{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T17:08:00Z","timestamp":1789146480848,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,8]]},"group-title":"Biochemistry","reference-count":81,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R35GM145283"],"award-info":[{"award-number":["R35GM145283"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32GM135134"],"award-info":[{"award-number":["T32GM135134"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["2022\/45\/B\/ST4\/03586"],"award-info":[{"award-number":["2022\/45\/B\/ST4\/03586"]}],"id":[{"id":"https:\/\/ror.org\/03ha2q922","id-type":"ROR","asserted-by":"publisher"}]},{"award":["2021\/41\/B\/NZ1\/03819"],"award-info":[{"award-number":["2021\/41\/B\/NZ1\/03819"]}],"id":[{"id":"https:\/\/ror.org\/03ha2q922","id-type":"ROR","asserted-by":"publisher"}]},{"award":["2020\/39\/B\/NZ1\/03054"],"award-info":[{"award-number":["2020\/39\/B\/NZ1\/03054"]}],"id":[{"id":"https:\/\/ror.org\/03ha2q922","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  RNAs regulate gene expression and cellular processes, often relying on specific conformations for function. RNA folding is hierarchical and sequence-dependent, with nearest-neighbor thermodynamic models commonly used to predict secondary structure. Current models were developed using optical melting experiments in 1 M NaCl, which does not represent the cellular environment. To address this, we developed a new model in Advanced Dulbecco\u2019s Modified Eagle Medium (Adv. DMEM), which mimics mammalian extracellular ionic composition. This\n                  <jats:italic>in vivo<\/jats:italic>\n                  -like model provides RNA folding parameters for helical base stacks and loop motifs. Optical melting experiments revealed helical stacks, particularly tandem G-U pairs, are less stabilizing in Adv. DMEM. Loop parameters were generally destabilizing but highly dependent on both sequence and loop type, with internal loops displaying idiosyncratic behavior. Structure prediction benchmarking revealed minimal differences overall, except for tRNAs, which showed improved prediction reliability and enhanced cloverleaf stability. Notably, tRNAs lack internal loops, suggesting further studies in Adv. DMEM could refine secondary structure predictions. This\n                  <jats:italic>in vivo<\/jats:italic>\n                  -like parameter set is included in the RNAstructure software package. Grounding these parameters in a physiologically relevant environment, we improve the biological relevance of RNA secondary structure predictions and establish a foundation for studying RNA folding under\n                  <jats:italic>in vivo<\/jats:italic>\n                  conditions.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"749202v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.04.749202","type":"posted-content","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T04:40:19Z","timestamp":1788928819000},"source":"Crossref","is-referenced-by-count":0,"title":["Nearest Neighbor Parameters for Estimating RNA Folding Stability with\n                  <i>In Vivo<\/i>\n                  -like Conditions"],"prefix":"10.64898","author":[{"given":"Olivia M.","family":"Hiltke","sequence":"first","affiliation":[{"name":"Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester Medical Center, Rochester, New York 14526, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elzbieta","family":"Kierzek","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martina","family":"Prochota","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta","family":"Rachwalak","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megan","family":"Miaro","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester Medical Center, Rochester, New York 14526, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thandolwethu","family":"Shabangu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester Medical Center, Rochester, New York 14526, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylwia","family":"Gorczynska","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryszard","family":"Kierzek","sequence":"additional","affiliation":[{"name":"Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12\/14, 61-704 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2907-6557","authenticated-orcid":false,"given":"David H.","family":"Mathews","sequence":"additional","affiliation":[{"name":"Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester Medical Center, Rochester, New York 14526, USA"},{"name":"Department of Biostatistics & Computational Biology, University of Rochester Medical Center, Rochester, New York 14526, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091109152467000_2026.09.04.749202v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2449-8"},{"key":"2026091109152467000_2026.09.04.749202v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/nature11233"},{"key":"2026091109152467000_2026.09.04.749202v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/35103511"},{"key":"2026091109152467000_2026.09.04.749202v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3681"},{"key":"2026091109152467000_2026.09.04.749202v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2012.12.024"},{"key":"2026091109152467000_2026.09.04.749202v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/418222a"},{"key":"2026091109152467000_2026.09.04.749202v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/299691a0"},{"key":"2026091109152467000_2026.09.04.749202v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/nature24049"},{"key":"2026091109152467000_2026.09.04.749202v1.9","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1003569"},{"key":"2026091109152467000_2026.09.04.749202v1.10","doi-asserted-by":"publisher","DOI":"10.1186\/2045-3701-2-1"},{"key":"2026091109152467000_2026.09.04.749202v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-019-0136-0"},{"key":"2026091109152467000_2026.09.04.749202v1.12","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1999.3001"},{"key":"2026091109152467000_2026.09.04.749202v1.13","doi-asserted-by":"publisher","DOI":"10.1007\/s000180050042"},{"key":"2026091109152467000_2026.09.04.749202v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2004.04.001"},{"key":"2026091109152467000_2026.09.04.749202v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(74)90560-9"},{"key":"2026091109152467000_2026.09.04.749202v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/newbio246040a0"},{"key":"2026091109152467000_2026.09.04.749202v1.17","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/15.22.9109"},{"key":"2026091109152467000_2026.09.04.749202v1.18","doi-asserted-by":"publisher","DOI":"10.1261\/rna.075341.120"},{"key":"2026091109152467000_2026.09.04.749202v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-024-02543-9"},{"key":"2026091109152467000_2026.09.04.749202v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2005.05.025"},{"key":"2026091109152467000_2026.09.04.749202v1.21","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-62703-709-9_3"},{"key":"2026091109152467000_2026.09.04.749202v1.22","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-62703-709-9_4"},{"key":"2026091109152467000_2026.09.04.749202v1.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2024.168549"},{"key":"2026091109152467000_2026.09.04.749202v1.24","doi-asserted-by":"publisher","DOI":"10.1021\/bi9809425"},{"key":"2026091109152467000_2026.09.04.749202v1.25","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0401799101"},{"key":"2026091109152467000_2026.09.04.749202v1.26","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-11-129"},{"key":"2026091109152467000_2026.09.04.749202v1.27","doi-asserted-by":"publisher","DOI":"10.1007\/s00214-005-0027-7"},{"key":"2026091109152467000_2026.09.04.749202v1.28","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-949-5_8"},{"key":"2026091109152467000_2026.09.04.749202v1.29","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt290"},{"key":"2026091109152467000_2026.09.04.749202v1.30","doi-asserted-by":"publisher","DOI":"10.1021\/bi3002709"},{"key":"2026091109152467000_2026.09.04.749202v1.31","doi-asserted-by":"publisher","DOI":"10.1101\/2021.10.16.464667"},{"key":"2026091109152467000_2026.09.04.749202v1.32","doi-asserted-by":"publisher","DOI":"10.1017\/S003358351600007X"},{"key":"2026091109152467000_2026.09.04.749202v1.33","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad807"},{"key":"2026091109152467000_2026.09.04.749202v1.34","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad020"},{"key":"2026091109152467000_2026.09.04.749202v1.35","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz019"},{"key":"2026091109152467000_2026.09.04.749202v1.36","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx170"},{"key":"2026091109152467000_2026.09.04.749202v1.37","doi-asserted-by":"publisher","DOI":"10.1201\/9780429258770"},{"key":"2026091109152467000_2026.09.04.749202v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.saa.2020.119410"},{"key":"2026091109152467000_2026.09.04.749202v1.39","doi-asserted-by":"publisher","DOI":"10.1016\/S0040-4039(01)90461-7"},{"key":"2026091109152467000_2026.09.04.749202v1.40","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkg632"},{"key":"2026091109152467000_2026.09.04.749202v1.41","unstructured":"Borer, P.N . (1975) In Fasman, G. D. (ed.). 3rd ed. CRC Press, Cleveland, OH, Vol. 1, pp. 589\u2013595."},{"key":"2026091109152467000_2026.09.04.749202v1.42","unstructured":"Richards, E.G . (1975) In Fasman, G. D. (ed.). 3rd ed. CRC Press, Cleveland, OH, Vol. 1, pp. 596\u2013603."},{"key":"2026091109152467000_2026.09.04.749202v1.43","doi-asserted-by":"publisher","DOI":"10.1021\/bi9615710"},{"key":"2026091109152467000_2026.09.04.749202v1.44","doi-asserted-by":"crossref","unstructured":"Seabold, S. and Perktold, J . (2010), Proceedings of the Sth PYTHON in Science Conference (SCIPY 2010), pp. 92\u201396.","DOI":"10.25080\/Majora-92bf1922-011"},{"key":"2026091109152467000_2026.09.04.749202v1.45","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2649-2"},{"key":"2026091109152467000_2026.09.04.749202v1.46","doi-asserted-by":"publisher","DOI":"10.1002\/1097-0282(1999)52:4<157::AID-BIP1001>3.0.CO;2-E"},{"key":"2026091109152467000_2026.09.04.749202v1.47","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/26.1.156"},{"key":"2026091109152467000_2026.09.04.749202v1.48","doi-asserted-by":"publisher","DOI":"10.1261\/rna.1200508"},{"key":"2026091109152467000_2026.09.04.749202v1.49","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(84)90145-8"},{"key":"2026091109152467000_2026.09.04.749202v1.50","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/22.17.3508"},{"key":"2026091109152467000_2026.09.04.749202v1.51","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(89)90026-7"},{"key":"2026091109152467000_2026.09.04.749202v1.52","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/21.13.3055"},{"key":"2026091109152467000_2026.09.04.749202v1.53","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1996.0119"},{"key":"2026091109152467000_2026.09.04.749202v1.54","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/22.17.3502"},{"key":"2026091109152467000_2026.09.04.749202v1.55","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/26.1.351"},{"key":"2026091109152467000_2026.09.04.749202v1.56","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/26.1.177"},{"key":"2026091109152467000_2026.09.04.749202v1.57","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/26.1.148"},{"key":"2026091109152467000_2026.09.04.749202v1.58","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/28.1.169"},{"key":"2026091109152467000_2026.09.04.749202v1.59","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkg006"},{"key":"2026091109152467000_2026.09.04.749202v1.60","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkn766"},{"key":"2026091109152467000_2026.09.04.749202v1.61","doi-asserted-by":"publisher","DOI":"10.1261\/rna.053694.115"},{"key":"2026091109152467000_2026.09.04.749202v1.62","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btl246"},{"key":"2026091109152467000_2026.09.04.749202v1.63","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkq792"},{"key":"2026091109152467000_2026.09.04.749202v1.64","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21633"},{"key":"2026091109152467000_2026.09.04.749202v1.65","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0686-2"},{"key":"2026091109152467000_2026.09.04.749202v1.66","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(95)59047-1"},{"key":"2026091109152467000_2026.09.04.749202v1.67","doi-asserted-by":"publisher","DOI":"10.1021\/bi011441d"},{"key":"2026091109152467000_2026.09.04.749202v1.68","doi-asserted-by":"publisher","DOI":"10.1021\/bi0029548"},{"key":"2026091109152467000_2026.09.04.749202v1.69","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx512"},{"key":"2026091109152467000_2026.09.04.749202v1.70","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btz222"},{"key":"2026091109152467000_2026.09.04.749202v1.71","doi-asserted-by":"publisher","DOI":"10.1016\/0042-6822(59)90043-1"},{"key":"2026091109152467000_2026.09.04.749202v1.72","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0016957"},{"key":"2026091109152467000_2026.09.04.749202v1.73","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(91)90499-v"},{"key":"2026091109152467000_2026.09.04.749202v1.74","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.5b00767"},{"key":"2026091109152467000_2026.09.04.749202v1.75","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.7b00325"},{"key":"2026091109152467000_2026.09.04.749202v1.76","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60327-429-6_1"},{"key":"2026091109152467000_2026.09.04.749202v1.77","doi-asserted-by":"publisher","DOI":"10.1186\/1748-7188-6-26"},{"key":"2026091109152467000_2026.09.04.749202v1.78","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkh449"},{"key":"2026091109152467000_2026.09.04.749202v1.79","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21596"},{"key":"2026091109152467000_2026.09.04.749202v1.80","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.04.023"},{"key":"2026091109152467000_2026.09.04.749202v1.81","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkag362"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.09.04.749202","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T16:15:37Z","timestamp":1789143337000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.09.04.749202"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,8]]},"references-count":81,"URL":"https:\/\/doi.org\/10.64898\/2026.09.04.749202","relation":{},"subject":[],"published":{"date-parts":[[2026,9,8]]},"subtype":"preprint"}}