Synthego's modified sgRNA are synthesized with 2'-O-methyl analogs and 3' phosphorothioate internucleotide
linkages in the first three nucleotides at both the 5' and 3' end of the RNA molecule.
The sugar analog a-L-threofuranosyl nucleotide (TNA for threose nucleic acid) (Figure 5, 2) has been examined if the oligonucleotides containing this shorter internucleotide building unit can also form GQs .
As the amino group replaced the 3'-OH, the internucleotide linkage at this site was 2'-5' instead of the natural 3'-5' (this modification of the backbone is named isoDNA; see also later).
The [T.sub.2][G.sub.4][T.sub.2] that assembles into a parallel-stranded tetramolecular GQ was found to be an inhibitor of HIV infection, especially if the phosphodiesters internucleotide linkages were replaced with P-S linkages (see Section 3.3), and its IC50 value was 0.3 [micro]M .
The backbone modifications, such as the thiophosphoryl (P-S) internucleotide linkages, increased the resistance to cellular nucleases, which is important for the aptamer activity .
A sulfur atom replaces one of the nonbonded oxygen atoms of the phosphodiester (P-O) internucleotide linkage (Figure 12, 1) in the phosphorothioate (P-S) linkage (Figure 12, 2).
In the methylphosphonate (P-Me, Figure 12, 3) internucleotide linkages, a methyl group replaces one of the nonbonded oxygen atoms, and thus the internucleotide linkage becomes uncharged, neutral.
The modified oligodeoxynucleotide according to the invention has the sequence 5'-TxAxTxTxCxCxGxTxCxAxT-3'-O--PO.sub.2 --O--R (SEQ ID NO:1), wherein X is an internucleotide
linkage of type o or s, with the proviso that x is an s linkage at least 4 times and at most 9 times, and o means a phosphodiester internucleoside linkage, s means a phosphorothioate internucleoside linkage, R means a C.sub.8 -C.sub.21 alkyl group, --(CH.sub.2 --CH.sub.2 O)n-(CH.sub.2).sub.m --CH.sub.3, or --CH.sub.2 --CH(OH)CH.sub.2 O--(CH.sub.2).sub.q --CH.sub.3 wherein n is an integer from 1 to 6, m is an integer from 0 to 20 and q is an integer from 7 to 20 and A is 2'-deoxyadenosine, G is 2'-deoxyguanosine, C is 2'-deoxycytidine and T is thymidine.
Solid-phase synthesis of oligodeoxynucleotides containing phosphoramidite internucleotide
linkages and their specific chemical.
Additionally, we have modified the probes' 3,-termini by thymidine attached via a 3,-3,-phosphodiester internucleotide
bond (37-"inverted" thymidine) to achieve greater stability to exonucleases [15-17].
The newly issued patent is an extension of previously issued patents (United States Patent 5,652,355, 6,143,881 and 6,346,614) and claims oligonucleotides comprising a deoxyribonucleotide, a 2'-substituted ribonucleotide, and a phosphorothioate or phosphorodithioate internucleotide
linkage, where the 2'-substitution is either 2'-O-aryl ribonucleotide or a 2'-O-amino ribonucleotide.
United States Patent 6,143,881, "Hybrid Oligonucleotide Phosphorothioates and Pharmaceutical Composition Thereof," owned by the University of Massachusetts and exclusively licensed to Hybridon, claims hybrid, or mixed backbone, oligonucleotides having at least one phosphorothioate internucleotide
linkage, in addition to both deoxyribonucleotides and the 2'-substituted ribonucleotides 2'-methoxy or 2'-alkoxyalkoxy, such as 2'-methoxyethoxy.