Long DNA constructs to study helicases and nucleic acid translocases using optical tweezers. Methods in Enzymology, 673, 311-358.

Single molecule biophysics experiments for the study of DNA-protein interactions usually require production of a homogeneous population of long DNA molecules with controlled sequence content and/or internal tertiary structures. Traditionally, Lambda phage DNA has been used for this purpose, but it is difficult to customize. In this article, we provide a detailed and simple protocol for cloning large (~25 kbp) plasmids with bespoke sequence content, which can be used to generate custom DNA constructs for a range of single-molecule experiments. In particular, we focus on a procedure for making long single-stranded DNA (ssDNA) molecules, ssDNA-dsDNA hybrids and long DNA constructs with flaps, which are especially relevant for studying the activity of DNA helicases and translocases. Additionally, we describe how the modification of the free ends of such substrates can facilitate their binding to functionalized surfaces allowing immobilization and imaging using dual optical tweezers and confocal microscopy. Finally, we provide examples of how these DNA constructs have been applied to study the activity of human DNA helicase B (HELB). The techniques described herein are simple, versatile, adaptable, and accessible to any laboratory with access to standard molecular biology methods.

The study of DNA-protein interactions using single-molecule methods typically requires the production of a homogeneous population of long DNA constructs of several tens of kbps.This is particularly important in methods that combine fluorescence because light diffraction limits resolution to a few hundreds of nm, i.e., around 1 kbp.Among these methods the combination of OT with confocal microscopy is powerful because it allows simultaneous and correlative manipulation and visualization of single-molecule interactions in real time with sub-picoNewton force resolution and sub-nanometer localization (Balaguer et al., 2021;Gutierrez-Escribano et al., 2019;Hashemi Shabestari, Meijering, Roos, Wuite, & Peterman, 2017;Wasserman, Schauer, O'Donnell, & Liu, 2019).Moreover, these long DNA molecules often need to include specific DNA sequences, i.e., a protein-binding site, and/or to incorporate localized tertiary structures, such as a ssDNA flap or a hairpin that acts as a loading-site for a protein (Brutzer, Luzzietti, Klaue, & Seidel, 2010;Hormeno et al., 2021;Levikova, Klaue, Seidel, & Cejka, 2013;Luzzietti et al., 2011;Wilkinson, Carrasco, Aicart-Ramos, Moreno-Herrero, & Dillingham, 2020).Finally, they also need to be labeled at their ends with chemical modifications (typically biotins and/or digoxigenins) to allow tight non-covalent binding to functionalized surfaces for manipulation.Both the length and specificity requirements of these substrates have created technical challenges that the single-molecule community have dedicated considerable effort towards overcoming.
The problem of length has traditionally been solved by employing commercially available bacteriophage DNA, typically from Lambda (48.5 kbp) (S.B. Smith et al., 1996).However, with the use of Lambda, the problem of the sequence specificity remains.Therefore, several strategies have been developed for the re-design of this kind of substrate.Some require restriction enzyme cleavage and ligation (Li et al., 2021), but this approach is limited to the few unique restriction sites present along the Lambda sequence.In vitro site-specific recombination methods have also been described (Bell, Plank, Dombrowski, & Kowalczykowski, 2012;Thorpe & Smith, 1998), but these involve complex molecular cloning steps and are limited to specific non-coding regions in the Lambda genome.Moreover, the specialized recombinase enzymes required need to be produced in-house as they are not commercially available.In vivo recombineering methods have also been described (Brown, De La Torre, & Finkelstein, 2017;Kim, De La Torre, Leal, & Finkelstein, 2017) but the purification of recombinant Lambda DNA from lysogens is required.In summary, although these methods do allow the generation of customized long DNA molecules, they are relatively complex and inaccessible to many laboratories.
The most common vehicle for the facile design, production and purification of specific DNA sequences are plasmids.These circular molecules are very easy to store and amplify using commercially available cloning strains and plasmid purification kits.In addition, because plasmids are copied within bacteria which possess robust DNA repair mechanisms, the quality of the resulting DNA is very high and far superior to that obtained in a PCR reaction (Eckert & Kunkel, 1991).Importantly, plasmids are an excellent choice for producing unique and/or customized DNA constructs by exploiting the availability of synthetic DNA fragments which can be inserted by conventional cloning methods or by Gibson Assembly (Van Loenhout, De Grunt, & Dekker, 2012;Mueller, Spenkelink, van Oijen, & Lewis, 2020;Sánchez et al., 2021;Spakman, King, Peterman, & Wuite, 2020).However, while plasmid-based approaches allow the production of long dsDNA constructs containing the sequences of interest, they do not address the generation of long ssDNA or ssDNA-dsDNA hybrids molecules which are especially useful for the study of DNA helicases and translocases.So far, the strategy followed to produce long ssDNA and ssDNA-dsDNA hybrid constructs has been based on Lambda DNA and is subject to the limitations above with respect to sequence design (Belan, Barroso, et al., 2021;Belan, Moore, et al., 2021;Brouwer et al., 2017;Candelli et al., 2013Candelli et al., , 2014;;G. A. King et al., 2013;S. B. Smith et al., 1996).
In this article we provide a detailed and simple protocol, accessible to any molecular biology laboratory, for the design and cloning of large plasmids (up to ~25 kbp) with sequences of interest.These plasmids serve to generate long customized DNA constructs for different singlemolecule experiments.In particular, we describe a procedure to produce long ssDNA and ssDNA-dsDNA hybrid constructs.We also describe a procedure to fabricate a long DNA construct containing a ssDNA flap that is useful to study the activity of DNA helicases (Levikova et al., 2013;Wilkinson et al., 2020).These three long DNA constructs are ideal for determining polarity of movement (Gilhooly, Gwynn, & Dillingham, 2013) and kinetic parameters such as translocation velocity or pausing frequency and duration.Besides the cloning steps, we also describe how to modify the substrates for immobilization in OT set-ups and provide examples of their use for the study of a human DNA helicase.

 2. Molecular design of large DNA plasmids
We describe below the procedure to produce large customized plasmids of over 17 kbp, containing particular regions of interest.These plasmids comply with two key requirements for single-molecule optical tweezers experiments, the long length and the capacity to easily modify the sequence and incorporate chemical modifications or tertiary structures at a specific region.
Large plasmids were obtained by ligation of three DNA pieces (C1, C2, and C3, Fig. 1).C1 and C2 fragments are fabricated by PCR using Lambda DNA as the template and including suitable restriction sites in the PCR primers.The third key piece (C3) contains the sequence of interest (e.g., several copies of a parS recognition sequence (Balaguer et al., 2021)), and is obtained either by plasmid digestion or by PCR amplification.Importantly, the C3 fragment also includes both an antibiotic resistance gene, to allow correctly-transformed bacteria to be selected using the appropriate antibiotic, and an origin of replication, to allow the autonomous replication of the final plasmid.The three fragments are ligated and DH5α competent cells transformed by regular heat-shock procedure.Following this strategy, we have cloned plasmids up to 25 kbp in length.This strategy is very versatile.One can use any plasmid of choice to produce the C3 piece and select different sections of Lambda DNA for PCR-amplification depending on the restriction sites present in them.Additionally, one can vary the restriction sites included in the primers employed to perform the PCRs depending on the ones that are present in the final fragments that need to be ligated.Here, it is good practice to select compatible restriction sites which, once cleaved, produce overhangs that can be ligated to overhangs obtained by different restriction enzymes (e.g., complementary SalI and XhoI, or NotI and PspOMI enzymes).This allows one to avoid the potential complications caused if one of the restriction sites is present in the original sequence.Also, note that although we have employed Lambda DNA to produce the C1 and C2 pieces by PCR, any other template DNA could be used.
• Plasmid DNA of interest.

Procedure
1. Design primers including restriction sites suitable to get three ligation-compatible pieces of DNA.For instance, C1 fragment including NotI and SalI, C2 fragment including BamHI and NotI and, C3 fragment including SalI and BamHI.If C3 fragment is PCR produced, these sequences can be incorporated in the primers.Note that the selected restriction sites cannot be present anywhere else along each specific DNA fragment (Fig. 1).2. Dissolve primer stocks to 100 μM in nuclease free water.Store at -20 °C.

3.
Set up PCR reactions for C1 and C2 fragments on ice with a high-fidelity DNA polymerase enzyme such as Phusion high-fidelity DNA polymerase, following the manufacturer's instructions (Table 1).4. Transfer the tubes to the PCR thermocycler and start the PCR program ( 29.Analyze the restriction digestions in a 1% agarose gel as described in step 5 and look for appropriate sizes dependent on the restriction enzyme chosen 30.Perform DNA sequence analysis to any preliminary positive plasmid in a commercially available service.For sequencing, select oligonucleotides that allow you to sequence fragments of DNA that comprises junctions between fragments C1-C2, C2-C3 and C3-C1, and any region of interest that need to be present in the large final plasmid.31.Once the large plasmid is confirmed, it is ready to be used to prepare long DNA substrates for single molecule experiments (see Note 2).

Fabrication of long double-stranded DNA constructs from large plasmids
This section describes the general procedure to incorporate modified handles to the previously linearized large plasmid described in Section 2. This procedure results in a long dsDNA construct (>17 kb) suitable for single-molecule experiments that require immobilization of the molecule from their ends.These can be OT or MT experiments, but also flow-stretched experiments that require immobilization of the DNA on a surface for further TIRF imaging.The procedure we describe below is based on the cleavage of the plasmid by one or two restriction enzymes and, without further purification, the ligation to functionalized fragments of ~1 kbp (Fig. 2).These handles are typically produced by PCR and can incorporate either biotins or digoxigenins.This methodology is extremely efficient and versatile: depending on the plasmid sequence design, one can choose different restriction enzymes to locate the region of interest at a particular position with respect to the immobilized ends.
An alternative to ligation of PCR-labeled handles is the filling of 5'-overhangs with modified deoxynucleotides using the Klenow Fragment of DNA polymerase, which lacks 3'→5' exonuclease activity (Derbyshire et al., 1988) as performed by (van den Broek, Noom, & Wuite, 2005;Candelli et al., 2013Candelli et al., , 2014;;Sánchez et al., 2021;Spakman et al., 2020).This is a simple procedure often employed with Lambda DNA because its cos end sites are 12-nt long and therefore allow labeling with several biotins or digoxigenins (Balaguer et al., 2021;Brouwer et al., 2017;Forget, Dombrowski, Amitani, & Kowalczykowski, 2013;Gross, Farge, Peterman, & Wuite, 2010;S. B. Smith et al., 1996;Wasserman et al., 2019).However, we have found this procedure to be limited, compared to the use of long handles, in several aspects.On one hand, only 5'-overhangs (3'-recessed ends) can be filled in by Klenow Fragment exo-.On the other hand, the same kind of modified nucleotide, in terms of biotins or digoxigenins, is incorporated in both sides of the DNA construct, so differential end-labeling, if needed, will require extra steps.Additionally, DNA ends are labeled on different strands, and this makes it impossible to prepare in a single step pure ssDNA or hybrid ssDNA-dsDNA constructs (see next sections).For the same reason, molecules cannot be torsionally constrained, if required.Finally, the amount of labeled nucleotides per DNA end is very low compared to a PCR-produced handle as usually a restriction digestion overhang is 4-nt long.This reduces the force that can be tolerated in a pulling experiment and makes in-flux fishing more difficult in dual-trap OT experiments.
Other approaches to label DNA ends are based on the annealing and ligation of biotinylated oligonucleotides to the overhang generated after restriction digestion.Single-stranded regions are then filled with biotinylated deoxynucleotides by Klenow Fragment exo-, rendering a DNA construct with biotin labels at both strands on either end (Van Mameren et al., 2009).Slight modifications of this strategy allows one to obtain a final dsDNA construct labeled in both ends and on the same strand with the aim of generating ssDNA tethers (Candelli et al., 2013(Candelli et al., , 2014)).Drawbacks of these methods are the price of biotinylated nucleotides and potential difficulties in annealing and ligating a ssDNA-oligonucleotide to an only 4-nt overhang.To overcome this issue, Lambda DNA is usually employed because it has longer 12-nt cos ends (Camunas-Soler et al., 2013;Candelli et al., 2013Candelli et al., , 2014;;Madariaga-Marcos et al., 2018;Van Oijen et al., 2003;S. B. Smith et al., 1996).Similarly, expensive internally-labeled oligonucleotides can be replaced by tailed oligonucleotides produced by terminal transferase (Camunas-Soler et al., 2013;Madariaga-Marcos et al., 2018).The problem of direct ligation of ssDNA over dsDNA can be also overcome by the use of a biotinylated end-cap hairpin oligonucleotide that self-anneals to generate a cohesive dsDNA end (Paik & Perkins, 2011).The use of biotinylated end-cap hairpin oligonucleotides with a 12-nt overhang compatible to cos end sites of Lambda DNA has also been reported (G. A. King et al., 2013;G. A. King, Burla, Peterman, & Wuite, 2019;G. A. King, Peterman, & Wuite, 2016).These methods have the inconvenience of using rather expensive biotinylated oligonucleotides together with having a reduced amount of labeled nucleotides per DNA end.
As mentioned above, we opted for production of modified handles by PCR.This is an easy and inexpensive method with additional advantages.It incorporates a high frequency of labeled nucleotides per DNA end, and this is important for in-flow capture of DNA molecules in OT experiments.The ligation process is very efficient and allows one to easily modify the position of a region of interest by just selecting another pair of restriction sites.It allows for the generation of torsionally constrained or non-torsionally constrained tethers, simply by using either phosphorylated or dephosphorylated handles, respectively.Importantly, it facilitates simple preparation of pure ssDNA or hybrid ssDNA-dsDNA constructs as described in following sections.
• DNA plasmid as template for preparation of handles.
• Large DNA plasmid prepared as described in Section 2.

A) Preparation of the central part
The plasmid design and the position of its unique restriction sites will determine the choice of appropriate restriction enzymes to locate any region of interest at a particular position with respect to the immobilized ends.Additionally, depending on the final purpose of these DNA constructs, the large plasmid can be linearized by digestion with a single restriction enzyme or with two different ones, which will render two non-complementary overhangs (Fig. 2).In the case of digestion with two enzymes, it is recommended that the restriction sites are close to one another (within 200 bp), because this will avoid an extra purification step.For the same reason, it is also recommended to use restriction enzymes that can be heat inactivated.It is important to keep purification steps to a minimum to avoid undesired damage to the DNA and dilute samples.Finally, it is important to keep in mind that long DNAs can easily break due to shear forces created by pipetting.Use wide-bore pipette tips and mix solutions containing large DNAs gently, avoiding high-speed vortexing and repetitive pipetting.
1. Cut the plasmid of interest (1 µg) with the appropriate restriction enzyme/s (5 units of each enzyme) in 6.25 μL buffer provided by the manufacturer for 1 h 30 min at the recommended temperature.This will create the necessary overhangs for ligation with highly-biotinylated handles.In this particular example, we linearized the plasmid shown in The handles are DNA fragments of ~1 kb produced by PCR and labeled with biotins or digoxigenins.The procedure below uses the pSP73-JY0 (Fili et al., 2010) or pBlueScript SK+ (Stratagene) plasmids as a template, but any other plasmid or linear DNA could be used.

1.
Design primers including in one of them the desired restriction site that generates a sticky end for ligation to the central fragment of the DNA construct.In this particular example, we fabricated a NotI handle or a compatible PspOMI handle (Fig. 2, left).2. Dissolve primer stocks to 100 μM in nuclease free water.Store at -20 °C.3. Set up PCR reactions to prepare highly-labeled handles, biotins in the present example, with GoTaq® G2 Flexi DNA polymerase following the manufacturer's instructions (Table 5).If the same handle is ligated to both ends of the central part, just perform a single PCR reaction.4. Transfer the tubes to the PCR thermocycler and start the PCR program (Table 6).Set the lid at 100 °C to prevent condensation.5.It is recommended to analyze the PCR products by loading 5 μL of PCR sample into a 1% agarose gel as described in Section 2.3, step 5.If the expected band is observed, continue with the next step.6. Purify the PCR products with a commercial PCR purification kit, and then elute products in 30 μL of supplied EB. 7. Perform digestion of the PCR fragments with 20-30 units of the proper restriction enzyme for 1 h 30 min in its recommended buffer and at indicated temperature.8. Perform a final purification of the digested-PCR products with a commercial PCR Purification kit and elute in 30 μL of supplied EB. 9. Determine DNA concentration with a NanoDrop spectrophotometer.Store at 4 °C.
An additional step of dephosphorylation could be required depending on the specific final DNA constructs as shown in Sections 4 and 5.Additional steps could be required depending on the specific DNA constructs as shown in next sections.

D) Test of production of long dsDNA constructs in C-Trap
Standard quality control assays of the fabricated long dsDNA construct involve force-extension curves to characterize the contour length of the tethers.Although we have performed these studies using the C-Trap instrument from LUMICKS (Candelli, Wuite, & Peterman, 2011), the procedure should be easy adaptable to any other home-made or commercial optical tweezers.
Our equipment incorporates a confocal microscope that allows visualization of duplex DNA by SYTOX intercalation or labeling of proteins with organic dyes or quantum dots.Experiments were done using a microfluidic cell with five laminar flow separated channels (Fig. 3A).This design without physical barriers allows the direct transfer of the trapped DNA between different flow channels and the in-situ assembly and characterization of the DNA constructs.
Mechanical characterization of dsDNA molecules.
1. Prepare flow cell for experiments (cleaning and passivation) as recommended by manufacturer.2. Fill in syringes of flow cell channels as follow: Channel 1. 10 µL polystyrene beads (previously resuspended by gently vortexing) in 990 µL TEN buffer.
Channel 3: 1 mL of TEN buffer for regular dsDNA.
Channel 5: 1 mL of TEN buffer.3. Open channels 1-3 and start flowing their content by applying a pressure of 0.2-0.4bar. 4. Move optical traps to a mid-position into channel 1. Trap one bead in each trap.Adjust the laser power to achieve a trap stiffness of ~0.4 pN/nm. 5. Move the traps to channel 2 and capture a single DNA tether between the beads.To do that, steer the bead in the mobile trap back and forth with respect to the other bead while keeping the flow open.If the DNA concentration is adequate, the biotinylated construct will ensemble between the streptavidin-covered beads after a few attempts, and a response in the force sensor will be noticed when moving one bead with respect to the other.The presence of moderate salt in the buffer helps to capture DNA using this procedure.6. Move the trapped DNA to channel 3. Perform a force extension curve at 1 µm/s to discard double tethers and detect if the molecule is torsionally-constrained (TC) or nicked (Fig. 3B).
As expected for the protocol described above, most of the molecules were TC (Fig. 3B, TC dsDNA, stretching (red) and relaxation (light red) curves), but occasionally, we also observed nicked molecules (Fig. 3B, nicked dsDNA, stretching (black and green) and relaxation (grey) curves).To assure a homogeneous population of nicked DNA tethers some strategies can be considered (see Note 3). 7. Check the contour length of the tether by fitting to a polymer extension model.In this case, a fit to the extensible Worm-Like-Chain (eWLC) model (Odijk, 1995) (Fig. 3B, blue dashed line) provided a contour length of 24,940 bp with a persistence length, PdsDNA, of 19 nm (using a crystallographic length for dsDNA, L0, dsDNA, of 0.34 nm bp -1 , and a stretch modulus, SdsDNA, of 1500 pN).
Direct visualization of dsDNA molecules.

8.
Once confirmed the integrity of a dsDNA tether, fill in channel 4 with 200 µL of 100 nM SYTOX Green dissolved in TEN buffer.9. Move the molecule to channel 4 and hold the molecule at 20-40 pN.10.Take a scan with the confocal microscope using excitation light at 488 nm.The whole molecule should be visible as SYTOX Green is a dsDNA labelling dye (Fig. 3C).11.Alternatively, take a kymograph to capture florescence between the beads versus time (Fig. 3D).Standard parameters are a pixel size of 50 nm and a line scan speed of 50-100 ms.It is recommended to work with the minimum laser power (~2%) to avoid the stained DNA from breaking during the kymograph acquisition.The emission is detected employing a green filter 585/75 nm.
It is important to note that the use of Intercalant dyes like SYTOX are useful to visualize dsDNA and check proper production of molecules as described above, but they can interfere with the binding and activity of proteins, and therefore should be avoided in those experiments.

Notes
1. Tandem products (double length) are often produced in the final ligation step (Section 3.3part C) because restriction sites are often palindromic.To avoid the generation of tandem molecules, one can increase the excess of handles or use compatible sticky ends obtained by restriction with two different enzymes.Sites generated by compatible restriction enzymes lose the original restriction sequence once they are ligated.In this case, add the enzyme employed to linearize the plasmid to the ligation mix.Note however, that there are occasions where it is desirable to promote the generation of tandem molecules to obtain longer constructs.In this case, decrease the excess of handles (Step 1, Section 3.3-part C). 2. Concentration of cleaved handles (Step 1, Section 3.3-part C) might be lower than expected, if the restriction site contains a deoxythymidine.In this case, a Bio-dUTP might be incorporated instead of a dTTP during the PCR fabrication, and the modified restriction site will not be cut by the restriction enzyme.If the restriction site contains a Thymine, a higher excess of that handle will be required.This section describes a specific procedure to produce ssDNA-dsDNA hybrids.These constructs are useful to assess the substrate specificity (ssDNA or dsDNA) of a DNA-binding protein, to mimic a single ssDNA-dsDNA junction, such as those found in the nucleus at replication forks and R-loops, or to study the activity of DNA polymerases at the junction (Hoekstra et al., 2017).Moreover, ssDNA-dsDNA junctions are loading sites for some translocases that can initiate translocation from such a position (Tomko et al., 2010).Some proteins like CST bind preferentially to these junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action (Bhattacharjee, Wang, Diao, & Price, 2017).
The first attempts to produce ssDNA-dsDNA hybrids were based on the use of exonucleases (Lee, Balci, Jia, Lohman, & Ha, 2013;Wuite et al., 2000).However, these approaches are inconvenient for several reasons.They do not allow full control of the position or the orientation of the junction because they can degrade DNA from an internal nick and it is difficult to control the length of the final ssDNA fragment.This results in a non-homogeneous population of molecules that may not be suitable for single-molecule studies.Additionally, if digestion of the dsDNA is produced in situ, then inactivation of the enzyme and/or an extra cleaning step of the flow cell is required.
Alternative methods based on the separation of one of the duplex strands by applying a stretching force to its complementary partner strand are currently preferred.In these methods one needs to produce nicks in the strand to be removed.Most approaches use the asymmetric BbvCI restriction site (Heiter, Lunnen, & Wilson, 2005) that allows nicking of either the bottom or top strand using Nb.BbvCI or Nt.BbvCI.This nicking combined with end-labeling using biotinylated/non-biotinylated oligonucleotides and sometimes Klenow exo-enzyme, can produce DNA molecules labeled at both ends but on only one of the strands (Candelli et al., 2013).This method obviously requires the engineering of a plasmid to contain those asymmetric restriction sites and relies on the use of oligonucleotides despite the difficulty of ligating a ssDNA oligonucleotide to a dsDNA end that is usually 4-nt long.A more recent approach to introduce nicks and gaps in a long DNA substrate uses site-specific Cas9 nicking (Anand et al., 2022;Belan, Barroso, et al., 2021;Belan, Moore, et al., 2021).The length and position of the gaps is determined by the choice of the guide RNA sequences.If one is looking for gaps of a specificlength, this is a very attractive strategy, although, as commented by the authors, there are some limitations regarding the length of the gap.
In the approach described below, we ligate a dephosphorylated biotinylated handle and use nicking enzymes to create a single nick along the DNA construct.This creates a region flanked by two nicks (depicted with red and green dots in Fig. 4A, respectively).The ssDNA strand between the nicks is then removed in-situ by tension as described before (Candelli et al., 2013).This method is simple because it only requires the engineering of one BbvCI restriction site in one of the three fragments employed to generate the large plasmid (Fig. 4B) and the second nick is easily introduced by the use of a dephosphorylated handle.Additionally, our method does not require the use of biotinylated/non-biotinylated oligonucleotides.This gives complete control over the position and orientation of the ss-dsDNA junction.Moreover, the position of the junction with respect to one DNA end can be easily modified by linearizing the plasmid with different enzymes and ligating with different handles (Fig. 4C).

Design of the DNA plasmid
This method requires a large plasmid that has one BbvCI restriction site (Fig. 4B).An example of oligonucleotides employed for PCR-amplification of C1 and C2 fragments from Lambda DNA is shown in Table 7.In this particular example, the BbvCI site is contained in the C1 fragment.Production of the C3 fragment by plasmid digestion is described in Section 2. The general procedure to produce this plasmid as well as the equipment, buffers, strain and reagents are described in Section 2.

Assembly of the ssDNA-dsDNA construct
The equipment, buffers, reagents, and procedure for the assembly of this DNA construct, are described in Section 3.However, the following specific points need to be considered.

A) Preparation of the central part
The central part must be linearized with two different restriction enzymes to generate noncomplementary overhangs.The ss-dsDNA junction can be positioned with respect to the beads by selecting particular pairs of restriction enzymes (Fig. 4C).This is convenient because it allows one to have different proportions of ssDNA and dsDNA without the need to fabricate a new plasmid (Fig. 4B).The linearized central part is next ligated to two different highly biotinylated handles, with one of them being dephosphorylated.In this particular example, we chose to linearize the plasmid of Figure 4B with XhoI and NotI (Fig. 4C, option 1, right).

B) Preparation of the highly-biotinylated handles
1. Select the handle to be dephosphorylated (in this example, the XhoI handle).This handle should include the restriction site compatible with one end of the linearized central part.
Note that the 5´-sticky end of this handle and the nick after the digestion with the nicking BbvCI restriction enzyme must be on the same strand.Follow next step of dephosphorylation after step 9 of Section 3.3-part B.
2. Dephosphorylation reaction.Incubate the purified handle with 1 unit per 1 pmol of DNA ends of rSAP in buffer provided by the manufacturer for 2 h at 37 °C.Inactivate the enzyme by heating at 65 °C for 10 min.This will remove the 5´-phosphate from the 5´-end of that specific handle, but it still can be ligated to the central part.No further purification is required.

C) Ligation
Follow and adapt the procedure described in Section 3.3-part C.
1. Mix half of the central part fragment (0.5 µg) with 10X-15X excess of each highlybiotinylated handle (one of them dephosphorylated), and ligate with 100 units of T4 DNA ligase in 6.5 μL of buffer provided by the manufacturer.No further purification is required.
The other half of the central part can be kept at 4C for an additional preparation.It is recommended not to prepare a large volume of this construct because random nicking or damage of the central part DNA will lead to unwanted products or the loss of the molecule in the pulling process to generate the gap.

D) Nicking with Nb.BbvCI or Nt.BbvCI restriction enzyme
1 Digest the ligated DNA molecule from part C with the nicking enzyme Nb.BbvCI or Nt.BbvCI (Nt.BbvCI in this particular example).Incubate 6.5 µL of ligated product with 6 units of nicking enzyme in 13 µL of buffer provided by manufacturer for 1 h at 37 °C.Inactivate the enzyme by heating at 80 °C for 20 min.No further purification is required.

2.
Add EDTA pH 8.0 to 1 mM final concentration to preserve.Store the final DNA construct at 4 °C.The final concentration should be around 38 ng/µl.
The final DNA construct contains 2 nicks that define a region that can be further removed insitu by force as described below.

Test of production of ssDNA-dsDNA constructs in the C-Trap instrument
To illustrate the procedure, we fabricated a ssDNA-dsDNA tether that contains a ssDNA region of 2.3 knt and a dsDNA region of 15 kbp (Fig. 4C, option 1, right).The molecule was produced in situ by applying a force of ~40-50 pN in a low salt buffer as detailed in Section 3.3-part D (Fig. 4A and 4D).This assay also determines the contour length of the tethers.Additional tests using confocal imaging with fluorescent intercalant SYTOX Green were conducted.
Mechanical generation of ssDNA-dsDNA hybrids.
1. Prepare the C-trap fluid cell as described in Section 3.3-part D, with Channel 2: 2-4 µL of stock DNA diluted in 300 µL TEN buffer.Channel 3: 1 mL of low-salt TE buffer for generation of ss-dsDNA hybrid.2. Capture a duplex DNA molecule with a dual optical tweezers following the procedure described in Section 3.3-part D.
3. Check the contour length of the duplex DNA by performing a force-extension curve up to ~40 pN.In this case, it is convenient to use the Petrosyan model that provides an approximation for the extension-force dependence for the WLC model that can be easily adapted to both dsDNA and ssDNA (Petrosyan, 2017).The extension given by the Petrosyan equation correctly described the data using a contour length of 17,430 bp with a crystallographic rise per base pair, L0, dsDNA, of 0.34 nm bp -1 , and a persistence length, PdsDNA, of 50 nm (Fig. 4D, dark blue dashed line).4. Remove the nicked region by performing a force-extension curve up to ~40-50 pN.
Detachment of the nicked region is produced when subsequent pulling cycles show stable and longer extensions than the expected for dsDNA at forces above 6 pN (Dessinges, Lionnet, Xi, Bensimon, & Croquette, 2004).The extension given by a hybrid ssDNA-dsDNA model (Petrosyan, 2017) using 15,130 bp of dsDNA (L0, dsDNA = 0.34 nm bp -1 and PdsDNA = 50 nm) and 2,300 nt of ssDNA (L0, ssDNA = 0.69 nm nt -1 , and PssDNA = 2 nm) correctly described the data and is included in Fig. 4D (light blue dashed line).These data confirmed the correct generation of the designed hybrid ss-dsDNA molecule.
Direct visualization of ssDNA-dsDNA hybrids.

5.
Once confirmed the generation of the ss-dsDNA hybrid by force, fill in channel 4 with 200 µL of 100 nM SYTOX Green dissolved in TEN buffer.6. Move the molecule to channel 4 and hold it at 40 pN.7. Take a scan with the confocal microscope using excitation light at 488 nm.SYTOX is a dsDNA labelling dye and only the duplex DNA region should be visible (Fig. 4E).The junction between ssDNA and dsDNA is marked with an arrow.8.Alternatively, take a kymograph to capture florescence between the beads versus time as described in Section 3.3-part D. In our case, we observe a clear non-labeled region corresponding to the 2.3-knt ssDNA fragment (Fig. 4F). 

Case 2: A pure single-stranded DNA construct for single molecule experiments
This section describes the specific procedure to clone a large plasmid to produce a long pure single-stranded DNA construct for optical tweezers experiments.The procedure is very similar to that described in Section 4, but with positioning of the nicking site close to the intact handle (the one with a 5'-phosphate).This already defines a complete region of the DNA construct flanked by two nicks (depicted with red and green dots in Fig. 5A) located in the same strand that can be removed in-situ by force in a low-salt buffer (Candelli et al., 2013).Note that additional BbvCI sites, although not essential, would help the process, but they would need to have the same orientation to generate nicks on the same strand.A key advantage of this method is that it employs a customized large plasmid, and therefore particular sequences, e.g, a region that could fold into G-quadruplex structures or contain telomeric sequences, can easily be incorporated.These constructs are useful to study the mechanical properties of ssDNA or the activity of translocases (see Section 7) (Hormeno et al., 2021;Lee et al., 2013).

Design of the DNA plasmid
This method requires a large plasmid with at least one BbvCI restriction site (underlined in Fig. 5B) located next to the restriction sites employed to linearize the plasmid.We used the plasmid of Figure 4B as starting material and produced a new C1' fragment by PCR with the primers shown in Table 8.The C1' fragment was then digested with NotI and SalI and introduced into the previous plasmid between NotI and XhoI.This new plasmid is 5-kbp larger and contains three BbvCI sites with the same orientation (Fig. 5B).An alternative, if the plasmid of Figure 4B has not been previously cloned, is to follow the method described in Section 4.1, but replacing the C1 fragment with the C1´ fragment.The general procedure to produce this plasmid as well as the equipment, buffers, strain and reagents are described in Section 2.

Assembly of the pure ssDNA construct
The equipment, buffers, reagents, and procedure for the assembly of this DNA construct, are described in Section 3.However, the following specific points need to be considered.

A) Preparation of the central part
The central part is obtained by linearization of the plasmid shown in Figure 5B with NotI and XhoI (Fig. 5C).The linearized central part is next ligated to two different highly biotinylated handles, being one of them dephosphorylated (the NotI handle).This creates a nick in the upper strand (red dot).This construct contains an essential BbvCI restriction site next to the XhoI cohesive end, where the phosphorylated handle is ligated, and two additional BbvCI restriction sites with the same orientation, leading altogether to three nicks on the same strand after cleavage with the Nb.BbvCI enzyme (green dots).

B) Preparation of the highly-biotinylated handles
Follow the procedure described in Section 4.2-part B.

C) Ligation
Follow the procedure described in Section 4.2-part C.

D) Nicking with Nb.BbvCI or Nt.BbvCI restriction enzyme
Follow the procedure for nicking as described in Section 4.2-part D. In this particular example, we used Nb.BbvCI (Fig. 5C) leading to a final DNA construct with 4 nicks, which will facilitate the process of removal of the upper strand by pulling in the optical tweezers setup.

Test of production of pure ssDNA construct in the C-Trap instrument
To illustrate the procedure, we fabricated a DNA construct of 22.3 kbp (Fig. 5C) and nicked the substrate with Nb.BbvCI, as described above.Detachment of the nicked strand was achieved with a dual optical tweezers in situ by applying a force of ~80-90 pN in a low salt buffer, as detailed in Section 3.3-part D (Fig. 5A and 5D).This assay also determines the contour length of the tethers.Additional tests using confocal imaging with a labeled single-stranded binding protein (MB543 labeled hRPA, hRPA MB543 , with emission at 570 nm (Kuppa, Pokhrel, Corless, Origanti, & Antony, 2021)) were conducted.
Mechanical generation of pure ssDNA.
1. Prepare the C-trap fluid cell as described in Section 3.3-part D, with Channel 2: 2 µL of stock DNA diluted in 300 µL TEN buffer.Channel 3: 1 mL of low-salt TE buffer for generation of pure ssDNA.Channel 4: 1 mL of reaction buffer (20 mM Tris pH 7.5, 30 mM NaCl, 4 mM MgCl2, 5 mM DTT).
2. Capture a duplex DNA molecule with a dual optical tweezers following the procedure described in Section 3.3-part D. 3. Check the contour length of the duplex DNA by performing a force-extension curve up to 40 pN (black line).In this case, a fit to the eWLC model (Odijk, 1995) (Fig. 5D, dark blue dashed line) provided a contour length of 22,164 bp with a persistence length, PdsDNA, of 17 nm (using L0, dsDNA = 0.34 nm bp -1 , and SdsDNA = 1500 pN). 4. Completely remove the nicked upper strand by performing a force-extension curve beyond the overstretching transition up to ~80-90 pN.Detachment of the nicked strand is produced when subsequent pulling cycles show stable and longer extensions than those expected for dsDNA at forces above 6 pN (Fig. 5D, gray and red lines).A fit to the extensible Freely Jointed Chain (eFJC) model (S.B. Smith et al., 1996) for single-stranded DNA is included in Fig. 5D (light blue dashed line) and provided a contour length of 12 µm for the ssDNA tether (0.54 nm nt -1 ) and a Kuhn length of 1.8 nm (using S = 600 pN).
Direct visualization of pure ssDNA using fluorescently labeled RPA.

5.
Once confirmed the production of the ssDNA tether, introduce 200 µl of 15 nM hRPA MB543 in reaction buffer in channel 4. 6. Move the molecule to channel 4 and hold it at 20 pN. 7. Take a scan with the confocal microscope using excitation light at 532 nm.RPA is a ssDNA binding protein and will visually provide the coverage of the ssDNA tether (Fig. 5E).8.Alternatively, take a kymograph to capture florescence between the beads versus time as described in Section 3.3-part D. In our case, we observe a clear full-length labeled ssDNA tether (Fig. 5F).

 6. Case 3: A flap dsDNA construct for single molecule experiments
This section describes the specific procedure to clone a large plasmid to produce a flap dsDNA construct.This DNA construct contains a single-stranded DNA tail, the flap, flanked by duplex DNA.The flap can be 5'-or 3'-terminated and can provide a loading site for SF1-6 helicases (Gorbalenya & Koonin, 1993;Singleton, Dillingham, & Wigley, 2007).Similar substrates to the one described here have been employed to monitor the unwinding activity of different helicases (Hormeno et al., 2021;Levikova et al., 2013;Wilkinson et al., 2020).
The method described below is based on the generation of a single-stranded gap region by nicking enzymes acting on closely spaced restriction sites (Luzzietti et al., 2011;Luzzietti, Knappe, Richter, & Seidel, 2012;H. Wang & Hays, 2001).The gap can be filled in with an oligonucleotide to create a flap (our case below) or with other more complex tertiary structures such as hairpins, bulges, T-shape junctions, etc….The gap can be also used to incorporate oligomers carrying a desired modification such as damaged bases, a fluorophore, or biotinlabeled deoxythymidines to facilitate the attachment of streptavidin-conjugated quantum dots for example (Brutzer et al., 2010;Dekker et al., 2002;Hormeno et al., 2021;Levikova et al., 2013;Luzzietti et al., 2011;Wilkinson et al., 2020).This strategy needs precise positioning of the nicking restriction sites and therefore necessarily involves the production of plasmids.In the procedure described below, a large plasmid containing several closely-spaced BbvCI restriction sites was cloned based on the design of Luzzietti et al. (Luzzietti et al., 2011(Luzzietti et al., , 2012)).After cleavage, a single-stranded gap of 63 nt was generated and partially filled with a long oligonucleotide.To generate a 5'-terminated flap we designed an oligo with 37 deoxythymidines (dT) at the 5'-end and a homologous region of 26 nt.This leaves a ssDNA gap of 37 nt.To produce a 3'-terminated flap the homologous region should be at the 5'-end and include a nonhomologous region at the 3'-end.Note that in this case, the 5'-end should be phosphorylated to be ligated to the main body of the construct.This approach based on customized plasmids allows the combination of a flap structure with the presence of sequences of interest located along the dsDNA.The procedure below describes the production of a 5'-terminated flap substrate.

Design of the DNA plasmid
This method requires a large plasmid with several closely-spaced BbvCI restriction sites (Fig. 6A).First, fabricate a large plasmid similar to the one shown in Figure 4B with appropriate restriction sites.Then, introduce a poly-BbvCI region into this plasmid.To do this, we used the plasmid reported in Wilkinson et al. (Wilkinson et al., 2020) and extracted a region with 5 BbvCI sites by XhoI-SalI cleavage.This fragment of 1,428 bp was introduced once in the XhoI site of the large plasmid shown in Figure 4B, resulting in new, large modified plasmid of 18,772 bp (Fig. 6A).Any plasmid with a BbvCI region would serve this purpose, but if this is not available, one can employ complementary oligonucleotides to generate a region with several BbvCI restriction site repetitions and introduce that region into the large plasmid.In any case, the orientation of the insert should be determined because this will ultimately have consequences for the orientation of the flap (see Section 2.3).Additional copies of BbvCI restriction sites along the plasmid in either orientation should not be a problem, as isolated nicks should be repaired in the final ligation step (see below).The general procedure to produce this plasmid as well as the equipment, buffers, strain and reagents are described in Section 2.

Assembly of the flap dsDNA construct
The equipment, buffers, reagents, and procedure for the assembly of this DNA construct, are described in Section 3.However, the following specific points need to be considered.

A) Preparation of the central part
The central part can be linearized with a single restriction enzyme.The choice of this enzyme will define the position of the poly-BbvCI region and ultimately the position of the flap with respect to the beads (see Note 1).This flexibility facilitates the production of different constructs with different proportions of dsDNA available for unwinding from the flap without the need to fabricate a new plasmid (Fig. 6B).In our particular example, we chose to linearize the plasmid with SalI.
Generation of the ssDNA gap by nicking with Nb.BbvCI or Nt.BbvCI restriction enzyme.
1. Select the nicking enzyme.In our example, we chose Nt.BbvCI.Digest 0.5 µg of the linearized plasmid with 6 units of Nt.BbvCI in 12.5 μL of buffer provided by the manufacturer at 37 °C for 2 h.This will create 5 nicks in the poly BbvCI region on the same strand.If desired, linearization and nicking of the DNA plasmid can be combined in a single step.2. Heat inactivate the enzyme for 20 min at 80 °C.This should also release the nicked fragments from the body of the molecule.3. Store the nicked central part at 4 °C, without further purification.
Annealing of the oligonucleotide that constitutes the flap to the gap.

4.
Design an oligonucleotide with a poly(dT) of desired length, the flap, and a homologous region to the gap.In our example, we designed a 63-nt oligonucleotide, that after the annealing of 26 nt into the Nt.BbvCI-gap, leaves a flap of 37-poly (dT) nucleotides labeled with an Alexa 488 fluorophore (A488) in the 5'-end (Table 9 and Note 2) and a gap of 37 nt.
To avoid this gap, another complementary oligonucleotide should be designed (blocking oligonucleotide).This oligonucleotide must be phosphorylated in its 5´-end.In this example, we designed an oligonucleotide of 32 nt that would leave a small gap of 5 nt after annealing (Table 9 and Note 2). 5. Dissolve oligonucleotide stocks to 100 μM and prepare a 12.5 μM stock aliquots in nuclease free water.Store at -20 °C.If the oligonucleotide is fluorescently-labeled, minimize the light exposure.6. Mix half of the sample produced at step 3 (0.25 µg) with a 200X excess of poly(dT)-flap and 200X excess of blocking oligonucleotide in 10 µL of annealing buffer (50 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl).Heat for 5 min at 80 °C, and slowly cool down to 30 °C at a -0.5 °C min -1 rate (Hormeno et al., 2021;Wilkinson et al., 2020).7. Store the long flap DNA central part at 4 °C, without further purification.

B) Preparation of the highly-biotinylated handles
Follow the procedure described in Section 3.3-part B.

C) Ligation
Follow and adapt the procedure described in Section 3.3-part C.
1. Mix the long flap DNA central part (0.25 µg) with 15X excess of highly-biotinylated handle (XhoI in this example), and ligate with 50 units of T4 DNA ligase in 12.5 μL of buffer provided by the manufacturer in the presence of SalI restriction enzyme to prevent tandem products (see Section 3.4).No further purification is required.2. Add EDTA pH 8.0 to 1 mM final concentration to preserve.Store the final flap DNA construct at 4 °C.The final concentration should be around 20 ng/µl.
The final dsDNA construct contains a 5'-ssDNA flap at a defined position that could act as a loading site for a protein of interest (see section 7).

Test of production of a flap dsDNA construct in C-Trap
To illustrate the procedure, we fabricated a dsDNA construct of 18.7 kbp containing a 5′terminated 37-nt poly(dT) ssDNA flap with an Alexa 488 fluorophore in the 5'-end positioned at 12.3 kbp from one end (Fig. 6B).Initially, a force extension curve was performed to determine the contour length of the tethers.

Mechanical characterization and visualization of a flap dsDNA construct.
This procedure is identical to the one described in Section 3.3-part D, with slight differences.
The force-extension curve should lead to molecules around 18,700 bp.In our example (Fig. 6C), a fit to the eWLC model provided a contour length of 18559 bp with a persistence length, PdsDNA, of 20 nm (using L0, dsDNA = 0.34 nm bp -1 , and a stretch modulus, SdsDNA, of 1500 pN).Similarly, a confocal scan and kymograph following the procedure of Section 3.3-part D but in the absence of SYTOX Green, can be obtained to identify the presence of the flap in the DNA construct (Fig. 6D and 6E).A blue dot consistent with the position of the fluorophore-labelled flap was identified.

Notes
1.In the process of linearization of the plasmid, it is recommended to choose a restriction enzyme that generates compatible sticky ends with another restriction enzyme.This should avoid the formation of tandem molecules in the ligation step if the original restriction enzyme is present in the ligation reaction (see Section 3.4).In this manuscript we have used the pairs XhoI/SalI and NotI/PspOMI.2. Nicking with Nb.BbvCI will require the design of different poly(dT) tailed and blocking oligonucleotides to anneal into that gap.
 7. Long DNA constructs for the study of human HELB in C-trap experiments The specific long DNA constructs described in previous sections were used as substrates to study the activity of human DNA helicase B (HELB).HELB has a central domain sharing homology with the Superfamily 1 (SF1) helicase RecD.RPA protein and DNA binding activities of HELB have been linked to interactions with this central domain (Hazeslip, Zafar, Chauhan, & Byrd, 2020).We and others have shown that HELB possesses single-stranded DNA dependent ATPase activity and 5'-to-3' helicase activity (Hormeno et al., 2021;Saikrishnan, Griffiths, Cook, Court, & Wigley, 2008;Taneja et al., 2002).
We performed translocation and unwinding experiments using the three kinds of substrates fabricated in this manuscript.The activity of the protein was measured as changes in extension of the DNA tethers or by direct imaging with the confocal microscope coupled to our dual optical tweezers.To visualize HELB protein in confocal fluorescent experiments, we labeled HELB with quantum dots (QDs).Direct conjugation of proteins with biochemically modified QDs has become very useful for single-molecule studies (Nelson, Ali, & Warshaw, 2011).The only requirement is that the protein has a tag (i.e., biotin, Strep-tag, His-tag), to serve as the attachment point for the QDs.The use of QDs has multiple advantages, such as a higher photo stability compared to organic dyes, a higher brightness, and a wide variety of commercially available QDs with different excitation/emission properties or surface coatings.The procedures detailed below describe the process of conjugation of HELB with QDs, and the experiments with the confocal microscope.

B) Mechanical characterization of the specific DNA tether.
This procedure is identical to that described for the different types of molecules fabricated: hybrid ss-dsDNA, pure ssDNA, or flap dsDNA (Sections 4.3, 5.3, and 6.3).Fill in channel 4 and 5 with reaction buffer.

C) Direct visualization of HELB-QD protein on the specific DNA tether.
1. Once confirmed the capture in the C-trap of the appropriate DNA tether, fill in channel 4 with 200 µl of fluorescent 5-20 nM HELB-QD in reaction buffer supplied with 2 mM ATP. 2. Move the molecule to channel 4 and hold it at 8-25 pN. 3. Take a kymograph to capture florescence between the beads versus time as described in Section 3.3-part D, using excitation light at 488 nm.Labeled HELB particles should be visible mainly in the blue channel employing the blue filter 512/25 nm.Force and position data are recorded simultaneously.Typical line velocity was 50 ms line -1 .

Hybrid ss-dsDNA construct
A hybrid ss-dsDNA construct is useful to study translocation on ssDNA and potential unwinding activities beyond the ss-dsDNA junction by DNA helicases and translocases.
As an example, we built a hybrid substrate of 2.3 knt ssDNA and 15 kbp dsDNA (Section 4.2) and held the construct at 25 pN force, to avoid the formation of secondary structures on the singlestranded region.At this force, about 1/3 of the distance between the beads corresponds to single-stranded DNA and 2/3 to dsDNA.Once the substrate was successfully produced and its integrity checked (Section 4.3), we moved it to the protein channel and started recording a kymograph following the procedure described in Section 3.3-part D. These experiments showed that HELB binds to the ssDNA region and confirmed that it translocates unidirectionally from 5'to-3' (towards the dsDNA junction) as reported in (Hormeno et al., 2021) (Fig. 7A).HELB was also able to overcome the junction and unwind the duplex DNA, although occasionally we also observed stalling events at the junction.The unwinding activity was detected by an increase in the extension of the tether at 25 pN, because the extension of single-stranded DNA is larger than dsDNA at that force (Dessinges et al., 2004).The binding and translocation of other HELB proteins on the exposed single-stranded region was also observed.

Pure ssDNA construct
A pure ssDNA construct was produced to study the translocation activity of HELB following the procedure described in Section 5.2.Then, we moved the tether to a channel containing 5 nM HELB-QD and 2 mM ATP and kymographs were recorded at 50 ms line -1 .As expected, HELB particles always moved in the same direction on each particular ssDNA molecule (Fig. 7B).This assay cannot define protein translocation polarity -a hybrid substrate is best for this -but it allowed us to determine a translocation rate of 72 ± 40 nt s -1 within a force range of 13-30 pN (Hormeno et al., 2021).

Flap dsDNA construct
An Alexa 488 labeled 37 nt-flap dsDNA construct was produced to study unwinding activity of HELB following the procedure described in Section 6.2.HELB showed a complex behavior on this kind of substrate.We observed that the protein could unwind the duplex starting from the 5'-tail but it was also able to translocate without preventing re-annealing (Fig. 7C), in a process that resembles a translocation property of the AddAB helicase (Yeeles, van Aelst, Dillingham, & Moreno-Herrero, 2011).Slight modifications of the procedure to fabricate the flap substrate (Section 6.2) should allow us to incorporate other potential loading structures, like overlapping 5' and 3'-flaps, or a T-junction.A detailed study will be subject of future work.

Summary and conclusions
The DNA construct itself is a central component of any single-molecule experiment aimed at understanding the mechanism of action of DNA-interacting proteins.Moreover, the detection of the activity of multiple motor proteins is based on the mechanical properties of DNA either in its duplex or single-stranded form.The length and sequence of these substrates are two key elements that must be controlled during their design and fabrication.The length is a particularly important issue in assays that involve fluorescence because of the inherent spatial resolution limitations imposed by diffraction.
This chapter describes a general molecular design for customized large DNA plasmids of up to 25 kbp.These plasmids are used to prepare long DNA constructs by simple linearization with selected restriction enzyme/s and ligation to labeled handles.Along similar lines, large plasmids have recently been exploited to produce equivalent customized DNA constructs (Van Loenhout et al., 2012;Mueller et al., 2020;Sánchez et al., 2021;Spakman et al., 2020).However, the reported methods did not address in detail the procedures required to generate long ssDNA or ssDNA-dsDNA hybrid molecules with customized sequences, because most of them were based on Lambda DNA (Belan, Barroso, et al., 2021;Belan, Moore, et al., 2021;Brouwer et al., 2017;Candelli et al., 2013Candelli et al., , 2014;;G. A. King et al., 2013;S. B. Smith et al., 1996).The method presented here is very flexible and easy to implement in any molecular biology laboratory with a minimum infrastructure.For instance, a new hybrid ss-dsDNA construct with a different proportion of single and double-stranded DNA could be easily generated by choosing other restriction sites to linearize the central part of the molecule.Similarly, following the same strategy, a particular sequence of interest could be positioned at a new location.In addition to pure ssDNA and hybrid ss-dsDNA molecules, we also present a procedure to fabricate long dsDNA molecules containing a flap ssDNA, which are of particular interest to study the activity of helicases.Overall, our method fulfilled the two key requirements of long length and sequence specificity for optical tweezers experiments.Indeed, one of the examples included multiple copies of EcoRI restriction sites (see Table 7), which can be used as helicase roadblocks using EcoRI E111G , a catalytically inactive mutant with exceptionally tight binding (Balaguer et al., 2021;Brüning, Howard, Myka, Dillingham, & McGlynn, 2018;K. King, Benkovic, & Modrich, 1989).The three substrates presented here were quality-controlled in force extension and fluorescence imaging experiments and validated for use in single molecule experiments using the human HELB protein, demonstrating their enormous potential to study DNA helicases and translocases.A) Individual DNA tethers are assembled in channels 1-3 separated by laminar flow containing streptavidin-coated beads, the biotinylated DNA construct and buffer, respectively.Channel 3 is filled with low-salt TE buffer if hybrid ssDNA-dsDNA or pure ssDNA constructs are prepared or with TEN buffer if the DNA will not be subjected to force-induced melting, as for dsDNA or flap DNA constructs.Force-extension curves are performed in this channel to discard double tethers and confirm the single-tether has the expected contour length.In this channel, pure ssDNA or hybrid ssDNA-dsDNA constructs are also formed.Once the quality of the DNA construct has been checked, the traps are moved to channels 4 and 5 for labeling with SYTOX or for protein loading and imaging.B) Force-extension curve of a torsionally-constrained, and nicked 25 kbp dsDNA (TC dsDNA, stretching (red) and relaxation (light red) curves; nicked, stretching (black and green) and relaxation (grey) curves).Blue dashed line is a fit to the eWLC model.C) 2D confocal scan of the tethered dsDNA construct stained with 100 nM SYTOX.The experiment was performed at 40 pN.D) Representative kymograph of the dsDNA construct stained with 100 nM SYTOX.The experiment was performed at 30 pN.Fig. 4 Fabrication of a long hybrid ssDNA-dsDNA construct.A) Scheme illustrating in situ formation of a ss-dsDNA hybrid tether corresponding to DNA construct 1 (panel C, right side), using dual-trap optical tweezers.A functionalized dsDNA molecule with two nicks, one due to Nt.BbvCI nicking and the other to ligation to a dephosphorylated handle (depicted with green and red dots, respectively), is captured with the optical traps.Then, the fragment of ssDNA between the two nicks is removed by force-induced melting of the duplex.B) Schematic representation of the DNA plasmid employed to produce the ss-dsDNA hybrid constructs.The indicated restriction sites are unique ones among others not included.The unique BbvCI restriction site is necessary to produce the ss-dsDNA junction.C) Different ssDNA-dsDNA constructs, among others, can be prepared by digestion of the plasmid shown in (B) with different pairs of two close restriction enzymes and later ligation with two different highly biotinylated handles, with one of them being dephosphorylated (marked with a red dot).The nicking with Nb.BbvCI (left side) or Nt.BbvCI (right side) in combination with force-induced melting, would allow the creation of two different oriented ss-dsDNA junctions at well-defined positions for the same dsDNA fragment.Not only the junction but also the sequence of interest if present in C3 fragment, are differently positioned with respect to the beads.D) Forceextension curves of DNA construct 1 (panel C, right side) that allows the transition from pure dsDNA (black) to a ssDNA-dsDNA hybrid construct (red and green).Dark blue and light blue dashed lines are representations of the Petrosyan equation for dsDNA and hybrid ss-dsDNA, respectively (see main text).E) 2D confocal scan of a tethered ss-dsDNA construct stained by 100 nM SYTOX.White arrow denotes the ss-dsDNA junction.The experiment was performed at 40 pN.F) Representative kymograph of the hybrid construct stained with 100 nM SYTOX.The black arrow denotes the ss-dsDNA junction.The experiment was performed at 44 pN.Fig. 5 Fabrication of a long pure ssDNA construct.A) Scheme illustrating in situ formation of a pure ssDNA tether corresponding to the DNA construct shown in panel C, using dual-trap optical tweezers.A functionalized dsDNA molecule with four nicks, three due to Nb.BbvCI nicking (green dots) and the fourth due to ligation to a dephosphorylated handle (red dot), is captured with the optical traps.The essential BbvCI nick is the one next to the phosphorylated handle.The complete upper strand is removed by force-induced melting of the duplex.B) Schematic representation of the large DNA plasmid employed to produce a pure ssDNA construct.In this particular example, a new C1´ fragment (light green fragment) has been included in the plasmid shown in Figure 4B, in between NotI and XhoI.The essential BbvCI site (underlined) is absolutely required at that position.Additional copies of BbvCI can be present, but they need to be in the same orientation as the essential site to obtain nicks in the same strand.C) Scheme of the DNA construct obtained after digestion of plasmid shown in (B) with NotI and XhoI and ligation with handles.Once the final nicked construct is melted by force as described in (A), the upper strand is completely released.Dephosphorylated handle is depicted with a red dot, and Nb.BbvCI nicks with green dots.The essential BbvCI restriction site is underlined.D) Force-extension curves indicate the transition from dsDNA (black) to ssDNA (gray and red).Fits to the eWLC and eFJC models are included (see main text).E) 2D confocal scan of a tethered ssDNA covered by 15 nM hRPA MB534 .The experiment was performed at 17 pN.F) Kymograph of the same experiment shown in (E).Biotinylated-human HELB is firstly conjugated with streptavidin-coated QDs and then its activity is studied using the fabricated DNA constructs.The 5'-to-3' configuration is supported by bulk experiments shown (Hormeno et al., 2021).In each case, the lower panel shows the corresponding distance between the beads and force.A) Representative kymograph of HELB-QDs movement (blue) on hybrid ss-dsDNA construct shown in Fig. 4C (DNA construct 1, right side) in the presence of 2 mM ATP at 25 pN.HELB requires a ssDNA region to bind the DNA and then translocates in the 5'-to-3' direction towards the ss-dsDNA junction.Once it reaches the junction, HELB is also able to overcome the junction and unwind the duplex DNA, although sometimes we also observed stalling events at the junction.B) Representative kymograph of HELB-QDs translocation (blue) on the pure ssDNA construct shown in Fig. 5C in the presence of 2 mM ATP under 16 pN of tension.All HELB molecules move in the same direction.C) Representative kymograph of HELB-QDs movement on the 37 nt-and Alexa 488 labeled-flap dsDNA construct shown in Fig. 6B.We observed that the protein could unwind the duplex starting from the 5'-tail but cannot prevent re-annealing of the strands, as the movement is not accompanied by a concomitant increase in tether length.

Figure 1
with NotI (Fig. 2, left).2. Heat-inactivate the restriction enzyme/s by incubating at the temperature and time recommended by the manufacturer.3. Store at 4 °C.Do not purify the central fragment at this stage.The final concentration should be around 160 ng/µl.Additional steps are required for fabrication of the central part of the flap DNA construct (Section 6).

1.
Mix 1 µg of the central part of the DNA construct (Step 3, Section 3.3-part A) with 10X-15X excess of each highly-biotinylated handle/s (Step 9, Section 3.3-part B), and ligate with 200 units of T4 DNA ligase in 12.5 μL of buffer provided by the manufacturer.Transfer the tube to the PCR thermocycler and incubate at 16 °C for 15 h followed by heat inactivation at 65 °C for 20 min (see Note 1 and Note 2). 2. Add EDTA pH 8.0 to 1 mM final concentration to preserve.Store the final long DNA construct at 4 °C, without further purification.The final concentration should be around 80 ng/µl.

3 .
Homogeneous population of nicked DNA tethers instead of torsionally constrained molecules can be obtained by linearizing the large plasmid with two different restriction enzymes as shown in Figure 2, right, e.g., XhoI and NotI, and ligation with two different labeled handles, being one of them dephosphorylated.4. Case 1: A hybrid ssDNA-dsDNA construct for single-molecule experiments

A)
Conjugation of biotinylated-HELB with streptavidin-coated QDs 1. Incubate a 1:5 molar ratio solution of the biotinylated protein and streptavidin-coated QDs (Qdot ™ 525 Streptavidin, Invitrogen) for 30 min on ice.Keep the volume small, 5-10 µL.Gently mix two or three times during the incubation.2. Add 10 µL 1 mM biotin (Sigma) to neutralize the streptavidin molecules of the QDs not bound to the protein.Incubate 10 min on ice.3. Dilute the protein-QD mixture in the reaction buffer (20 mM Tris pH 7.5, 30 mM NaCl, 4 mM MgCl2, 5 mM DTT) supplied with 2 mM ATP, to a final volume of 200-300 µL.Keep this volume low to avoid wasting proteins.Typically, the final concentration is 5-20 nM protein-QD.

Fig. 1
Fig. 1 Fabrication of large DNA plasmids.Scheme of the cloning of a large plasmid by ligation of C1, C2 and C3 fragments.C1 and C2 fragments are obtained by PCR-amplification of Lambda DNA, and C3 fragment is obtained by digestion or PCR amplification of a plasmid that contains a sequence of interest.In this example, the indicated restriction sites (except for BamHI) are unique sites.

Fig. 2
Fig. 2 Fabrication of long DNA constructs.The large plasmid shown in Figure 1 can be linearized by single or double digestion (left and right side, respectively) for later ligation with highlybiotinylated handles.Sticky ends obtained by restriction digestion with NotI or PspOMI are compatible, and therefore a biotinylated handle obtained by digestion with either NotI or PspOMI can be ligated to the NotI-sticky end of the central part.The same is true for sticky ends obtained after digestion with either XhoI or SalI.This can help to avoid generating tandem (double-length) tethers as mentioned in Section 3.4.Fig. 3 Optical tweezers and confocal experiments to test the production of dsDNA constructs.A) Individual DNA tethers are assembled in channels 1-3 separated by laminar flow containing streptavidin-coated beads, the biotinylated DNA construct and buffer, respectively.Channel 3 is filled with low-salt TE buffer if hybrid ssDNA-dsDNA or pure ssDNA constructs are prepared or with TEN buffer if the DNA will not be subjected to force-induced melting, as for dsDNA or flap DNA constructs.Force-extension curves are performed in this channel to discard double tethers and confirm the single-tether has the expected contour length.In this channel, pure ssDNA or hybrid ssDNA-dsDNA constructs are also formed.Once the quality of the DNA construct has been checked, the traps are moved to channels 4 and 5 for labeling with SYTOX or for protein loading and imaging.B) Force-extension curve of a torsionally-constrained, and nicked 25 kbp dsDNA (TC dsDNA, stretching (red) and relaxation (light red) curves; nicked, stretching (black and green) and relaxation (grey) curves).Blue dashed line is a fit to the eWLC model.C) 2D confocal scan of the tethered dsDNA construct stained with 100 nM SYTOX.The experiment was performed at 40 pN.D) Representative kymograph of the dsDNA construct stained with 100 nM SYTOX.The experiment was performed at 30 pN.

Fig. 6
Fig. 6 Fabrication of a long DNA construct containing a ssDNA flap.A) Schematic representation of the large plasmid employed to produce a flap DNA construct.A new fragment (purple) that contains five closely-spaced BbvCI restriction sites (yellow) has been introduced into the plasmid shown in Figure 4B, in between XhoI restriction site.B) Scheme of the DNA construct obtained after digestion of the plasmid with SalI and ligation with compatible XhoI handles.The construct includes a 5′-ssDNA flap sequence of 37 poly(dT)-nts labeled with Alexa 488 fluorophore on the 5´end.C) Force-extension curves of the flap dsDNA construct (stretching (black) and relaxation (gray) curves).The blue dashed line is a fit to the eWLC (see main text).D) 2D confocal scan of a tethered fluorophore-labelled flap dsDNA construct.The dsDNA molecule is free of nucleic acid stain, and the blue dot is consistent with the position of the Alexa488 fluorophore in the flap.The experiment was performed at 15 pN.E) Kymograph of the same experiment shown in (D).

Fig. 7
Fig. 7 Examples of the use of the DNA constructs to study HELB helicase in C-trap experiments.Biotinylated-human HELB is firstly conjugated with streptavidin-coated QDs and then its activity is studied using the fabricated DNA constructs.The 5'-to-3' configuration is supported by bulk experiments shown(Hormeno et al., 2021).In each case, the lower panel shows the corresponding distance between the beads and force.A) Representative kymograph of HELB-QDs movement (blue) on hybrid ss-dsDNA construct shown in Fig.4C(DNA construct 1, right side) in the presence of 2 mM ATP at 25 pN.HELB requires a ssDNA region to bind the DNA and then translocates in the 5'-to-3' direction towards the ss-dsDNA junction.Once it reaches the junction, HELB is also able to overcome the junction and unwind the duplex DNA, although sometimes we also observed stalling events at the junction.B) Representative kymograph of Figure 1

Force
Figure 5

Table 2 )
. Set the lid at 100 °C to prevent condensation.

24. Transfer the tubes to the PCR thermocycler and start the PCR program (Table 4). Set the lid at 100 °C to prevent condensation. 25. Analyze the PCR products in a 1% agarose gel. Mix 10 μL of PCR sample with 2 μL of
For bacterial work, sterile conditions should be maintained.Unless you have access to a laminar flow cabinet, you can work on an open bench near the flame generated by a Bunsen burner or the small Butane/propane gas bottle.The updraft from the heat should create a relatively sterile field with which to work.Clean your work space with 70% ethanol, wear gloves and "flame" the openings of bottles to remove any contaminant.2. The final large plasmid can be used to clone a new large plasmid by introducing a new DNA fragment of interest as shown in Sections 5 and 6. 1.