MeltR software provides facile determination of nucleic acid thermodynamics

Sieg, Jacob P.; Arteaga, Sebastian J.; Znosko, Brent M.; Bevilacqua, Philip C.

doi:10.1016/j.bpr.2023.100101

Cited by 7 publications

(14 citation statements)

References 31 publications

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“…Melting points of double stranded RNA 7mers (Figure 4, analogue to [38] ) with native uridine and m1Ψ containing oligos were determined at concentrations between 0.9 and 12 μM (single stranded oligo concentration, Figures S17 and S18). The program MeltR [39] was used to calculate the slope of 1/T m versus ln(C t ) versus and then to derive the thermodynamic parameters of RNA dimer formation (Table 2, Figures S19 and S20). It can be seen that the melting energies of the studied sequence are slightly more favourable for the m1Ψ modified oligomer compared with the native oligomer with uridine.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

RNA oligomers at atomic resolution containing 1‐methylpseudouridine, an essential building block of mRNA vaccines

Nievergelt,

Berliat,

McAuley

et al. 2024

ChemMedChem

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All widely used mRNA vaccines against COVID‐19 contain in their sequence 1‐methylpseudouridine (m1Ψ) instead of uridine. In this publication, we report two high resolution crystal structures (at up to 1.01 and 1.32 Å respectively) of one such double‐stranded 12‐mer RNA sequence crystallized in two crystal forms. The structures are compared with similar structures which do not contain this modification. Additionally, the structure of 1‐methyl‐pseudouridine itself was determined.

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Section: Resultsmentioning

confidence: 99%

“…[a] calculated with MeltR, [39] using its method 2 (1/T m versus ln[C t ]), [b] calculated with nearest neighbour parameters from Xia et al; [41] [c] calculated with nearest neighbour parameters from Mauger et al; [21] [d] calculated with nearest neighbour parameters from Hudson et al [38]…”

Section: Discussionmentioning

confidence: 99%

RNA oligomers at atomic resolution containing 1‐methylpseudouridine, an essential building block of mRNA vaccines

Nievergelt,

Berliat,

McAuley

et al. 2024

ChemMedChem

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“…We note that other recent work is available for improved fitting of optical melting data. 58 P−Z and G−Z Stacking Free Energy Change Nearest Neighbor Fits. A custom Python program was written to fit the P−Z nearest neighbor parameters.…”

Section: ■ Discussionmentioning

confidence: 99%

“…An example of the output from our Matlab routines is provided as Figure S1 and the code is available at GitHub at . We note that other recent work is available for improved fitting of optical melting data …”

Section: Methodsmentioning

confidence: 99%

DNA Structure Design Is Improved Using an Artificially Expanded Alphabet of Base Pairs Including Loop and Mismatch Thermodynamic Parameters

Pham,

Miffin,

Sun

et al. 2023

ACS Synth. Biol.

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We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A−T and G−C to include the pair between 2-amino-8-(1′-β-D-2′deoxyribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one and 6amino-3-(1′-β-D-2′-deoxyribofuranosyl)-5-nitro-(1H)-pyridin-2-one, abbreviated as P and Z. To obtain the thermodynamic parameters needed to include P−Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit free energy and enthalpy nearest neighbor folding parameters for P−Z pairs and G−Z wobble pairs. We find G−Z pairs have stability comparable to that of A−T pairs and should therefore be included as base pairs in structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include the P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing it with P−Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases in which Eterna-player solutions were provided. P−Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P−Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

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“…Given the following definitions: Since the concentration of O1 and O2 are equal by preparation, we can substitute [O2] and [O1:O2] in the first equation to obtain a quadratic equation in terms of [O1]: The temperature dependence of hybridization can then be captured within the temperature dependence of K as follows: With R being the gas constant (1.987 x 10 −3 kcal/mol/K) and assuming that ΔH and ΔS are temperature independent in the range of interest, so that the unbound fraction of O1 ( f = [O1]/[O1] tot ) in a melting experiment is simply: The fluorescent signal coming from our melting oligonucleotides is an average of the signals coming from single stranded O1 oligonucleotides and double stranded O1 oligonucleotides, weighted for their respective emissions, whose temperature dependent behavior can be assumed as being linear for simplicity 30 . To fit our data, instead of manually subtracting baselines from our traces to convert them into fractions, we opted to automatically incorporate them in our fitting routine 31 for better reproducibility, so that the raw fluorescent signals (F) were fitted as: Where F ss (T) = m ss + q ss is the signal coming from single stranded O1 and F ds (T) = m ds + q ds is the signal coming from double stranded O1.…”

Section: Methodsmentioning

confidence: 99%

RNA complexes with nicks and gaps: thermodynamic and kinetic effects of coaxial stacking and dangling ends

Todisco,

Radakovic,

Szostak

2024

Preprint

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Multiple RNA strands can interact in solution and assume a large variety of configurations dictated by their potential for base pairing. Although duplex formation from two complementary oligonucleotides has been studied in detail, we still lack a systematic characterization of the behavior of higher order complexes. Here we focus on the thermodynamic and kinetic effects of an upstream oligonucleotide on the binding of a downstream oligonucleotide to a common template, as we vary the sequence and structure of the contact interface. We show that coaxial stacking in RNA is well correlated with but much more stabilizing than helix propagation over an analogous intact double helix step (median ΔΔG37°C ± 1.7 kcal/mol). Consequently, approximating coaxial stacking in RNA with the helix propagation term leads to large discrepancies between predictions and our experimentally determined melting temperatures, with an offset of ≈ 10°C. Our kinetic study reveals that the hybridization of the downstream probe oligonucleotide is impaired (lower kon) by the presence of the upstream oligonucleotide, with the thermodynamic stabilization coming entirely from an extended lifetime (lower koff) of the bound downstream oligonucleotide, which can increase from seconds to months. Surprisingly, we show that the effect of nicks is dependent on the length of the stacking oligonucleotides, and we discuss the binding of ultrashort (1 ~ 4 nt) oligonucleotides that are rele-vant in the context of the origin of life. The thermodynamic and kinetic data obtained in this work allow for the prediction of the formation and stability of higher order multi-stranded complexes.

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MeltR software provides facile determination of nucleic acid thermodynamics

Cited by 7 publications

References 31 publications

RNA oligomers at atomic resolution containing 1‐methylpseudouridine, an essential building block of mRNA vaccines

RNA oligomers at atomic resolution containing 1‐methylpseudouridine, an essential building block of mRNA vaccines

DNA Structure Design Is Improved Using an Artificially Expanded Alphabet of Base Pairs Including Loop and Mismatch Thermodynamic Parameters

RNA complexes with nicks and gaps: thermodynamic and kinetic effects of coaxial stacking and dangling ends

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