Oligo Modified Linker Attachment Chemistry

Linker Modified Oligonucleotides

Bio-Synthesis offers hundreds of useful linker modifications for oligonucleotide to be attach to other biopolymer. Choosing the right one can be confusing. Begin by choosing a modification category based on the required function:

• Are you attaching the oligonucleotide to thiol modified surfaces or be incorporated into polyacrylamide gels during polymerization -> Acrydite modification .
• Do you need oligonucleotide substrate for T4 RNA ligase in the absence of ATP -> Adenylation.

All modifications we offered can be used to link an oligonucleotide with another molecule or a surface.

Linker spacer arms for incorporation between the reactive group or diagnostic moiety and the oligonucleotide have been prepared. The shorter carbon chain linkers (C3: (CH2)3) can be used in instances where the proximity of the oligonucleotide poses no problems. The longer chain linkers (C6 and C12) are typically used when the oligonucleotide must be spaced far enough from the corresponding undesired interactions.

Amino Modification

Primary amines are reactive with a number of useful molecules such as isothiocyanates, NHS esters, or activated carboxylates. Amino oligo modifications are useful for the attachment of fluorescent reporter groups, biotin, digoxigenin and enzymes or antibodies. They are also used for immobilization of oligonucleotides on solid support.

Aldehyde Modification

Aldehyde modified oligonucleotide is another approach to introduce reactive group to 5'-end of an oligo. Aldehyde modifier is an electrophilic substitution. The aldehyde moiety reacts with a variety of substituted hydrozinos and semicarbazides to form stable hyrozones and semicarbazides, respectively (12). The 5'-Aldehydes can also react with amines to form Schiff's base, but the Schiff's base must be reduced to form a stable linkage.

Two phosphoramidites that incorporate an aldehyde moiety on the 5'–terminus are available. The amidites introduce the aldehyde via a benzaldehyde functional group but vary in the linker length (C6 vs C2). Both aldehyde modifiers are incorporated using standard solid phase synthesis methodologies. However, the 5'-aldehyde modifier C2 is protected and the aldehyde must be freed using detritylation conditions with 80% acetic acid or 2% aqueous trifluoroacetic acid after purification.

Thiol Modifiers

A thiol group can be used to attach an oligonucleotide to a variety of fluorescent and nonfluorescent moieties or surfaces. Oligos containing thiol modifiers are shipped in their oxidized (disulfide) form and require chemical reduction by dithiothreitol (DTT) or Tris (2-carboxyethyl) phosphine (TCEP) prior to use. Dithiol can be inserted into an oligonucleotide at the 5’ position, the 3’ position or internally. Each insertion results in two SH groups available for coupling with ligands or surfaces (linkage to gold surfaces is one popular application). The dithiol phosphoramidite (DTPA) modification can be inserted in series so that 2, or even 3, groups can be positioned adjacent to each other to increase efficiency of ligand/surface interactions.

Azide (NHS Ester)

Bio-Synthesis's azide modification uses an NHS ester functional group to attach an azide moiety at the 5’, 3’, or any internal position in an oligonucleotide. This azide moiety may subsequently be used to attach alkyne modified groups using the click reaction.

Alkynes

Alkyne modifiers are used to react with azide-labeled functional groups to form stable bonds through the click reaction. 5’ Hexynyl is the simplest and most popular way to introduce a 5’ terminal alkyne group. 5-Octadinynyl dU is a modified base with an 8-carbon linker terminating in an alkyne group and is the preferred way to insert alkynes at internal positions within a sequence. This modification is also available for 3’ or 5’ attachment.

Cholesteryl-TEG

Cholesterol can be conjugated to oligonucleotides and can facilitate uptake into cells. It has been used as a transfection aid for antisense oligos and siRNAs, both in vitro and in vivo.

Biotins

Biotin is one of the most popular attachment chemistry options. For most applications standard 5’ Biotin fulfills all needs. Standard biotin does not require additional purification and may be ordered at all scales. 5’ Dual Biotin inserts two adjacent biotin moieties in a sequence. This can slightly increase affinity to streptavidin. Biotin dT allows placement of a biotin internally without disrupting nucleotide spacing. Biotin-TEG helps reduce steric hindrance in applications that require the use of magnetic beads. PC biotin contain a photocleavable spacer between the biotin group and the oligonucleotide when exposure to long-wave UV light in 300-350 nm spectral range, cleavage release the oigo with a 5'-phosphate group.

PC Linkers

Photocleavable PC linker can be incorporated into oligonucleotides at any position by standard automated DNA synthesis methodology. PC Linker Phosphoramidite has the added advantage in that photocleavage results in monophosphate fragments at both the 3'- and 5'-termini of the oligonucleotide fragments.

Oligo Linkers and Attachment Chemistry

Linkers and attachment chemistry modifications of oligonucleotides are separated into three groups, depending on the position of the modification introduced:

• 5' Modifications
• Internal Modifications
• 3' Modifications

Purification is recommended for all oligos greater than 40 bases in length and for many modified oligos. For demanding applications such as multiplex PCR, cloning, mutagenesis or antisense/RNAi methods, additional purification will significantly improve oligonucleotide performance. Purified oligos up to 60 bases with a purity guarantee receive QC by gel electrophoresis.

Browse our selection of linker and attachment chemistry modifications using the tabs below. When you are ready to design and order your oligonucleotide, simply click the order button . Alternatively, let us design the oligonucleotides for you.

Ordering & Contact Information

• Please contact us for additional information or send an email to info@biosyn.com.
• You may also request an online quote.
• To contact us by phone, please Call 972-420-8505 (USA) 800-227-0627 (INTL.) or Fax 972.420.0442
• Orders may be placed using a purchase order (PO) or by credit card through our secure online ordering system.
• When using a credit card ( ) it will be billed under "Bio-Synthesis, Inc."