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The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.

Adams NM, Olmsted IR, Haselton FR, Bornhop DJ, Wright DW - Nucleic Acids Res. (2013)

Bottom Line: Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity.Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity.Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.

ABSTRACT
Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼10(5) target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

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Comparison of the BSI binding response and net hybridization upon adding the 15-mer, 22-mer, 44-mer, 88-mer or four consecutive 22-mer DNA probes to the RNA target. (A) The probe length that produces optimal BSI signal is 22 nt. Four short 22-mer DNA probes have improved signal over one 88-mer spanning the same target sequence. A scrambled negative control sequence produced negligible signal. (B) Net hybridization of the four consecutive 22-mers is significantly greater than any of the four probe lengths.
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gkt165-F2: Comparison of the BSI binding response and net hybridization upon adding the 15-mer, 22-mer, 44-mer, 88-mer or four consecutive 22-mer DNA probes to the RNA target. (A) The probe length that produces optimal BSI signal is 22 nt. Four short 22-mer DNA probes have improved signal over one 88-mer spanning the same target sequence. A scrambled negative control sequence produced negligible signal. (B) Net hybridization of the four consecutive 22-mers is significantly greater than any of the four probe lengths.

Mentions: The design space of oligonucleotide probes targeting the ∼1300 nt RSV N gene RNA biomarker sequence is extremely large. Oligonucleotides of virtually any length and sequence complementary to the target could be used, and a variety of chemically modified nucleotides could be substituted for natural nucleotides. Because an exhaustive study of all the potential probe designs is not feasible, we conducted a systematic evaluation of a subset of oligonucleotide probe design parameters. The first probe investigated was RSVN(242–263), a 22-mer DNA probe with a sequence chosen based on previous success as a primer for PCR studies aimed at amplifying RSV N gene cDNA. BSI measurements using the RSVN(242–263) 22-mer probe produced a linear response proportional to the concentration of the RSV N gene RNA with a LOD of 3.73 nM target RNA (Figure 2A and Table 1). As a negative control, a scrambled sequence of the same 22-mer was evaluated under the same conditions and yielded negligible signal. Postulating that probe length would impact BSI signal, probes RSVN(242–256), RSVN(242–285) and RSVN(242-329) were tested, which are 15, 44 and 88 nt in length, respectively, and start from the same position in the target RNA as the 22-mer probe. Each of these probe lengths resulted in slightly less signal and poorer detection limits than the 22-mer probe. To relate the BSI signal to a net increase in base pairs, the relative hybridization of these probes to the RNA target was determined using a SYBR Green assay (Figure 2B). The signal generated from the intercalation of SYBR Green dye in the 15-mer, 44-mer and 88-mer probe:target hybrids was slightly greater than that of the 22-mer probe:target hybrid. Interestingly, we discovered that by dividing the 88-mer probe into four contiguous 22-mer probes, the slope of the BSI response was significantly increased, resulting in a LOD of 2.04 nM target RNA, or ∼4.1 × 105 molecules. This enhancement in sensitivity was reflected by the increase in the net hybridization of the four consecutive 22-mers compared to the 15-mer, 22-mer, 44-mer and 88-mer probes (Figure 2 and Table 1). These results indicate that of the probes tested, the optimal length is 22 nt for BSI detection and that the BSI signal can be enhanced using multiple probes of that length.Figure 2.


The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.

Adams NM, Olmsted IR, Haselton FR, Bornhop DJ, Wright DW - Nucleic Acids Res. (2013)

Comparison of the BSI binding response and net hybridization upon adding the 15-mer, 22-mer, 44-mer, 88-mer or four consecutive 22-mer DNA probes to the RNA target. (A) The probe length that produces optimal BSI signal is 22 nt. Four short 22-mer DNA probes have improved signal over one 88-mer spanning the same target sequence. A scrambled negative control sequence produced negligible signal. (B) Net hybridization of the four consecutive 22-mers is significantly greater than any of the four probe lengths.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643578&req=5

gkt165-F2: Comparison of the BSI binding response and net hybridization upon adding the 15-mer, 22-mer, 44-mer, 88-mer or four consecutive 22-mer DNA probes to the RNA target. (A) The probe length that produces optimal BSI signal is 22 nt. Four short 22-mer DNA probes have improved signal over one 88-mer spanning the same target sequence. A scrambled negative control sequence produced negligible signal. (B) Net hybridization of the four consecutive 22-mers is significantly greater than any of the four probe lengths.
Mentions: The design space of oligonucleotide probes targeting the ∼1300 nt RSV N gene RNA biomarker sequence is extremely large. Oligonucleotides of virtually any length and sequence complementary to the target could be used, and a variety of chemically modified nucleotides could be substituted for natural nucleotides. Because an exhaustive study of all the potential probe designs is not feasible, we conducted a systematic evaluation of a subset of oligonucleotide probe design parameters. The first probe investigated was RSVN(242–263), a 22-mer DNA probe with a sequence chosen based on previous success as a primer for PCR studies aimed at amplifying RSV N gene cDNA. BSI measurements using the RSVN(242–263) 22-mer probe produced a linear response proportional to the concentration of the RSV N gene RNA with a LOD of 3.73 nM target RNA (Figure 2A and Table 1). As a negative control, a scrambled sequence of the same 22-mer was evaluated under the same conditions and yielded negligible signal. Postulating that probe length would impact BSI signal, probes RSVN(242–256), RSVN(242–285) and RSVN(242-329) were tested, which are 15, 44 and 88 nt in length, respectively, and start from the same position in the target RNA as the 22-mer probe. Each of these probe lengths resulted in slightly less signal and poorer detection limits than the 22-mer probe. To relate the BSI signal to a net increase in base pairs, the relative hybridization of these probes to the RNA target was determined using a SYBR Green assay (Figure 2B). The signal generated from the intercalation of SYBR Green dye in the 15-mer, 44-mer and 88-mer probe:target hybrids was slightly greater than that of the 22-mer probe:target hybrid. Interestingly, we discovered that by dividing the 88-mer probe into four contiguous 22-mer probes, the slope of the BSI response was significantly increased, resulting in a LOD of 2.04 nM target RNA, or ∼4.1 × 105 molecules. This enhancement in sensitivity was reflected by the increase in the net hybridization of the four consecutive 22-mers compared to the 15-mer, 22-mer, 44-mer and 88-mer probes (Figure 2 and Table 1). These results indicate that of the probes tested, the optimal length is 22 nt for BSI detection and that the BSI signal can be enhanced using multiple probes of that length.Figure 2.

Bottom Line: Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity.Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity.Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.

ABSTRACT
Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼10(5) target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

Show MeSH
Related in: MedlinePlus