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
N4-Et-dC-CE Phosphoramidite	10-1068-95	50 µmole	125.00
	10-1068-90	100 µmole	225.00
	10-1068-02	0.25g	675.00

Locked Nucleic Acid (LNA™) Phosphoramidites

Locked Nucleic Acid (LNA) was first described¹ by Wengel and co-workers in 1998 as a novel class of conformationally
restricted oligonucleotide analogues, possessing very high affinity and excellent specificity toward complementary DNA and RNA². LNA molecules have been used to improve design and efficacy of oligonucleotides directed at regulating gene expression, for example, antisense and siRNA. Incorporating LNA into oligonucleotides has positive effects on delivery to cells, stability, non-specific effects, toxicity, and pharmacokinetics. LNA can also be used to develop more efficient diagnostic tools, for example, probes in genotyping³ and in DNA array design⁴. Because of their increased affinity, LNA containing probes can be shortened and this is particularly attractive for miRNA expression research⁵. These properties are detailed in several reviews⁶.

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 (Figure 1).

FIGURE 1: STACKING OF CAP ON 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 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.