An RNA microchip containing immobilized oligoribonucleotides with protective groups at 2'-O-positions.
To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.
Mikheikin
Andrey L
AL
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation.
Surzhikov
Sergey A
SA
Zasedateleva
Olga A
OA
Vasiliskov
Vadim A
VA
Pan'kov
Sergey
S
Grechishnikova
Irina V
IV
Kisselev
Lev L
LL
Zasedatelev
Alexander S
AS
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D013816:Thermodynamics
D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes; D009843:Oligoribonucleotides; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D012313:RNA; D004247:DNA; D004722:Endoribonucleases; C022543:ribonuclease T(2)