Modified Bases Modifications
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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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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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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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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Dideoxycytidine (ddC) is a 3’ chain terminator that prevents 3’ extension by DNA polymerases.
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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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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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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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Locked Nucleic Acids (LNA’s)
LNA bases have a modification to the ribose backbone that locks the base in the C3'-endo position, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an oligo at any position except the 3'-end. Applications have been described ranging from antisense oligos to hybridization probes to SNP detection and allele specific PCR. Due to the large increase in Tm conferred by LNAs, they also can cause an increase in primer dimer formation as well as self-hairpin formation. We therefore recommend limiting the number of LNAs incorporated into a single oligo to 10 bases or less.
NOTE: TM calculations for sequences containing LNA are only estimates, as complete parameters for these calculations have yet to be published. Any applications requiring extremely accurate prediction of the TM for these probes should be reviewed with a technical support representative from Exiqon, Inc. (http://www.exiqon.com) Click here for more information
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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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.
Hydroxmethyl dC is a recently discovered modified base with a probable epigenetic role.
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UNA Unlocked Nucleic Acid
Unlocked Nucleic Acids (UNAs) are acyclic RNA analogs without a C2'-C3' bond in the ribose ring. While base pairing occurs with UNAs, the missing bond increases the flexibility of the molecule, decreasing duplex thermostability. Each UNA substitution decreases oligonucleotide melting temperature (Tm) by 5–10°C, with multiple insertions having an additive effect [Jensen et al. (2008) Nucleic Acids Res Symp Series 52:133–134]. UNA substitutions, therefore, can be used to modulate Tm, and thus hybridization specificity. UNAs also block exonuclease function, and can be placed at sequence ends to stabilize oligonucleotides. Additionally, UNA placement at select internal sites of an siRNA can reduce miRNA-type off-target effects [Vaish et al. (2011) Nucl Acids Res, 39:1823–1832]; [ Bramsen et al. (2010) Nucl Acid Res, 38(17):5761–5773].
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Iso-dC and Iso-dG
Iso-dC and Iso-dG are chemical variants of cytosine and guanine, respectively. Iso-dC will hydrogen bond with Iso-dG but not with dG. Similarly, Iso-dG will base pair with Iso-dC but not with dC. Incorporation of these bases into DNA effectively expands the genetic alphabet and permits synthesis of oligonucleotides that have increased specificity and decreased mismatch hybridization potential. For example, an oligonucleotide containing Iso-dC can be designed so that it will hybridize to a complementary oligo containing Iso-dG but will not hybridize to any naturally occurring nucleic acids sequence. Oligonucleotides that contain either or both iso-bases require IE-HPLC purification.
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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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