2.2.1 DNA oligonucleotides
All DNA primers used throughout this thesis were purchased from Metabion (Martinsried, Germany) and ordered as 100 µM stock solutions. For longer time storage the primers were kept at 4 °C in a refrigerator. All DNA primers were used for PCR reactions and designed using the software tool Oligo Analyzer 3.1 (Integrated DNA Technologies, Munich, Germany).
2.2.1.1 Primers for cloning
Primers that were needed for cloning experiments are listed in Appendix J. They were phosphorylated before usage (2.15.2) or were designed carrying the respective palindromic sequence for a restriction endonuclease (2.15.4). Primers that were used for mutagenesis PCR (2.15.8.2) have a much longer nucleotide sequence harboring point mutations in the middle of the primer sequence.
2.2.1.2 Primers for DNA sequencing
To verify DNA sequences of generated plasmid constructs Sanger sequencing was performed at eurofins mwgΙoperon (2.15.10) using primer sequences listed in Appendix K.
2.2.1.3 Primers for RNA analyses
Quantitative PCR reactions (2.15.8.3) of human mRNA or non-coding RNA levels were done using primer sequences listed in Table 1.
22
Table 1: Primers for RNA analyses.
RNA transcript Name of
DNA primer Sequence 5’ 3’ Product
length [bp]
hsa 5S rRNA qPCR 5S rRNA fwd 5‘- TCT CGT CTG ATC TCG GAA GC qPCR 5S rRNA rev 5‘- AGC CTA CAG CAC CCG GTA TT 88 hsa ß-Actin qPCR Actin fwd 5'- CCA ACC GCG AGA AGA TGA
qPCR Actin rev 5'- CCA GAG GCG TAC AGG GAT AG 97 hsa MYC qPCR MYC fwd 5‘- CCT TGC AGC TGC TTA GAC
qPCR MYC rev 5’- GAG TCG TAG TCG AGG TCA T 116 hsa E2F3 qPCR E2F3 fwd 5’- GAG ACT GAA ACA CAC AGT CC
qPCR E2F3 rev 5’- CCT GAG TTG GTT GAA GCC 98 hsa PIM1 qPCR PIM1 fwd 5'- ATC AGG GGC CAG GTT TTC T
qPCR PIM1 rev 5'- GGG CCA AGC ACC ATC TAA T 73 hsa pri-mir-17-92 qPCR pri-17-92 fwd 5’- CAT CTA CTG CCC TAA GTG CTC CTT
qPCR pri-17-92 rev 5’- GCT TGG CTT GAA TTA TTG GAT GA 68
2.2.1.4 Primers for cDNA generation of miRNAs
To generate cDNA (2.15.7) of human miRNAs specific stem-loop miRNA primers were designed according to Chen et al. (178). A list of all used primers is depicted in Table 2. The 6/7 nt indicated by lower case letters are reverse complementary to the 3’-end of the miRNAs listed in Appendix L.
Table 2: Primers for cDNA generation of miRNAs.
MiRNA Name of
DNA primer Sequence 5’ 3’
hsa-miR-15a loop miR-15a RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACc aca aac
hsa-miR-16-1 loop miR-16-1 RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACc gcc aat
hsa-miR-17/20a loop miR-17/20a RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACc tac ctg
hsa-miR-24-1 loop miR-24-1 RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACc tgt tc
hsa-miR-26a loop miR-26a RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACa gcc tat
hsa-miR-33a loop miR-33a RT 5'- TGG ATA TCC ACA CCA GGG TCC GAG GTA TTC GGT GTG GAT ATC CAt gca atg
hsa-miR-33b loop miR-33b RT 5’- GUC GUA UGG UCA CGA GGG UCC GAG GUA UUC CGU GAC CAU ACG ACg caa ug
hsa-miR-144 loop miR-144 RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACa gta cat
hsa-miR-374a loop miR-374a RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACc act tat
has-miR-423 loop miR-423 RT 5'- GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACa aag tct
23 2.2.1.5 Primers for qPCR of miRNAs
Quantitative PCR reactions (2.15.8.3) were conducted to determine human miRNA levels in several cell lines. The primers are based on the human miRNA sequences which are listed in Appendix L. To obtain roughly the same melting temperature of the individual primers in subsequent qPCR reactions, a 5’-overhang was designed that is indicated by lower case letters in Table 3.
Table 3: Primers for qPCR of miRNAs.
MiRNA Name of DNA primer Sequence 5’ 3’ Product
length [bp]
hsa-miR-15a miR-15a QT forward 5'- cgc gcT AGC AGC ACA TAA TG 61 hsa-miR-16-1 miR-16-1 QT forward 5'- cgc gcT AGC AGC ACG TAA AT 61 hsa-miR-17 miR-17 QT forward 5'- cgc gcC AAA GTG CTT ACA GTG 62 hsa-miR-20a miR-20a QT forward 5'- gcc gcg cTA AAG TGC TTA TAG TG 64 hsa-miR-24-1 miR-24-1 QT forward 5'- cgc gcT GGCTCA GTT CAG CAG 62 hsa-miR-26a miR-26a-1 QT forward 5'- cgc gcT TCA AGT AAT CCA GG 61 hsa-miR-33a miR-33a QT forward 5'- cgc gcG TGC ATT GTA GTT G 60 hsa-miR-33b miR-33b QT forward 5’- cgc gcG TGC ATT GCT GTT G 60 hsa-miR-144 miR-144 QT forward 5'- gcg cgc gcT ACA GTA TAG ATG 62 hsa-miR-374a miR-374a QT forward 5'- gcc gcg cTT ATA ATA CAA CCT G 63 has-miR-423 miR-423 QT forward 5'- cTG AGG GGC AGA GAG CG 58 reverse primer miR-33a 5'- CAC CAG GGT CCG AGG T - reverse primer miR-33b 5‘- CAC GAG GGT CCG AGG TA - uni reverse primer QT 5'- GTG CAG GGT CCG AGG T -
2.2.2 RNA oligonucleotides
RNA oligonucleotides used in this thesis were purchased from different companies as indicated in the following chapters. They were ordered lyophilized and diluted in double-distilled water if not indicated otherwise. For long time storage RNA oligonucleotides were kept at -20 to -80 °C.
2.2.2.1 SiRNAs
Used siRNAs were purchased from Metabion (Martinsried, Germany), Ambion® Life Technologies (Darmstadt, Germany) or Dharmacon Thermo Fisher Scienific (Lafayette, CO, USA) and listed in Table 4. All siRNA oligonucleotides were designed as double-stranded DICER1 processing products (sense and antisense orientation) with an overhang of two
24 deoxythymidines (dT) positioned on their 3’-end. The sense strand is specific and fully complementary (location either in the coding sequence or the 3’-UTR) to the human mRNA of interest.
Table 4: SiRNAs.
MRNA of
interest Name of siRNA Sequence 5’ 3’
VR1 mRNA VR1 siRNA (179) sense 5'- GCG CAU CUU CUA CUU CAA CdTdT antisense 5'- GUU GAA GUA GAA GAU GCG CdTdT
PIM1 mRNA
PIM1 siRNA (1130)
sense 5'- GAU AUG GUG UGU GGA GAU AdTdT antisense 5'- UAU CUC CAC ACA CCA UAU CdTdT PIM1 siRNA
(1491)
sense 5'- GGA ACA ACA UUU ACA ACU CdTdT antisense 5'- GAG UUG UAA AUG UUG UUC CdTdT siRNA 33a sense 5'- AAA AUG CAC AAA CAA UGC AdTdT
antisense 5'- UGC AUU GUU UGU GCA UUU UdTdT luc mRNA siRNA luc sense 5'- CUU ACG CUG AGU ACU UCG AdTdT
antisense 5'- UCG AAG UAC UCA GCG UAA GdTdT MYC mRNA siRNA MYC sense 5'- CAG GAA CUA UGA CCU CGA CUA dTdT
antisense 5’- UAG UCG AGG UCA UAG UUC CUG dTdT
E2F3 mRNA siRNA E2F3 sense 5’- ACA GCA AUC UUC CUU AAU AdTdT antisense 5’- UAU UAA GGA AGA UUG CUG UdTdT
SiRNAs were ordered lyophilized and were diluted in siRNA buffer (30 mM HEPES KOH pH 7.4, 100 mM KCl, 2 mM Mg2Cl, 50 mM NH4Ac) to reach concentrations up to 2 µg/µL.
After determination of siRNA concentration (2.14.1) they were prepared for long time storage at -80 °C.
2.2.2.2 MiRNA Mimics
Modified miRNA mimics, called miRIDIAN miRNA mimics, were purchased from Dharmacon Thermo Fisher Scientific (Lafayette, CO, USA). MiRIDIAN miRNA mimics are intended to mimic native miRNAs and are generated as double-stranded RNA molecules which are chemically enhanced with a proprietary design. Their sequences are based on the human endogenous, mature miRNAs listed in the miRBase Sequence Database 16.0 (180). All miRNAs were dissolved in ddH20 and prepared for storage at -80 °C. Used miRNAs are listed below (Table 5):
25
Table 5: MiRNA mimics.
MiRNA of
interest Order Number Sequence 5’ 3’
hsa-miR-33a (C-300509-07) 5'- GUG CAU UGU AGU UGC AUU GCA hsa-miR-33b (C-300958-03) 5'- GUG CAU UGC UGU UGC AUU GC hsa-miR-15a (C-300482-03) 5'- UAG CAG CAC AUA AUG GUU UGU G hsa-miR-16-1 (C-300483-03) 5'- UAG CAG CAC GUA AAU AUU GGC G Negative
control mimic #1 (CN-001000-01) no sequence available
hsa-miR-33a - leader 5'- GUG CAU UGU AGU UGC AUU GCA
passenger 5’- TGC AAT GCA ACT ACA ATG CAC
hsa-miR-33b - leader 5'- GUG CAU UGC UGU UGC AUU GC
passenger 5'- CAG UGC CUC GGC AGU GCA GCC C
In case of hsa-miR-33a and -33b the DICER1 processing product of pre-miR-33a and pre-miR-33b as published in the miRBase Sequence Database 14.0 (2.18.6) was additionally purchased by eurofins mwg|operon (Table 5), but lacking the 5’-terminal phosphate. The leader and passenger strand were dissolved in siRNA annealing buffer (30 mM HEPES KOH pH 7.4, 100 mM KCl, 2 mM Mg2Cl, 50 mM NH4Ac) respectively. After measuring RNA concentration (2.14.1) equimolar amounts of each strand were mixed and heated to 95 °C.
Annealing of both strands was done for 1 h at 37 °C to receive the double-stranded DICER1 processed miRNA product.
2.2.2.3 MiRNA hairpin inhibitors
MiRNA hairpin inhibitors, called miRIDIAN hairpin inhibitors, were purchased from Dharmacon Thermo Scientific (Lafayette, CO, USA). MiRIDIAN hairpin inhibitors are chemically enhanced RNA molecules that are designed to inhibit endogenous miRNA functions. Thus their purpose is to discover and investigate natural functions of miRNAs (181). Their sequences are based on the human endogenous mature miRNAs listed in the miRBase Sequence Database 16.0 (180). All miRIDIAN hairpin inhibitors were dissolved in double-distilled water and prepared for long-term storage at -80 °C. Used miRIDIAN hairpin inhibitors are depicted in Table 6:
Table 6: MiRIDIAN hairpin inhibitors.
MiRNA of
interest Order number Sequence 5’ 3’
hsa-let-7a (IH-300473-07) 5'- UGA GGU AGU AGG UUG UAU AGU U hsa-miR-17 (IH-300485-06) 5'- CAA AGU GCU UAC AGU GCA GGU AG hsa-miR-20a (IH-300491-05) 5'- UAA AGU GCU UAU AGU GCA GGU AG
26 2.2.3 LNA-based oligonucleotides
As a second class of miRNA inhibitors RNA oligonucleotides containing an LNA (Locked Nucleic Acid) modification were designed. This modification mimics an RNA structure because it contains an O2’-C4’-methylene-linked bicyclic ribose unit which locks its conformation in the C3’-endo conformation and thus increases the stability of this oligonucleotide (113), (114). The LNA based oligonucleotides were purchased from RiboTask (Odense, Denmark). They were ordered as lyophilized dry powder, dissolved in double-distilled water and stored at -20 °C.
2.2.3.1 LNA miRNA inhibitors
All LNA-modified oligonucleotides showed fully complementarity to the human miRNAs of interest to gain miRNA inhibition. The ordered LNA oligonucleotides (Table 7) harbor a natural phosphodiester (PO) backbone with one exception containing a modified phosphorothioate (PS) backbone. They have introduced the LNA modification at some or all positions of the oligonucleotide, except for isolated 2’-oxymethyl pyrimidine residues, marked as [mU] and [mC] (Table 7), that are introduced to lower self-pairing of the individual oligonucleotides.
Table 7: LNA miRNA inhibitors.
MiRNA of interest
Name of
LNA oligonucleotide Sequence 5’ 3’
hsa-miR-17-5p
hsa-miR-20a LNA (PS) miR-17/20a 5'- GCA CT[mU] TG
hsa-miR-17-5p LNA miR-17 5'- CTG TAA GCA CT[mU] TG hsa-miR-20a LNA miR-20a 5'- CTA TAA GCA [mC]T[mU] TA hsa-let-7a LNA let-7a (8-mer) 5'- CTA CCT CA
hsa-let-7a LNA let-7a (10-mer) 5'- TAC TAC CTC A hsa-let-7a LNA let-7a (12-mer) 5'- CCT ACT ACC TCA hsa-let-7a LNA let-7a (14-mer) 5'- AAC CTA CTA CCT CA - LNA anti-RNase P (182) 5'-CAA GCA GCC UAC CC