RNA oligos - Nucleocapsid project
Naming and Storing Oligos
Oligos are ordered in several tubes (SIGMA has the option to order 5 aliquots).
Tubes are stored dry at room temperature (in Gernot office).
Fluorescent oligos are dissolved (fresh, millipore water) at a concentration of 10 uM (please write the concentration on the tube). Prepare aliquots of the dissolved Oligos - store stocks at -80°C (physics -80° freezer).
The non-labeled oligos for this study are also stored at a concentration of 100 uM.
If the oligos are dissolved at lower concentration - please mark it on the tube!!! -
Storage of Oligos: -80°C MST freezer; MST tower 3
Boxes for the dry oligos (CoV-RNA dry) and extra box for the dissolved stocks (CoV-RNA).
Naming of Oligos
short name: CoV-RNA 1...XXX (and number). Labeling on top of the tube: Only the number
trivial - ordering name: XXXX
dsRNA hybrids are named according to the numbers of the individual RNAs. for example: #RNA2+3
fluorescent RNA - N-MST binding studies
Region 6 RNA of CoV2 1-1000 to test whether NP binds preferentially to ssRNA, dsRNA or structured RNA
first set of oligos - ordered at SIGMA
5 aliquots each; store fluorescent RNA as stocks in aliquots.
CoV-RNA 1 - cy5 CoV2 498-536 [Cyanine5] CUG CAC CUC AUG GUC AUG UUA UGG UUG AGC UGG UAG CAG
CoV-RNA 2 - cy5 Cov2 498-520 ss [Cyanine5] CUG CAC CUC AUG GUC AUG UUA UGG UU
CoV-RNA 3 - rv to CoV2 498-520 AAC CAU AAC AUG ACC AUG AGG UGC AG
CoV-RNA12 - cy5 RNA12_Meister_9nt: Cy5-GUGGACUGC (MWG 12-20; 347ul - 100uM)
#RNA1: cy5 CoV2 498-536
#RNA2: cy5 Cov2 498-520 ss
#RNA2+3: cy5 Cov2 498-520 ss hybridized to rv to CoV2 498-520
Subdivide the structured RNA Oligo - 2 oligos with only the loop
CoV-RNA 4 - cy5 CoV2 505- 524+x CAU GGU CAU GUU AUG GUU GAC CAU G
CoV-RNA 5 - cy5 CoV2 498d538 CUG CAC CUC AUG GUC AUG UGG UAG CAG
RNA16 - cy3-AAA CUU UAAU (10nt in length, cy3 labelbing, ardered at MWG 8.5.21) - in order to make dsRNA with RNA15 (same sequence as RNA8+RNA14)
RNA length dependence
Oligos for ssRNA
based on dsRNA - cy5 Cov2 498-520 (length 26nt)
CoV-RNA 2 (26nt length): cy5 Cov2 498-520 ss
CoV-RNA 6 (15nt length): cy5 Cov2 498-15nt [Cyanine5] CUG CAC CUC AUG GUC
CoV-RNA3-Cy5 - rv to CoV2 498-520 AAC CAU AAC AUG ACC AUG AGG UGC AG (26nt) - ordered at SIGMA in 0.2uM scale 5-1-2021 - 4.2nmol per tube
CoV-RNA3-Cy3 - rv to CoV2 498-520 AAC CAU AAC AUG ACC AUG AGG UGC AG (26nt) - ordered at SIGMA in 0.05uM scale 14-6-2021
5'end of CoV2 genome
CoV-RNA 8 (10nt length): CoV2 Cy5RNA 1-7 Cy5-AUUAAAGUUU (10nt - is in reality from 1 to 10)
CoV-RNA12 (9nt length). CoV-RNA12: Cy5-CUA AAC G (0.05uM scale ordered at MWG 8.12.20) !!!RNA12 is 9nt Meister RNA - check naming!!!!
Oligos for dsRNA - fluorescent
26nt length dsRNA
#RNA2+3: cy5 Cov2 498-520 ss hybridized to rv to CoV2 498-520
15nt length dsRNA
CoV-RNA 7 - rv to CoV2 498-15nt GAC CAU GAG GUG CAG
#RNA6+7: cy5 Cov2 498-15nt hybridized to rv to CoV2 498-15nt
10nt length dsRNA
CoV-RNA 8 (10nt length): CoV2 Cy5RNA 1-7 Cy5-AUU AAA GUU U hybridized to CoV-RNA14 AAA CUU UAAU (this dsRNA gradually looses fluorescence after hybridisation ??!! - for this try Cy3 labeling -> see RNA16+RNA15)
RNA16 (Cy3-AAA CUU UAAU) hybridized to RNA15 (AUU AAA GUU U)
Oligos for dsRNA - non-fluorescent
RNA12 - GUG GAC UGC hybridized to RNA13 - GCA GUC CAC (melting temp - about 37°C)
non-fluorescent RNA Oligos
CoV-RNA 3 - rv to CoV2 498-520 AAC CAU AAC AUG ACC AUG AGG UGC AG (26nt) - ordered at SIGMA in 1uM scale 5-1-2021 - 43.8nmol per tube (366ug)
CoV-RNA9 -RNA9 CoV2 1-15 ATTAAAGGTTTATAC (15nt - 1uM scale - ordered at MWG 26.11.20 - for MST)
CoV-RNA10 -RNA10 CoV2 UCUCUAAACG (10nt - 5’ untranslated region of the genomic SARS-CoV-2 RNA acc. to: pmid: 33264373) (1uM scale ordered at MWG 8.12.20 - only: 259ul - 100uM)
CoV-RNA11 -RNA11 CoV2 CUAAACG (7nt - 5’ untranslated region of the genomic SARS-CoV-2 RNA acc. to: pmid: 33264373) (1uM scale ordered at MWG 8.12.20 - only 305ul - 100uM)
RNA12 - RNA12_Meister_9nt: GUGGACUGC (got from Veronika Graus - Meisterlab) 200ul 800uM - storage: -80MST MST3 CoV-RNA
RNA12 - Sigma - RNA12_Meister_9nt: GUG GAC UGC - ordered from SIGMA 10uM scale 12-2020: per tube 564nmol (1619ug) sufficient material for high concentration
RNA13 - GCA GUC CAC - ordered from SIGMA 31.3.21 0.2 scale - hybridizes with RNA12
RNA14 - AAA CUU UAAU - ordered from SIGMA 13.4.21 0.2 scale - hybridizes with RNA8
RNA15 - AUU AAA GUU U (is the non-labeled RNA8; 10 nt in length) (ordered at MWG 8.5.21 - to make cy3 dsRNA with RNA16)
Chaperone assay
RNA oligos
RNA3 (Cy3, 26nt) hybridized to RNA6 (Cy5, 15nt) used by Julia Seidel
CoV-RNA 7 - rv to CoV2 498-15nt GAC CAU GAG GUG CAG hybridized to CoV-RNA 2 - cy5 Cov2 498-520, 26nt [Cyanine5] CUG CAC CUC AUG GUC AUG UUA UGG UU
DNA oligos
Chap-assay C (cy5, 18nt) hybridized to Chap-assay B (non-modified, 42nt) - ordered at MWG
Chap-assay C (cy5, 18nt) TTATTAACCCTCACTAAA
Chap-assay B (non-modified, 42nt)TTTCGGCCTCATCAGGTCATCGCTTTAGTGAGGGTTAATAAA
LLPS assay
RNA
ssRNA: Cy5 RNA3 (26nt)
dsRNA: RNA2+Cy5-RNA3 hybrid (26nt)
dsRNA+ssRNA extension: Cy5-RNA6 + RNA3 hybrid (26/15nt ds)
DNA
dsDNA+ssDNA extension: Cy5-Chap-assay C + Chap-assay B
special oligos
internal hybridisation dsRNA
dsRNA8-14 - [CY5] (AUU AAA GUU U) [Spd] [Spd] [Spd] (AAA CUUUAA U) (ordered at MWG - 8.5.21) self forming duplex; for binding assays in MST