Unrestricted accessibility of short oligonucleotides to RNA.
The propensity of RNA to fold into higher-order structures poses a major barrier to the use of short probes (<15 nucleotides) by preventing their accessibility. Introduction of the pseudo-complementary bases 2-aminoadenine (nA) and 2-thiouracil (sU) and the destabilizing base 7-deazaguanine (cG) into RNA provides a partial solution to this problem. While complementary in hydrogen bonding groups, nA and sU cannot form a stable base pair due to steric hindrance, and are thus pseudo-complementary. Each, however, recognizes the regular T/U and A complements, allowing pairing with oligonucleotides. Short pseudo-complementary RNAs can be prepared by in vitro transcription. Relative to standard transcripts, the modified transcripts possess reduced secondary structure and increased accessibility to short (8-mer) probes in the locked nucleic acid (LNA) configuration. They also hybridize to complementary probes with increased specificity and thermostability. Practical application of this strategy to oligonucleotide-based hybridization assays will require engineering of RNA polymerase for more efficient utilization of pseudo-complementary nucleoside triphosphates.
Gamper
Howard B
HB
Department of Biochemistry and Molecular Pharmacology, Room 222BLSB, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania, 19107, USA. Howard.Gamper@jefferson.edu.
Arar
Khalil
K
Gewirtz
Alan
A
Hou
Ya-Ming
YM
D015075:2-Aminopurine / Q000031:analogs & derivatives / Q000378:metabolism; D020029:Base Pairing / Q000502:physiology*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D019271:Hypoxanthine / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D015347:RNA Probes / Q000378:metabolism*; D013696:Temperature; D013889:Thiouracil / Q000378:metabolism
D009841:Oligonucleotides; D015347:RNA Probes; D019271:Hypoxanthine; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D013889:Thiouracil; D006147:Guanine; D012313:RNA; C066856:7-deazaguanine