Page 11 - Oligonucleotide Therapeutics Solution Guide

Characteristic analysis
Quality Control

Tm Analysis UV-2600i+TMSPC-8

Features
Tm Analysis of Nucleic Acid Drugs Tm analysis system, TMSPC-8, is connected to a spectrophotometer and measures the Tm of oligonucleotides by heating and cooling. The rate of Modification
temperature increasing/decreasing can be selected from 12 levels of ± 0.1, 0.2, 0.5, 1, 2 and 5 °C. There are 2 types of 8 micro-multi-cells with
optical path length of 10 mm (minimum sample volume of 100 μL) and 1 mm (minimum sample volume of 10 μL), and these cells allow to measure Target selection
click here 8 types of samples simultaneously in parallel.

• The Melting Temperature (Tm) is calculated by two methods, the Average
Method and the differential method.
benefits • Multiple samples can be analyzed in parallel with eight microcells.
• It is possible to calculate thermodynamic parameter analysis as well as Excision
Tm measurement. Unprotected Oligomer synthesis

Methods and Results Table 2 Tm analysis results
Tm (°C)
Sample M13 primer Methods Tm Analysis Software
Forward 5’-CGACGTTGTAAAACGACGGCCAGGA-3’ Ascending process Descending process
Reverse 5‘-TCCTGGCCGTCGTTTTACAACGTCG-3’ As shown in Fig. 3, a typical melting curve has a region corresponding to a double-stranded or single-stranded structure at the low temperature side
Conc., Volume The sample was prepared to have 0.5 to 1 Abs at 260 nm and Average method 77.75 77.31 (pre-transition region) and at the high temperature side (post-transition region)
a salt concentration of 100 mmol / L. 35 µL was added to a cell
having an optical path length of 1 mm. Differential method 77.92 77.85 with a transition region where the absorbance varies greatly. Tm is defined as
Preparation The sample is an equal mixture of Forword and Reverse, heated the temperature at which the mole fractions of the double and single strands Pretransition Transition region Post transition
at 95 ˚C for at least 10 minutes, and then annealed by cooling at are equal. region region
a rate of 2 °C / min. Purification
Tm
Analytical As shown in Table 1 One way to calculate Tm from a melting curve is called the average method.
Conditions
The baselines (tangents) in the pre- and post-transition regions are determined
Results Table 2 shows the results calculated by two types of calculation
methods, the average method and the differential method. In the by the least squares method, and the intersection point between the median Double strand
average method, tangents are drawn for each section selected line of the two tangents and the absorbance curve is the Tm. For more details Abs at 260 nm
in the pre-transition region and post-transition region, and the on this method, refer to "The Average Method Analysis" below. In addition,
Tm is the intersection of the midlines of the two tangents and Single strand
the absorbance curve. As shown in Fig. 1, the Tm is calculated by the method of calculating the first derivative of each point in the specified
arbitrarily setting the pre-transition region and the post-transition temperature range for the absorbance curve, and setting the Tm value as the
region. In the differential method, a first-order differential maximum value of the first derivative is called the differential method.
operation is performed for each point within the set partition,
and the position showing the maximum value is calculated as
the Tm value. As shown in Fig. 2, the judgment area was set The Tm analysis software calculates the Tm. In the average method, the Tm is Temperature
arbitrarily and the Tm value was obtained. calculated by setting a total of four points of the start point and the end point
Figure 3 Example of Melting curve
of the baseline in each of the before transition region and the after transition Quality Control
region. Characteristic analysis
Table 1 Analysis Conditions
Figure 1 Example of analysis by the midline method
Spectrophotometer: UV-2600i The differential method calculates the Tm by setting the range of the temperature region. Since the absorbance changes depending on the
Option: TMSPC-8 temperature even when measuring only the buffer solution, this software has a temperature blank correction function that subtracts the buffer
Cell: eight microcells solution data from the data of the sample in advance. In order to further improve the accuracy of the analysis, it is also equipped with a two-
Optical pathlength: 1 mm wavelength correction function that determines the difference in absorbance by measuring at any two wavelengths.
Wavelength: 260 nm
The Average Method Analysis
Blank Wavelength: 320 nm
Temperature range: 25 ~ 95 °C DDS
Temperature rise: 1 °C The average method is analyzed on the assumption that a simple two-state equilibrium relationship is established between the duplex and the
Ramp rate: 1 °C/ min single strand. The absorbance can be expressed by the following equation, where ε ds and ε ss are the molar molecular absorption coefficients of the
double and single chains, respectively. Pharmacokinetics
Abs = ( ε ds α + ε ss （1-α )) CL
C is the total strands concentration, L is the optical path length, and α is the mole fraction of the duplex. Tm is defined as the temperature at which
alpha equals 1/2. In strict two-state treatment, ε ds and ε ss have constant values regardless of temperature, but they are not actually constant values
and change in proportion to temperature (T), so it is necessary to introduce a term proportional to T.
Figure 2 Example of analysis by differential method ε ds = a ds + b ds T ε ss = a ss + b ss T
The Tm analysis software uses these equations to calculate Tm values as follows:.
Conclusions 1.The user determines a start point and an end point of the baseline in the forward transition region and the backward transition region. Other
2.The software automatically calculates ads, bds, and bss by regression calculation.
Tm Analysis System can calculate Tm for double-stranded DNA and RNA for quality and stability evaluation. Also, by using Tm calculated
3. The software automatically calculates the midline between these two baselines and calculates the intersection of the midline and the absorbance
at various concentrations, more detailed hybridization analysis is possible. curve as the Tm value.

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