C-5 propyne derivatives and G-Clamp

Substitution of C-5 propynyl-dC (pdC) for dC and C-5 propynyl-dU (pdU) for dT are effective strategies to enhance base pairing. Using these base substitutions, duplex stability and melting temperatures are raised by the following amounts: C-5 propynyl-C 2.8° per substitution; C-5 propynyl-U 1.7° per substitution. AP-dC (G-clamp) substitutes for dC and is another very important modified nucleoside that enhances hybridization by 7.5° per substitution. The ability of these modified bases to enhance binding while maintaining specificity has proven useful in antisense research and in the synthesis of high affinity probes. AP-dC is also a fluorescent nucleoside and should find uses in DNA structural research.

Item	Catalog No.	Pack	Price ($)
pdC-CE Phosphoramidite	10-1014-90	100 µmole	85.00
	10-1014-02	0.25g	245.00
	10-1014-05	0.5g	490.00
pdU-CE Phosphoramidite	10-1054-90	100 µmole	65.00
	10-1054-02	0.25g	195.00
	10-1054-05	0.5g	390.00
AP-dC-CE Phosphoramidite	10-1097-95	50 µmole	230.00
	10-1097-90	100 µmole	460.00
	10-1097-02	0.25g	1175.00

bases affecting duplex stability

C-5 methyl pyrimidine nucleosides are known to stabilize duplexes relative to the non-methylated bases. Therefore, enhanced binding can be achieved using 5-methyl-dC in place of dC, duplex melting temperature being increased by 1.3°. Improved stacking in this case is believed to be brought about by elimination of water molecules from the duplex. 2,6-Diaminopurine 2'-deoxyriboside (2-amino-dA) forms an additional hydrogen bond with Thymidine, thereby leading to duplex stabilization with a melting temperature increase of 3°. Our 2-amino-dA monomer exhibits fast and effective deprotection in ammonium hydroxide and it is stabilized to depurination during synthesis. Sequences with high GC content may contain mismatches and still hybridize because of the high stability of the G-C base pair. The N4-ethyl analogue of dC (N4-Et-dC) hybridizes specifically to natural dG but the stability of the base pair is reduced to about the level of an AT base pair.

AP-dC (G-clamp) enhances oligo hybridization since the AP-C....G base pair contains 4 hydrogen bonds, which makes the interaction much stronger than the regular C....G base pair with its 3 hydrogen bonds.

Item	Catalog No.	Pack	Price ($)
5-Me-dC-CE Phosphoramidite	10-1060-90	100 µmole	50.00
	10-1060-02	0.25g	120.00
5-Me-dC-CPG	20-2060-01	0.1g	50.00
1 µmole columns	20-2160-41	Pack of 4	200.00
0.2 µmole columns	20-2160-42	Pack of 4	120.00
2-Amino-dA-CE Phosphoramidite	10-1085-95	50 µmole	70.00
(2,6-diaminopurine)	10-1085-90	100 µmole	125.00
	10-1085-02	0.25g	250.00

Sequences with high GC content may contain mismatches and still hybridize because of the high stability of the G-C base pair. The N4-ethyl analogue of dC (N4-Et-dC) hybridizes specifically to natural dG but the stability of the base pair is reduced to about the level of an AT base pair.

Coupling N6-Me-dA (10-1003) and N4-Et-dC (10-1068) with 1H-tetrazole leads to a trace of branching at the secondary amine positions, while DCI leads to around 15% branching. In collaboration with Berry and Associates, the acetyl protected monomers were prepared. Acetyl protection was chosen since it would block branching reactions. Oligonucleotides synthesized using these monomers proved to be compatible with all popular deprotection strategies from UltraMild to UltraFast. When the acetyl protected monomers were compared with the unprotected monomers using DCI as activator, branching was reduced from 15% to zero.

Locked Nucleic Acid (LNA™) Phosphoramidites

Locked Nucleic Acid (LNA) phosphoramidites have been discontinued.

Caps for Increased Duplex Stability and Base-Pairing Fidelity at Termini

New cap structures allow for the preparation of hybridization probes with increased affinity for complementary sequences. The monomers used to prepare capped oligonucleotides are phosphoramidites that can be readily introduced via automated DNA synthesis at the end of solid phase syntheses. The caps favor the formation of stable Watson-Crick duplexes by stacking on the terminal base pair (Figures 1 and 2).

FIGURE 1: STACKING OF CAP ON TERMINAL BASE PAIR	FIGURE 2: STACKING OF Uaq CAP ON 3’ TERMINAL BASE PAIR

Melting point increases of over 10 °C per modification can be realized for short duplexes.1,2 The caps fit canonical Watson-Crick base pairs and do not stack well on mismatched base pairs. This leads to increased base pairing selectivity at the terminal and the penultimate position of oligonucleotides featuring the caps. Base pairing fidelity is usually low at the termini, where fraying occurs frequently in the absence of caps. The beneficial effects of the caps are also realized when longer target strands are bound, so there is no need for blunt ends for the duplexes formed.1,2 The caps, when attached to the 5’ terminus of an oligonucleotide, also facilitate purification as their lipophilicity leads to prolonged retention on reversed phase columns or cartridges. Finally, capping of termini may discourage the degradation of oligonucleotides by exonucleases.

3’-Uaq Cap CPG, a Uridine support modified with a 2’- anthraquinone residue, is the most effective oligonucleotide cap known to date.3,4 For short hybrid duplexes between DNA probes and RNA target strands, the increase in Tm is up to 18 °C and the modification is effective in increasing the Tm of DNA:DNA, RNA:RNA, and DNA:RNA hybrid duplexes. 3’-Uaq Cap also increases probe specificity by depressing the melting point of terminal mismatches.