Affinity Plus Modified Bases
Locked nucleic acids—Affinity Plus modified bases
Affinity Plus modified bases are locked nucleic acids. Addition of locked nucleic acid monomers help to increase and can be used to fine-tune sequence melting temperature (Tm). These bases also confer significant in vitro and in vivo nuclease resistance. Learn more about the benefits of including Affinity Plus bases in your oligo and probe sequences by visiting our Locked nucleic acid technology page. When ordering, denote Affinity Plus bases in your sequence using +A, +G, +C, +T. You can include up to 20 Affinity Plus bases in Affinity Plus DNA & RNA Oligonucleotides, and up to 6 Affinity Plus bases in Affinity Plus qPCR Probes.
2’-O-methoxy-ethyl Bases (2’-MOE)
2’-MOE bases are often used for antisense oligos (ASO), aptamers, and siRNA. Compared to standard RNA bases 2’-MOE bases offer increased resistance to nuclease degradation, reduced toxicity, and increased affinity for binding to complimentary RNA. For more information on antisense technologies and strategies please see: Antisense oligonucleotides.
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2'-O-Methyl RNA Bases
2'-O-Methyl RNA Bases
2'-O-Methyl RNA is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification. Oligonucleotides can be directly synthesized that contain 2'-O-Methyl RNA. This modification increases Tm of RNA:RNA duplexes but results in only small changes in RNA:DNA stability. It is stabile with respect to attack by single-stranded ribonucleases and is typically 5 to 10-fold less susceptible to DNases than DNA. It is commonly used in antisense oligos as a means to increase stability and binding affinity to the target message. To include a 2’ O-methyl modification in your RNA sequence, simply place a lowercase "m" in front of the base; for example, mAmGmCmU.
2' Fluoro bases have a fluorine modified ribose which increases binding affinity (Tm) and also confers some relative nuclease resistance when compared to native RNA. These modifications are commonly employed in ribozymes and siRNAs to improve stability in serum or other biological fluids.
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2-Aminopurine can substitute for dA in an oligonucleotide. It is a naturally fluorescent base that is sensitive to the local environment making it a useful probe for monitoring the structure and dynamics of DNA hairpins and for detecting the base stacking state of a duplex. 2-Aminopurine can be destabilizing and slightly lower the Tm.
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5-Bromo-deoxyuridine is a photoreactive halogenated base that can be incorporated into oligonucleotides to crosslink them to DNA, RNA or proteins with exposure to UV light. Crosslinking is maximally efficient with light at 308 nm.
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DeoxyUridine (dU) can be substituted for dT in DNA oligonucleotides. The base can be removed by the enzyme uracil-N-deglycosylase (UNG) which renders the oligo susceptible to strand scission. One common use of this strategy is to eliminate amplified DNA and prevent cross-contamination.
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This modified base can form three hydrogen bonds when base-paired with dT and can increase the Tm of short oligos by as much as 1-2°C per insertion. This effect, however, is complex and is dependent on sequence context.
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Dideoxycytidine (ddC) is a 3’ chain terminator that prevents 3’ extension by DNA polymerases.
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Historically, the first universal base employed was 2’-deoxyInosine (dI). DeoxyInosine is a naturally occurring base that, while not truly universal, is less destabilizing than mismatches involving the four standard bases. Hydrogen bond interactions between dI and dA, dG, dC and dT are weak and unequal, with the result that some base-pairing bias does exist with dI:dC > dI:dA > dI:dG > dI:dT. When present in a DNA template, deoxyInosine preferentially directs incorporation of dC in the growing nascent strand by DNA polymerase.
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Hydroxymethyl dC is a recently discovered modified base with a probable epigenetic role.
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Inverted dT can be incorporated at the 3’-end of an oligo, leading to a 3’-3’ linkage which inhibits both degradation by 3’ exonucleases and extension by DNA polymerases.
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Placing Inverted Dideoxy-T at the 5’ end of a sequence will prevent unwanted 5’ ligations.
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5-Methyl deoxyCytidine when substituted for dC will increase the Tm by as much as 0.5°C per insertion. In addition, the presence of 5-Methyl dC in CpG motifs can prevent or limit unwanted immune responses that otherwise occur if oligos are administered in vivo, which is of particular importance in antisense applications.
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5-Nitroindole is currently the best universal base available. It does not favor any particular base-pairing (i.e., it does not support base-specific hydrogen bond formation), but does contribute to duplex stability through base-stacking interactions. Therefore, it is not as destabilizing to the duplex as mismatches between the standard bases. 5-Nitroindole directs random incorporation of any specific base when used as a template for DNA polymerase and partially blocks enzyme processivity.
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Super T (5-hydroxybutynl-2’-deoxyuridine) is a duplex-stabilizing modified base that increases oligonucleotide Tm. Oligonucleotides containing Super T can be extended normally by polymerases, including Taq polymerase,making Super T a useful modified base for designing short primers or probes for low-complexity, A-T rich sequences
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Super G (8-aza-7-deazaguanosine) is a modified base that eliminates naturally occurring, non-Watson-and-Crick secondary structures associated with guanine-rich sequences. Oligonucleotides containing Super G can be extended normally by polymerases, including Taq polymerase, making Super G a useful modified base for designing guanine-rich primers and probes. In addition, unlike standard guanine bases, Super G does not quench fluorophores, potentially improving probe performance
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