Effects of sodium ions on melting temperatures of DNA–DNA and LNA–DNA duplexes

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Duplexes were investigated that had a T•A base pair in the X•Y mismatch site. All duplexes had the same base sequence, 5′-ggtcctttcttggtg-3′/3′-ccaggaaagaaccac-5′, where LNA modifications were introduced at various positions (). Solid lines were calculated using a published salt correction ()

Fluorescence emission spectra of target oligomer, 5′-gcgaggpggctt-3′, with single 2-aminopurine (p) reveal the magnitude of stacking interactions

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Duplexes containing a H-bonded t•p base pair are compared with mismatched g•p base pair duplexes. Both unmodified DNA probes (blue lines) and probes with a LNA triplet at the mismatch site (red lines) were studied. UV melting experiments in 1 M Na buffer showed that LNA triplets increased mismatch discrimination for these sequences

Dependence of melting temperature and mismatch discrimination on oligomer length

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Average melting temperatures were calculated for 50% g•c DNA duplex oligomers that did not contain any mismatched base pairs as well as oligomers with single g•t or a•c mismatches. Predictions assumed total single strand concentration of 400 nM in 1 M Na buffer

SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

<div><p>Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine <i>Hras</i> mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes <i>in vivo</i> molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.</p></div

Difference of Δ values between LNA and DNA probes for various mismatches

<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Sequence Set 1 (panel A and B), and Sets 2 and 3 (panel C) are plotted. Positions of LNA residues and set names are shown in Figure 1. A positive difference indicates improved mismatch discrimination for the LNA probe relative to the DNA probe. Dashed lines denote the range (±0.8°C), which is within the experimental error of the measurements

Single cell Acceptor photobleach analysis.

<p>The Anchor (FRET donor, AF488) and mutant Probe (FRET acceptor, TR) were co-transfected to the same cell and fluorescence was measured before (upper panel) and after (lower panel) red photobleaching. As a result of photobleaching, the acceptors red signal decreases. When energy transfer occurred, the donor green emission increased after the acceptors photobleach. <b>A.</b> In 308 cells, which endogenously carry the Hras point mutation, an increase in green donors signal was observed after acceptor photobleach whereas in <b>B.</b> Hras wt MEF cells, no increase in donors signal was observed.</p

FRET probes are SNP specific <i>in vitro</i> at 37°C.

<p>Multicomponent plots indicating fluorescent signals from the anchor (blue line) and the allele specific probe (red line) as a course of temperature increase (37–75°C). <b>A</b> – Anchor/<b>A, T</b> allele specific probe or O – no probe/<b>A, U</b> – target RNA oligo complement to A and T probes respectively. Perfect match between the probe and target results in an energy transfer between the donor anchor and acceptor probe. When both the donor anchor and acceptor probe are bound to target at 37°C, their proximity causes energy transfer resulting in red fluorescence. As a course of temperature increase, the oligos denaturate from target (51°C), energy transfer ceases and only the donor fluorescence can be detected, causing a typical “8” shaped FRET plot to be generated (<b>A, B</b>). A single mismatch in target sequence results in low affinity of the oligos therefore no FRET response is detected (<b>C, D</b>). No target control (<b>E</b>), no donor control (<b>F</b>), no acceptor control (<b>G</b>).</p

Hras oligo probes sequences and modifications.

<p>Key: <b>A,C,U/T,G –</b>2′OMe RNA<b>, a,c,u,g –</b> RNA<b>, <u>A,C,T,G</u></b> - LNA residues<b>, Bold font -</b> SNP site<b>, TR –</b> Texas Red<b>, AF –</b> Alexa Fluor<b>, “*” -</b> phosphorothioate <b>(PS)</b> internucleotide linkage<b>, PSEnd-</b> incidates phosphorothioate bonds only on terminal linakges of the molecule<b>, targ-</b> target sequence.</p

Influence of chemical modifications on target specificity and FRET intensity.

<p>Three differentially modified FRET couples were incubated with: no target (left bar), mismatch target (middle bar) or full match target (right bar) and FRET was measured. Targ – target, Anc - Anchor Alexa Fluor 488 (AF488), PS- ODNs heavily modified with PS bonds, PSEnd- PS modifiIcation only at the edge of the ODN, pT- mutant specific probe T marked with either Texas Red (TR) or Alexa Fluor 594 (<b>AF594</b>) as acceptor fluorophore.</p

Schematic of RBMBs and the methodology used to detect individual RNA transcripts in living cells.

<p>(A) RBMBs are hairpin-forming oligonucleotide probes that are labeled with a reporter dye, quencher, and reference dye. The close proximity of the reporter dye and quencher in the absence of target RNA results in a low fluorescent state. Upon hybridization to complementary RNA, the fluorescent dye and quencher are forced apart, resulting in the restoration of fluorescence. The reference dye remains unquenched regardless of the conformation of the RBMB. The double-stranded domain with a 3′-UU overhang drives nuclear export. (B) To detect individual RNA transcripts, cells were engineered to stably express RNA with 96-tandem repeats of the RBMB target site in the 3′-untranslated region. Binding of up to 96 RBMBs to each RNA transcript results in discrete bright fluorescent spots that can be readily visualized and tracked in real-time by wide-field fluorescence microscopy.</p