Melting temperatures for rsDNA: Theory vs experiment.

Comparison of measured and predicted Tm for rsDNA solutions. Symbols: experimental Tm obtained from the melting curves of 8N, 12N and 20N (Fig 3), as function of salt, cNaCl. Conditions are specified in the legend. Dashed lines: theoretical predictions of Eq (7).</p

Comprehensive Description of the theoretical model.

Partition Function; Melting Curve Approximations for rsDNA solution; Energetic Parametrization based on α; Pairing Statistics with αi = 0; Effects of the Ionic Strength on the Pairing Statistics.Fig A: Melting Curves of rsDNA, according to the Low T Approximation, High T Approximation and Unification Ansatz. (PDF)</p

Probability of defectless duplexes in rsDNA: Salt dependence.

Fraction of paired 8A*8B* as a function of ϕ, expressing their dilution in 8N, at T = 15°C for cNaCl = 0.15M (blue dots) and cNaCl = 1.0M (red dots). Dashed lines: theoretical predictions, with the shaded regions obtained from the experimental uncertainty on the pairing energy between A*B*, (see S2 Text).</p

Double strand vs. random sequence DNA melting.

Ensemble melting curve as a function of temperature. Green dots: measured ensemble melting of 12N at crsDNA = 0.04 g/l. Shading marks experimental uncertainty resulting from the average over 8 experimental replicas. Dashed lines: theoretical melting predicted for equimolar solutions of two complementary 12mers at cDNA = 0.02 g/l each (dashed blue line) and cDNA = 0.04/412 ≈ 2.4 10−9 g/l each (dashed red line). 12N θe(T) exhibits a behavior intermediate between the two. Dashed lines are obtained by averaging many melting curves of complementary 12mers. cNaCl = 1M in all curves.</p

Ensemble melting of rsDNA.

Measured ensemble melting curves of rsDNA at crsDNA = 0.04 g/L for 12N (panel a, open circles), 20N (panel a, full diamonds) and 8N at crsDNA = 25 g/L (panel b, open squares), at various salt concentrations: cNaCl = 0.15M (blue), cNaCl = 0.45M (green) and cNaCl = 1M (red). Shading marks experimental uncertainty resulting from the average over 6–8 experimental replicas for 8N and 12N, whereas, for 20N, just one experiment is shown as described in the text. Dashed lines, with same color code, are the theoretical predictions of Eq (7).</p

Probability of defectless duplexes in rsDNA: Theory vs experiment.

Fraction of paired 8A*8B* in 8N, θA*B*, as determined via CQ experiments (dots and light shading, as in Fig 4) and from the model (dashed lines and dark shading), at cNaCl = 0.15M and for several ϕ values (colors, see legend). Black data, lines and shadings: melting in 8A*+8B* solutions, in the absence of rsDNA. Shaded regions of the theoretical predictions are obtained from the experimental uncertainty on the pairing energy between A* and B** (see S2 Text).</p

Materials and methods.

Characterization of rsDNA synthesis; Measurement of rsDNA concentration; UV Absorbance: Experimental Setup; Analysis of UV Absorbance Data; Characterization of A* and B* Fluorescence; Contact-Quenching Data Analysis; Free Energy of A*B* Duplex. Fig A: HPLC traces of 12N compared with two different 12mers. HPLC traces of 8N, 12N and 20N. Fig B: Quartz microfluidic. Quantum Northwest Peltier. Fig C: Temperature calibration of T measured by a thermistor in contact with the microfluidic cell vs. the internal control Tpeltier. Fig D: Steps in the analysis of absorbance data A(T) of 12N used to extract the melting curves θe. Fig E: Fluorescence Emission Spectra of labeled DNA systems: A*, B*, A*B*, A*B and AB*. Fig F: Absorbance Spectra of labeled DNA systems: A*, B*, A*B*, A*B and AB*. Fig G: Simplified representation of the 12A*B* duplex, showing the relative size of DNA duplex, linker and fluorescent moieties FAM and TexasRed. Fig H: fluorescence intensity of TexasRed vs. T in a solution of 12A* + 12B*. Fig I: Normalized fluorescence intensity of TexasRed in a solution of 12A*, 12B* and 12B. Fig J: Average normalized fluorescence of 8A*+8B*. The fit enables determining the linear drift at low temperatures, corresponding to the signal of fully paired A*B*. Fig K: Tm vs A*B* concentration measured by CQ for the following systems. (PDF)</p

Pairing statistics in rsDNA.

Theoretical predictions of the fraction of perfect and defected rsDNA duplexes (as given by Eq (9)), parametrized by α. (a): the six most probable duplex motifs in 12N. (b): fraction of duplexes with a total number of unpaired bases |α|, in 8N (dotted lines), 12N (continuous lines), 20N (dashed lines). cNaCl = 1M and crsDNA = 25g/l.</p

Further results.

Evidence of Out-of-Equilibrium Conditions; Perfect pairing probability with different energies and number of nucleobases types. Fig A: Evidence of out-of-equilibrium behavior for 12N. T dependence of the fraction of duplexed strands θe measured while heating and cooling at 1°C/min. Fig B: Evidence of out-of-equilibrium behavior for 20N. Absorbance vs. T measured upon heating after one month equilibration at 4°C. Fig C: θA*B*, fraction of paired 12A* − 12B* in 12N, determined from the model and via CQ experiments with different cooling rates. Fig D: computed with different values of fCG in 8N. Fig E: computed with different values of nb in 12N. (PDF)</p

Description of the system.

(a): Solutions of random-sequence DNA (rsDNA) oligomers of length L are mixtures made of 4L distinct molecules, obtained by all the combinations of the four nucleobases, which are present at any position in the sequence with equal probability. (b): Each rsDNA oligomer can interact with 4L different rsDNA oligomers, leading to a 4L × 4L interaction matrix. Each dot in the matrix represents the most energetically favorable pairing between the two selected rsDNA oligomers, among all the possible mutual shifts. (c) Each position in the interaction matrix corresponds to a specific duplex motif, characterized by pairing errors which are here described by the parameters in α. The most probable duplex in the matrix is highly defected, as the last example in the panel.</p