN and force range is 15-95 pN in this work.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Noller, H. F., Lancaster, L., Mohan, S., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+structural+dynamics+in+translocation:+yet+another+functional+role+for+ribosomal+RNA.">Ribosome structural dynamics in translocation: yet another functional role for ribosomal RNA.</a> Quarterly Review of Biophysics. 50, 12 (2017).</li><li>Zhou, J., Lancaster, L., Donohue, J. P., Noller, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+ribosome+hands+the+A-site+tRNA+to+the+P+site+during+EF-G-catalyzed+translocation.">How the ribosome hands the A-site tRNA to the P site during EF-G-catalyzed translocation.</a> Science. 345, 1188-1191 (2014).</li><li>Grentzmann, G., Ingram, J. A., Kelly, P. J., Gesteland, R. F., Atkins, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual-luciferase+reporter+system+for+studying+recoding+signals.">A dual-luciferase reporter system for studying recoding signals.</a> RNA. 4, 479-486 (1998).</li><li>Fang, Y., Treffers, E. E., Li, Y., Tas, A., Sun, Z., van der Meer, Y., de Ru, A. H., van Veelen, P. A., Atkins, J. F., Snijder, E. J., Firth, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+-2+frameshifting+by+mammalian+ribosomes+to+synthesize+an+additional+arterivirus+protein.">Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.</a> Proceedings of National Academy of Sciences of the United States of America. 109, 2920-2928 (2012).</li><li>Yan, S., Wen, J. D., Bustamante, C., Tinoco, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+excursions+during+mRNA+translocation+mediate+broad+branching+of+frameshift+pathways.">Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways.</a> Cell. 160, 870-881 (2015).</li><li>Shirokikh, N. E., Alkalaeva, E. Z., Vassilenko, K. S., Afonina, Z. A., Alekhina, O. M., Kisselev, L. L., Spirin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+ribosome-mRNA+complexes+at+different+translation+stages.">Quantitative analysis of ribosome-mRNA complexes at different translation stages.</a> Nucleic Acids Research. 38, 15 (2010).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+pathways+of+-1+translational+frameshifting.">Dynamic pathways of -1 translational frameshifting.</a> Nature. 512, 328-332 (2014).</li><li>De Silva, L., Yao, L., Wang, Y., Xu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-defined+and+sequence-specific+noncovalent+binding+forces+of+DNA.">Well-defined and sequence-specific noncovalent binding forces of DNA.</a> Journal of Physical Chemistry B. 117, 7554-7558 (2013).</li><li>Yin, H., Xu, S., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+DNA+rulers+reveal+an+"mRNA+looping"+intermediate+state+during+ribosome+translocation.">Dual DNA rulers reveal an "mRNA looping" intermediate state during ribosome translocation.</a> RNA Biology. 15, 1392-1398 (2018).</li><li>Tsai, T. W., Yang, H., Yin, H., Xu, S., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+"-1"+and+"-2"+ribosomal+frameshiftings+revealed+by+force+spectroscopy.">High-efficiency "-1" and "-2" ribosomal frameshiftings revealed by force spectroscopy.</a> ACS Chemical Biology. 12, 1629-1635 (2017).</li><li>Altuntop, M. E., Ly, C. T., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+study+of+ribosome+hierarchic+dynamics+at+the+peptidyl+transferase+center.">Single-molecule study of ribosome hierarchic dynamics at the peptidyl transferase center.</a> Biophysical Journal. 99, 3002-3009 (2010).</li><li>Garcia, N. C., Yu, D., Yao, L., Xu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+atomic+magnetometer+at+body+temperature+for+magnetic+particle+imaging+and+nuclear+magnetic+resonance.">Optical atomic magnetometer at body temperature for magnetic particle imaging and nuclear magnetic resonance.</a> Optics Letters. 35, 661-663 (2010).</li><li>Yao, L., Xu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range,+high-resolution+magnetic+imaging+of+nanoparticles.">Long-range, high-resolution magnetic imaging of nanoparticles.</a> Angewandte Chemie International Edition. 48, 5679-5682 (2009).</li><li>Kurkcuoglu, O., Doruker, P., Sen, T. Z., Kloczkowski, A., Jernigan, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+structure+controls+and+directs+mRNA+entry,+translocation+and+exit+dynamics.">The ribosome structure controls and directs mRNA entry, translocation and exit dynamics.</a> Physical Biology. 5, 046005 (2008).</li><li>Qu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+uses+two+active+mechanisms+to+unwind+messenger+RNA+during+translation.">The ribosome uses two active mechanisms to unwind messenger RNA during translation.</a> Nature. 475, 118-121 (2011).</li><li>Jacks, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+ribosomal+frameshifting+in+HIV-1+gag-pol+expression.">Characterization of ribosomal frameshifting in HIV-1 gag-pol expression.</a> Nature. 331, 280-283 (1988).</li><li>Schuwirth, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+the+bacterial+ribosome+at+3.5+&#197;+resolution.">Structures of the bacterial ribosome at 3.5 &#197; resolution.</a> Science. 310, 827-834 (2005).</li><li>Jia, H., Wang, Y., Xu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+force+spectroscopy+reveals+ribosomal+motion+at+sub-nucleotide+steps.">Super-resolution force spectroscopy reveals ribosomal motion at sub-nucleotide steps.</a> Chemical Communications. 54, 5883-5886 (2018).</li></ol>

No potential conflict of interest was reported by authors.

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 rulers. The dual ruler assay is uniquely suited to probe both sides of mRNA during translocation. Because it is a physical approach that only relies on the formation of the duplexes, it is not limited by the mRNA sides or other biological constrains. This is an advantage compared to other techniques that are based on biochemical reactions. We have shown that by using a long linker molecule, the rulers can reach the targeted binding site to determine the mRNA position. Specifically, for the ribosome, the linker length is 50 T, which corresponds to 17 nm in length. This length is very close to the size of the ribosome17. The concentrations of the antibiotics are typical values from the literature. It is also possible to use our method to reveal the onset values for their functions.
There are two critical aspects for the dual ruler assay. One is the delicate functionalized surface for molecular immobilization and subsequent biological reactions. Every loading and washing step should be performed with minimum disturbance to the surface, so that the molecules immobilized on the surface will remain intact. For the DNA-mRNA hybridization process, sufficient time is necessary to ensure the completion of the process. It is also advised to use TAM10 buffer with extra 1 M NaCl to maintain a homeostatic system. The other critical aspect is the magnetization of the beads. Since excessive beads are used, there are plenty of free beads not immobilized on the surface. Therefore, when magnetizing the sample, the coated surface should approach and leave the magnet vertically. Any minor swing of the sample can possibly cause scratching damage on the surface.
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-nt resolution. Novel mechanistic information regarding ribosome translocation has been obtained. The method is generally applicable for molecular displacement of nucleic acids, which is widely encountered in molecular biology.
For future development and applications, we have shown that using acoustic radiation force to replace the centrifugal force will further improve the force resolution, reaching sub-nt regime18. We are also investigating multiplexed detection using atomic magnetometers to improve the detection efficiency. With these improvements, we expect our method detailed in this work will find broad applications in biological research.&#160;

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>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:217px;">Glass slides</td>
			<td style="width:239px;">Evaporated Coatings</td>
			<td style="width:161px;"></td>
			<td>60.0 &#215; 4.0 &#215; 0.3 mm3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Acetic acid</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">A6283-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">3-Aminopropyltriethoxysilane</td>
			<td style="width:239px;">UCT specialties</td>
			<td style="width:161px;">21400088</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Epoxy glue</td>
			<td style="width:239px;">Devcon</td>
			<td style="width:161px;">31345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Streptavidin</td>
			<td style="width:239px;">ThermoFisher</td>
			<td style="width:161px;">434301</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">EDTA</td>
			<td style="width:239px;">GIBCO</td>
			<td style="width:161px;">774750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">M6250-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:161px;">5427R</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Vortex mixer</td>
			<td style="width:239px;">VWR</td>
			<td style="width:161px;">VM-3000</td>
			<td></td>
		</tr>
		<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;">SR530</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Laser</td>
			<td style="width:239px;">Newport</td>
			<td style="width:161px;">TLB-6918-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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 sample scan and produced a signal trace, with the maximum signal when the sample is the closest to the sensor. Figure 1B shows the photo of the overall instrument. Figures 1C,D show the photos of the magnetization station and the centrifuge used for force application, respectively.
Principle of the dual DNA ruler assay is shown in Figure 2. The ribosome complex is immobilized on the surface via the 5&#39;&#160;end of the mRNA. Two DNA rulers are designed to probe the exact position of the ribosome, one for each side of the mRNA. The current immobilization scheme makes the 3&#39;&#160;side easily accessible for the probing DNA rulers. Therefore, DNA oligomers conjugated with magnetic beads can form duplexes with the uncovered mRNA. The 5&#39;&#160;side, however, is sterically hindered by the ribosome and the surface. A linker molecule is thus needed for the DNA to reach the uncovered mRNA on this side. By varying the linker length, we have determined that when the linker is longer than 50 T, we can detect a strong magnetic signal which indicates successful formation of DNA-mRNA duplexes (Figure 2B). The correlation of dissociation force to duplex length is achieved by varying the number of nt on the DNA that are complementary to the mRNA. Figures 2C,D show the correlation for the 3&#39;- and 5&#39;- sides, respectively. Because the force difference between duplexes of consecutive lengths is typically 12-20 pN and the force resolution is typically 2-4 pN, we routinely achieve single-nt resolution for the length of DNA-mRNA duplexes based upon their dissociation forces.
Figure 3 presents the results of normal translocation in the absence and presence of various antibiotics. The translocation is from MF-Pre to MF-Post (Figure 3A). The inset indicates that MF-Pre carries vacant tRNAfMet and MF-tRNAPhe at the P- and A-sites, respectively. MF-Post carries tRNAfMet and MF-tRNAPhe at the E- and P-sites, respectively, with a vacant A-site. Figure 3B shows that without antibiotics, the ribosome moves by 3 nt on both sides (moving toward the 3&#39;-end). This is because of the following reasons: (i) The DNA-mRNA duplexes at the 5&#39;&#160;side exhibit 12 bp and 15 bp binding forces in MF-Pre and MF-Post, respectively. (ii) Duplexes at the 3&#39;&#160;end exhibit a reversed change from 15 to 12 bp (Figure 3C). However, when both fusidic acid and neomycin are present, the force spectra show that the ribosome moves only by 1 nt at the 5&#39;&#160;side but 2 nt at the 3&#39;&#160;side (Figure 3D,E). This result indicates that the ribosome translocated via a stepwise mechanism, resulting a looped mRNA conformation that has an extra nt inside the ribosome, which has not been experimentally revealed before. This is consistent with reported theoretical simulation14. Alternatively, the ribosome may be stretched to cover 28 nt of mRNA, instead of the usual 27 nt15. After washing away the antibiotics, normal translocation occurs, as evidenced by 3 nt movements from both the 5&#697; and 3&#697; sides (purple trace in Figure 3D, E). The looped conformation does not form when only fusidic acid is used. The force spectra are consistent with the ribosome movement of 3 nt at both sides, similar to the situation for normal translocation (Figure 3F,G). The dual ruler assay also reveals that viomycin completely inhibited translocation, as shown by 0 nt movement on both sides (Figure 3H,I).
We have also investigated whether this looped conformation can form on &#34;-1&#34;&#160;frameshifting motifs. Here, the translocation from MFNF-Pre and MFNF-Post complexes takes place on the &#39;U6A&#39;&#160;motif, which has been found to be part of the &#39;-1&#39;&#160;frameshifting motif in HIV16. Figures 4A,B show that in the MFNF-Post, the DNA-mRNA duplex is shortened by either 2 or 3 nt at the 3&#39;-end; the duplex length increases by either 2 or 3 nt at the 5&#697;-ends. The results suggest the coexistence of both normal translocation and &#34;-1&#34;&#160;frameshifting, with the former correlating with the 3-nt movement and the latter correlating with the 2-nt movement. The dual ruler assay is able to clearly resolve the two populations. With both neomycin and fusidic acid, we observe that the ribosome moves by 2 nt at the 3&#39;-end but only 1 nt at the 5&#697;-end, respectively (Figure 4C,D). Therefore, the looped mRNA conformation also takes place on the slippery sequence. When only fusidic acid is present for the MFNF-Pre (Figure 4E,F), the result is the same as that for MFNF-Post in panels A and B (blue traces), indicating that fusidic acid alone is not sufficient to pause translocation or frameshift. Therefore, it confirms that both fusidic acid and neomycin are required for the unequal displacements for the two sides of mRNA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59918/59918fig1.jpg" /> 
Figure 1: Instruments for the FIRMS-based dual DNA ruler assay. (A) Schematic of the magnetic detection. 1: sample and its mount; 2: motor; 3: atomic sensor and magnetic shield. (B) Photo of the atomic magnetometer and sample handling system. The numbers indicate the actual corresponding components shown in the schematic. Note the magnetic shield is inside the cardboard box for improved thermal stability. (C) Magnetization station. (D) Centrifuge used for force application. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59918/59918fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59918/59918fig2.jpg" /> 
Figure 2: Dual ruler assay with single-nt resolution for studying ribosome translocation. (A) Schematic of the dual ruler assay, in which two DNA rulers are designed to respectively probe the 5&#697;- and 3&#697;- ends. The red line indicates the polyT linker. (B) Optimization of the linker length for Ruler-In. (C) FIRMS results to determine the dissociation forces of the duplexes between Ruler-Ins and mRNA.&#160; (D) Dissociation forces of the duplexes between Ruler-Outs and mRNA. The error bar is defined as the ratio between the instrument noise and the overall magnetic signal decrease (B0), with typical value of &#177;3-5%. Figure has been reproduced with permission9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59918/59918fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59918/59918fig3.jpg" /> 
Figure 3: Probing translocation steps under the influence of various antibiotics. (A) Probing scheme for the MF-Pre and MF-Post complexes. Inset indicates the schematic ribosomes in Pre and Post, which correspond to the solid and dash-lined ovals, respectively. (B, C) FIRMS results with no antibiotics. (D, E) Results with both fusidic acid and neomycin. (F, G) Results with fusidic acid only. (H, I) Results with viomycin only. Left panels: using Ruler-In to probe the 5&#39; side; right panels: using Ruler-Out to probe the 3&#39; side. The error bars are calculated the same way as those in the previous figure. Figure has been reproduced with permission9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59918/59918fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59918/59918fig4.jpg" /> 
Figure 4: FIRMS results of using dual rulers to probe frameshifting. (A, B) MFNF-Pre and MFNF-Post without antibiotics. (C, D) Results with both fusidic acid and neomycin. (E, F) Results with only fusidic acid. Left panels: using Ruler-In to probe the 5&#697; side; right panels: using Ruler-Out to probe the 3&#697; side. The error bars are calculated the same way as those in the previous figure. Figure has been reproduced with permission9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59918/59918fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution at JoVE.com

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. ...

dual-dna-rulers-to-study-mechanism-ribosome-translocation-with-single

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.2K Views.  University of Houston. 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.

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

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.