Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 159

4.4.2 Modifications of the 5

- and 3

-Termini

There are a number of reagents useful for attachment to the termini of synthetic oligonucleotides during
chemical synthesis.21,22For example, phosphoramidite-building blocks are available for the synthesis of
oligonucleotides bearing either 5
- or 3
-phosphate groups (Figure 4.19). An important class of modifiers
are reporter groups. For example, fluorophores are useful in fluorescence studies for quantification or
localisation of an oligonucleotide, in fluorescence resonance energy transfer (FRET) studies (Section
11.1.2) and in automated DNA sequencing (see Section 5.1.2). Another important reporter group is biotin,
which may be incorporated, for example, during solid-phase synthesis as a phosphoramidite reagent or on
the 3
-end of an oligonucleotide through attachment to the solid support (Figure 4.19). Biotinylated oligonu-
cleotides have the advantage that they can be separated from other biomolecules by the extremely tight inter-
action of biotin with streptavidin, for example, with streptavidin-coated beads or micro-titre plates.
Linkershave become increasingly important units for conjugation of oligonucleotides to other biomole-
cules, particularly those linkers that have the capability of generating a terminal amino, thiol or carboxylate
group following oligonucleotide synthesis and a subsequent linker deprotection reaction (Figure 4.20).

DMTO

S

O-R

O O

R = succinyl-CPG, 3'-PO 4 2-

R=P(OCH 2 CH 2 CN)NiPr 2 ,5'-PO 4 2-

HN NH

S

H

N

ODMT

O

O-R

O

R = succinyl-CPG, 3'-biotin

R=P(OCH 2 CH 2 CN)NiPr 2 , 5'-biotin or internal

Figure 4.19 Oligonucleotide terminal modifiers

N

HN

O Me

O

dR

N

NH

H

C

N

dR

O

NH 2 N

HN

X

O

O

dR

N

HN

S

O

Ribose

N

N N

N

Ribose

NH 2

a b c X=Br,I

d e

Figure 4.18 Nucleoside analogues used in structural studies involving oligonucleotides. (a) 5-propynyl-2
-deoxyuri-
dine, (b) 7-deaza-2
-deoxyguanosine, (c) 5-halo-2
-deoxyuridine, (d) 4-thiouridine, and (e) 2-amino-
purine riboside