Speeding up biomolecular interactions by molecular sledding

Reaction partners are functionalised with a DNA sliding peptide and the association between them is significantly speeded up in the presence of DNA in solution.


Preparation of the binding partners
Stock solution of binding partner B was prepared as follows. N-terminus Cy3 labelled pVIc (custom peptide synthesis by Bio-Synthesis, Inc), 50 µM, was derivatised at Cys10' in 25 mM PBS buffer (pH 7.3) by adding biotin-PEG-maleimide (5 kDa, Nanocs) to 50 µM. The reaction vial was wrapped in aluminium foil to protect from light and the reaction was allowed to proceed for 5 hours at room temperature on a nutator. The reagent was subsequently stored at +4 0 C and used as is without further purification over the course of one month. Successful conjugation was confirmed by MALDI/TOF mass spectrometry (figure S1).

Spectrofluorometer assay
The experiments were performed in the sliding buffer, containing 60% wt. glycerol, 10 mM HEPES (pH 7.0), 2 mM NaCl, 20 mM Ethanol, 50 µM EDTA. The reactions were run in a 3mL quartz cuvette (FP-1004, Jasco) on a Jasco FP-8300 spectrofluorometer at 20 o C, continuously stirred at 800 rpm. The excitation and emission wavelengths were 520 nm and 666 nm correspondingly. The reaction read-out was FRET signal detected as a function of time. The solutions of binding partners S and B were prepared 30 minutes before the experiment in the following way. Solution 1 (binding partner S), final volume 2 mL, was obtained by incubating Cy5-streptavidin (43-4316, Life Technologies) with a two-fold excess of biotin-PEG23-pVIc (custom peptide synthesis by Bio-Synthesis, Inc) in the sliding buffer to render Cy5-streptavidin molecules functionalised with pVIc. Solution 2 (binding partner B), final volume 1 mL, was prepared from the stock solution and contained biotin-PEG-pVIc-Cy3 in the sliding buffer. Prior to the experiments, DNA of the required length was added at a necessary concentration to both solutions. Solution 1 was transferred into the cuvette and FRET vs. time signal acquisition was started. Subsequently, solution 2 was rapidly injected into the cuvette with a syringe. The final reaction mix of a final volume 3 mL contained 37.5 nM Cy5-streptavidin (functionalised by 75 nM biotin-PEG23-pVIc), DNA at a chosen concentration and 150 nM binding partner B.

FRET traces analysis
The obtained FRET vs. time traces were initially processed using Spectra Analysis subprogram of the Spectra Manager V.2 software package by JASCO Inc. The data were subject to baseline correction, noise elimination and smoothing (binomial interpolation, 6 iterations). Subsequently, the traces were fit with a single exponential function ( ) = C (1 − e − τ ⁄ ) and reaction times τ were extracted from the fit.

Single-molecule experiments
The experiments were performed in the sliding buffer (see "spectrofluorometer assay" section) using flow-stretched λ-DNA as a scaffold for sliding. Binding partner S was used as is; binding partner B was first pre-incubated with Cy5 labelled streptavidin to be able to use a 641 nm (Cy5 excitation) laser instead of a 532 nm (Cy3 excitation) one, which drastically improved the signal-to-noise ratio and allowed us to detect clearly discernible sliding along DNA. The values for binding times 1 ≅ 0.3 and the 1D diffusion coefficient 1 ≅ 3 • 10 4 2 s ⁄ (adjusted for high viscosity sliding buffer) of binding partner S are based on the studies of P. C. Blainey et al. 1

Primer synthesis
Amino modified primers (provided by biomers.net GmbH, table S2) were functionalised by the peptides in a two-step reaction. 2,3 First, lyophilised amino modified primers (1.5 mM) were reacted with a 20-fold excess of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-Nhydroxysuccinimide ester (sulfo-SMCC) linker (Sigma Aldrich) in 0.1 M sodium phosphate buffer (pH 7.6). A stock solution of sulfo-SMCC in DMF was used. The reaction was allowed to proceed in a shaker at room temperature overnight. After incubation, the reaction mixtures were centrifuged three times using 3000 Da cut-off spin-columns (Sartorius stedim biotech) to wash out excess Sulfo-SMCC and other small molecules by Milli-Q water. The resulting solution was lyophilised and the conjugate was used for the next coupling step without further treatment. The coupling efficiency was ~50%. Lyophilized primer-SMCC conjugate was dissolved in 0.1 M sodium phosphate buffer (pH 7.6) to obtain a 200 μM solution. Subsequently, dry peptide of choice (pVIc, K or S, provided by CASLO) were added in a 5-fold molar excess. If the peptide did not dissolve completely DMF was added until a clear solution was visible. The reaction mixture was vortexed at room temperature overnight and the product was purified by reversed-phase chromatography (buffer A (pH 7.5): 0.1 M triethylammonium acetate (TEAT) containing 5% Acetonitrile; buffer B (pH 7.5): 0.1 M TEAT containing 65% Acetonitrile; column: RESOURCE RPC 1 mL; gradient: 0-60%, 50CV; wavelength: 260 nm). Finally, the buffer was exchanged to Milli-Q water by centrifugation in a 3000 Da cut-off spin-column. The products were analysed using polyacrylamide gel electrophoresis (PAGE) (figure S3) and MALDI-TOF mass spectrometry (figure S4). For further use the conjugates were lyophilised. In this reaction step, a coupling efficiency of 30 -40% was achieved.

PCR reactions
Real time PCR (qPCR) experiment was performed using Bio-Rad iQ5 Real-Time PCR System (Bio-Rad Laboratories, Richmond, USA). 20 μL reaction mixtures contained forward and reverse primers (modified or unmodified) at 0.125 μM, DNA template (10 ng for circular 8669-bp M13KO7 DNA template, 5 ng for short linear 1970-bp DNA template), SYBR Green I (1×), Qiagen fast cycling PCR kit (1×) and Q-solution (1×). All reactions (unless indicated otherwise) were performed in triplicate using the following cycling protocol: initial template melting 98 o C (5min); 30 cycles of [98 o C (1min), 55.6 o C (1s), 68 o C (30s)]; 68 o C (4min), 4 o C (hold). The SYBR Green I fluorescence was monitored at the end of each cycle. It should be noted that the primer annealing temperature used was the one calculated for the original unmodified primers. The modified primers may have different optimal annealing temperature but, nevertheless, for all primers the same annealing temperature was used not to introduce a variable in the experiments. To compare the kinetics of amplicon formation for PCR reactions with different primers we employed the threshold cycle (Ct) analysis. The Ct was set in the exponential phase of amplification using the built-in function of the software for Bio-Rad iQ5 Real-Time PCR System. To confirm that no unspecific amplification occurs and that primer annealing step is essential in the PCR protocol we performed several control experiments (figure S5). Figure S1. MALDI-TOF mass spectra of binding partner B. Binding partner B is a conjugate of biotin and Cy3 labelled pVIc. Both units are connected by a poly(ethylene glycol) (PEG) linker, which exhibits a degree of polymerization DP = 100. Due to the polydisperse nature of the PEG linker a broad mass peak corresponding to binding partner B is obtained. The calculated molecular weight (MW) of binding partner B is 6700 g/mol, while the the MW determined by MALDI/TOF is 6650 g/mol. After conjugation binding partner B was used without further purification because unconjugated Cy3-pVIc would not bind to streptavidin and the unconjugated biotin-PEG while still possessing the ability to bind to streptavidin, would not contribute to the FRET signal. To confirm that the rising FRET signal originates from the association between binding partners S and B we performed the control experiments for which we pre-incubated binding partner S with a 100x molar excess of biotin to occupy all binding pockets of streptavidin and prevent binding partner B from binding to them. The results for two cases are given: without DNA (black and red curves) and with 10 pM dsDNA of 2686 bp long (blue and green curves). A non-zero FRET signal in case of a 100x biotin excess might be explained by non-specific interactions between binding partners S and B.  Lane M: DNA ladder; Lane 1-3: amplicon from unmodified primers; Lane 4-6: amplicon from primer-K; Lane 7-9: amplicon from primer-pVIc; Lane 10-12: amplicon from primer-S. Figure S6. Amplicon formation. The formation of the amplicon of the correct length was confirmed using a 2% agarose gel.