Table 1. Melting point evaluation of TINA modified oligonucleotides.
| Antiparallel duplex sequences with/without para- and ortho-TINA | Melting point, Tm (°C) | ||||||
| E. coli rrs target sequence: match 5′ TGGGGAGCAAACAGGATT-ATTO495 3′ (D-624) | Effect of TINA on Tm | E. coli rrs target sequence: mismatch 5′ TGGGGAGCATACAGGATT-ATTO495 3′ (D-634) | Mismatch ΔTm | E. coli rrs target sequence: mismatch 5′ TGGGGAGCAATCAGGATT-ATTO495 3′ (D-809) | Mismatch ΔTm | ||
| Unmodified sequence (no TINA) | |||||||
| LC D-643 | ATTO590- AAT CCT GTT TGC TCC CCA | 68.9 | 61.6 | −7.3 | 61.1 | −7.8 | |
| Modification with ortho -TINA(Y) | |||||||
| LC D-644 | ATTO590-YAAT CCT GTT TGC TCC CCA | 72.8 | 3.9 | 65.0 | −7.8 | 64.9 | −7.9 |
| LC D-645 | ATTO590- AATYCCT GTT TGC TCC CCA | 69.5 | 0.6 | 60.9 | −8.6 | 60.2 | −9.3 |
| LC D-646 | ATTO590- AAT CCTYGTT TGC TCC CCA | 69.2 | 0.3 | 61.9 | −7.3 | 61.1 | −8.1 |
| LC D-647 | ATTO590- AAT CCT GTTYTGC TCC CCA | 68.8 | −0.1 | 64.8 | −4.0* | 61.6 | −7.2 |
| LC D-648 | ATTO590- AAT CCT GTT TGCYTCC CCA | 69.8 | 0.9 | 62.0 | −7.8 | 61.4 | −8.4 |
| LC D-649 | ATTO590- AAT CCT GTT TGC TCCYCCA | 72.0 | 3.1 | 64.1 | −7.9 | 63.8 | −8.2 |
| LC D-650 | ATTO590- AAT CCT GTT TGC TCC CCAY | 70.2 | 1.3 | 62.2 | −8.0 | 62.3 | −7.9 |
| LC D-653 | ATTO590-YAAT CCT GTTYTGC TCC CCA | 72.8 | 3.9 | 68.7 | −4.1* | 65.6 | −7.2 |
| LC D-655 | ATTO590-YAAT CCT GTT TGC TCC CCAY | 74.6 | 5.7 | 66.5 | −8.1 | 66.1 | −8.5 |
| LC D-660 | ATTO590-YAAT CCT GTTYTGC TCC CCAY | 72.8 | 3.9 | 68.4 | −4.4* | 65.6 | −7.2 |
| Modification with para -TINA(X) | |||||||
| LC D-665 | ATTO590-XAAT CCT GTT TGC TCC CCA | 73.7 | 4.8 | 66.3 | −7.4 | 65.6 | −8.1 |
| LC D-666 | ATTO590- AATXCCT GTT TGC TCC CCA | 67.9 | −1.0 | 58.7 | −9.2 | 58.1 | −9.8 |
| LC D-667 | ATTO590- AAT CCTXGTT TGC TCC CCA | 65.2 | −3.7 | 57.9 | −7.3 | 57.8 | −7.4 |
| LC D-668 | ATTO590- AAT CCT GTTXTGC TCC CCA | 63.1 | −5.8 | 59.6 | −3.5* | 57.4 | −5.7 |
| LC D-669 | ATTO590- AAT CCT GTT TGCXTCC CCA | 64.2 | −4.7 | 55.6 | −8.6 | 55.0 | −9.2 |
| LC D-670 | ATTO590- AAT CCT GTT TGC TCCXCCA | 67.7 | −1.2 | 58.9 | −8.8 | 58.8 | −8.9 |
| LC D-671 | ATTO590- AAT CCT GTT TGC TCC CCAX | 67.7 | −1.2 | 60.8 | −6.9 | 60.4 | −7.3 |
| LC D-674 | ATTO590-XAAT CCT GTTXTGC TCC CCA | 69.6 | 0.7 | 65.8 | −3.8* | 63.8 | −5.8 |
| LC D-679 | ATTO590- AAT CCT GTTXTGC TCC CCAX | 64.4 | −4.5 | 60.6 | −3.8* | 57.8 | −6.6 |
| LC D-676 | ATTO590-XAAT CCT GTT TGC TCC CCAX | 76.8 | 7.9 | 68.8 | −8.0 | 68.5 | −8.3 |
| LC D-681 | ATTO590-XAAT CCT GTTXTGC TCC CCAX | 69.5 | 0.6 | 65.7 | −3.8* | 63.9 | −5.6 |
| Modification with para (X)- and ortho (Y)-TINA | |||||||
| LC D-748 | ATTO590-XAATYCCT GTT TGC TCC CCA | 73.2 | 4.3 | 65.3 | −7.9 | 65.0 | −8.2 |
| LC D-749 | ATTO590-XAAT CCTYGTT TGC TCC CCA | 74.6 | 5.7 | 67.6 | −7.0 | 66.3 | −8.3 |
| LC D-750 | ATTO590-XAAT CCT GTTYTGC TCC CCA | 73.7 | 4.8 | 69.9 | −3.8* | 67.3 | −6.4 |
| LC D-751 | ATTO590-XAAT CCT GTT TGCYTCC CCA | 74.8 | 5.9 | 67.4 | −7.4 | 66.7 | −8.1 |
| LC D-754 | ATTO590- AAT CCT GTTYTGC TCC CCAX | 69.8 | 0.9 | 64.9 | −4.9* | 61.6 | −8.2 |
| LC D-756 | ATTO590-XAAT CCT GTTYTGC TCC CCAX | 73.9 | 5.0 | 70.0 | −3.9* | 67.7 | −6.2 |
| Shortened sequence with para (X)- or ortho (Y)-TINA | |||||||
| LC D-661 | ATTO590- AAT CCT GTT TGC TCC C | 66.0 | 58.0 | −8.0 | 57.7 | −8.3 | |
| LC D-662 | ATTO590-YAAT CCT GTT TGC TCC C | 70.3 | 4.3 | 61.9 | −8.4 | 61.5 | −8.8 |
| LC D-663 | ATTO590- AAT CCT GTT TGC TCC CY | 69.0 | 3.0 | 60.5 | −8.5 | 60.4 | −8.6 |
| LC D-664 | ATTO590-YAAT CCT GTT TGC TCC CY | 73.9 | 7.9 | 65.4 | −8.5 | 65.1 | −8.8 |
| LC D-682 | ATTO590-XAAT CCT GTT TGC TCC C | 71.1 | 5.1 | 63.2 | −7.9 | 62.9 | −8.2 |
| LC D-683 | ATTO590- AAT CCT GTT TGC TCC CX | 68.6 | 2.6 | 60.1 | −8.5 | 59.8 | −8.8 |
| LC D-684 | ATTO590-XAAT CCT GTT TGC TCC CX | 75.6 | 9.6 | 66.5 | −9.1 | 66.3 | −9.3 |
Change in Tm and ΔTm of Watson-Crick based antiparallel duplexes stabilized by para-TINA (X) and/or ortho-TINA (Y) molecules. Tm was determined using 0.5 µM of each strand in 50 mM phosphate buffer, pH 7.0, with 100 mM NaCl and 0.1 mM EDTA. Tm was defined as the peak of the first derivate using both annealing and dissociation curves. Base mismatches are underlined and marked in bold black. *Mismatch adjacent to TINA.